HUMAN HEPATOCYTE GROWTH FACTOR MUTANT AND USES THEREOF
20220064242 · 2022-03-03
Inventors
Cpc classification
A61P25/28
HUMAN NECESSITIES
C07K14/4753
CHEMISTRY; METALLURGY
A61P9/10
HUMAN NECESSITIES
A61K48/005
HUMAN NECESSITIES
International classification
Abstract
Disclosed is a human hepatocyte growth factor (hHGF) mutant. Also disclosed are a nucleic acid molecule encoding the mutant, a carrier containing the nucleic acid molecule, and a host cell containing the nucleic acid molecule or the carrier. At the same time disclosed are a pharmaceutical composition comprising the hHGF mutant or the nucleic acid molecule encoding the mutant, and the uses of the hHGF mutant or the nucleic acid molecule encoding the mutant.
Claims
1. A mutant of human hepatocyte growth factor (hHGF), which, compared with a natural hHGF, comprises a mutation as follows: the amino acid of the natural hHGF at a position corresponding to the 130.sup.th position of SEQ ID NO:1 is mutated into an amino acid with a basic side chain.
2. The mutant according to claim 1, which has one or more characteristics selected from the following: (1) the amino acid with a basic side chain is selected from arginine, histidine and lysine; (2) the natural hHGF has an amino acid sequence as shown in SEQ ID NO: 1; (3) the mutant has an amino acid sequence selected from SEQ ID NOs: 2, 3 and 4; (4) the mutant is modified; (5) the hHGF mutant is prepared by a recombinant expression method, or prepared by a chemical synthesis method; (6) the mutant is chemically modified; and (7) the mutant is modified by PEGylation.
3. An isolated nucleic acid molecule, which comprises a nucleotide sequence encoding the mutant according to claim 1.
4. A vector, which comprises the isolated nucleic acid molecule according to claim 3.
5. A host cell, which comprises the isolated nucleic acid molecule according to claim 3 or a vector comprising the isolated nucleic acid molecule.
6. A method for preparing the mutant according to claim 1, which comprises (i) culturing a host cell under a suitable condition, wherein the host cell comprises a nucleic acid molecule encoding the mutant and is able to express the mutant; and (ii) recovering the mutant from a cell culture of the host cell.
7.-12. (canceled)
13. The isolated nucleic acid molecule according to claim 3, wherein the isolated nucleic acid molecule has one or more characteristics selected from the following: (1) the nucleic acid molecule has a nucleotide sequence that is codon-optimized according to the preference of a host cell; and (2) the nucleic acid molecule has a nucleotide sequence selected from SEQ ID NOs: 6, 7 and 8.
14. The vector according to claim 4, wherein the vector has one or more characteristics selected from the following: (1) the vector is selected from the group consisting of plasmid, phagemid, cosmid, artificial chromosome, bacteriophage, and viral vector; (2) the vector is selected from the group consisting of naked plasmid, yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), P1-derived artificial chromosome (PAC), lambda phag, M13 phage, retroviral vector, adenovirus vector, adeno-associated virus vector, herpes virus vector, poxvirus vector, baculovirus vector, papilloma virus vector, and papovavirus vector; (3) the vector is a lentiviral vector or herpes simplex virus vector; (4) the vector is a vector for gene therapy; and (5) the vector is a pSN vector containing the nucleic acid molecule.
15. The host cell according to claim 5, wherein the host cell has one or more characteristics selected from the following: (1) the host cell is a prokaryotic cell or eukaryotic cell; (2) the host cell is selected from the group consisting of E. coli cell, yeast cell, insect cell, plant cell and animal cell; and (3) the host cell is a mouse cell or human cell.
16. The method according to claim 6, wherein the method comprises the following steps: (1) constructing of an expression vector, wherein the expression vector comprising a nucleic acid sequence encoding the mutant; (2) introducing the expression vector into a host cell, and culturing the host cell under a condition that allows protein expression; and (3) isolating and recovering the mutant from a cell culture of the host cell.
17. The method according to claim 16, wherein the method is characterized by one or more of the following: (1) the mutant has the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4; (2) the host cell is a CHO cell; and (3) the mutant is isolated and recovered from the cell culture of the host cell by anion exchange chromatography and heparin affinity chromatography.
18. A pharmaceutical composition, which comprises one or more of the following, together with a pharmaceutically acceptable carrier and/or excipient: (1) the mutant according to claim 1; (2) an isolated nucleic acid molecule encoding the mutant; and (3) a vector comprising the isolated nucleic acid molecule.
19. A method for treating a disease that can benefit from an activity of a natural hHGF in a subject, which comprises administering to a subject in need thereof a therapeutically effective amount of the following: (1) the mutant according to claim 1; (2) an isolated nucleic acid molecule encoding the mutant; (3) a vector comprising the isolated nucleic acid molecule; (4) a pharmaceutical composition comprising the mutant or the isolated nucleic acid molecule or the vector; or (5) any combination thereof.
20. The method according to claim 19, wherein the method is characterized by one or more of the following: (1) the disease is selected from the group consisting of ischemic disease, metabolic syndrome, diabetes and complication thereof, restenosis, and nerve damage; (2) the disease is selected from the group consisting of coronary artery disease (CAD), peripheral artery disease (PAD), restenosis after surgery, restenosis after perfusion, neurodegenerative diseases, traumatic nerve injury, and peripheral neuropathy; (3) the disease is myocardial infarction, lower extremity artery ischemia, amyotrophic lateral sclerosis (ALS), Parkinson's disease, dementia, or diabetic peripheral neuropathy; (4) the subject is a mammal; and (5) the subject is a human.
21. A method for promoting the growth and/or migration of an endothelial cell, which comprises administering to an endothelial cell or subject in need thereof an effective amount of the following: (1) the mutant according to claim 1; (2) an isolated nucleic acid molecule encoding the mutant; (3) a vector comprising the isolated nucleic acid molecule; (4) a pharmaceutical composition comprising the mutant or the isolated nucleic acid molecule or the vector; or (5) any combination thereof.
22. The method according to claim 21, wherein the endothelial cell is an umbilical vein endothelial cell.
23. A method for promoting the formation of a blood vessel, which comprises administering to a subject in need thereof an effective amount of the following: (1) the mutant according to claim 1; (2) an isolated nucleic acid molecule encoding the mutant; (3) a vector comprising the isolated nucleic acid molecule; (4) a pharmaceutical composition comprising the mutant or the isolated nucleic acid molecule or the vector; or (5) any combination thereof.
24. The method according to claim 23, wherein the blood vessel is a microvessel.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0092]
DESCRIPTION OF SEQUENCE INFORMATION
[0093] The information of the sequences involved in the present invention is provided as follows.
TABLE-US-00001 (amino acid sequence of natural hHGF) SEQ ID NO: 1 QRKRRNTIHEFKKSAKTTLIKIDPALKIKTKKVNTADQCANRCTRNKGLPFTCKAFVFDKARKQC LWFPFNSMSSGVKKEFGHEFDLYENKDYIRNCIIGKGRSYKGTVSITKSGIKCQPWSSMIPHEHSF LPSSYRGKDLQENYCRNPRGEEGGPWCFTSNPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHT ESGKICQRWDHQTPHRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCA DNTMNDTDVPLETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLREN YCRNPDGSESPWCFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYMGNLSQTRSGLTCSMW DKNMEDLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTTPTIVN LDHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRDLKDY EAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLPNYGCTIPE KTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAGAEKIGSGPCEGD YGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKIILTYKVPQS (amino acid sequence of 130Arg-hHGF) SEQ ID NO: 2 QRKRRNTIHEFKKSAKTTLIKIDPALKIKTKKVNTADQCANRCTRNKGLPFTCKAFVFDKARKQC LWFPFNSMSSGVKKEFGHEFDLYENKDYIRNCIIGKGRSYKGTVSITKSGIKCQPWSSMIPHEHRF LPSSYRGKDLQENYCRNPRGEEGGPWCFTSNPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHT ESGKICQRWDHQTPHRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCA DNTMNDTDVPLETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLREN YCRNPDGSESPWCFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYMGNLSQTRSGLTCSMW DKNMEDLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTTPTIVN LDHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRDLKDY EAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLPNYGCTIPE KTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAGAEKIGSGPCEGD YGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKIILTYKVPQS (amino acid sequence of 130His-hHGF) SEQ ID NO: 3 QRKRRNTIHEFKKSAKTTLIKIDPALKIKTKKVNTADQCANRCTRNKGLPFTCKAFVFDKARKQC LWFPFNSMSSGVKKEFGHEFDLYENKDYIRNCIIGKGRSYKGTVSITKSGIKCQPWSSMIPHEHHF LPSSYRGKDLQENYCRNPRGEEGGPWCFTSNPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHT ESGKICQRWDHQTPHRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCA DNTMNDTDVPLETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLREN YCRNPDGSESPWCFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYMGNLSQTRSGLTCSMW DKNMEDLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTTPTIVN LDHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRDLKDY EAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLPNYGCTIPE KTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAGAEKIGSGPCEGD YGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKIILTYKVPQS (amino acid sequence of 130Lys-hHGF) SEQ ID NO: 4 QRKRRNTIHEFKKSAKTTLIKIDPALKIKTKKVNTADQCANRCTRNKGLPFTCKAFVFDKARKQC LWFPFNSMSSGVKKEFGHEFDLYENKDYIRNCIIGKGRSYKGTVSITKSGIKCQPWSSMIPHEHKF LPSSYRGKDLQENYCRNPRGEEGGPWCFTSNPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHT ESGKICQRWDHQTPHRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCA DNTMNDTDVPLETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLREN YCRNPDGSESPWCFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYMGNLSQTRSGLTCSMW DKNMEDLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTTPTIVN LDHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRDLKDY EAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLPNYGCTIPE KTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAGAEKIGSGPCEGD YGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKIILTYKVPQS (nucleotide sequence encoding natural hHGF) SEQ ID NO: 5 caaaggaaaa gaagaaatac aattcatgaa ttcaaaaaat cagcaaagac taccctaatc 60 aaaatagatc cagcactgaa gataaaaacc aaaaaagtga atactgcaga ccaatgtgct 120 aatagatgta ctaggaataa aggacttcca ttcacttgca aggcttttgt ttttgataaa 180 gcaagaaaac aatgcctctg gttccccttc aatagcatgt caagtggagt gaaaaaagaa 240 tttggccatg aatttgacct ctatgaaaac aaagactaca ttagaaactg catcattggt 300 aaaggacgca gctacaaggg aacagtatct atcactaaga gtggcatcaa atgtcagccc 360 tggagttcca tgataccaca cgaacacagc tttttgcctt cgagctatcg gggtaaagac 420 ctacaggaaa actactgtcg aaatcctcga ggggaagaag ggggaccctg gtgtttcaca 480 agcaatccag aggtacgcta cgaagtctgt gacattcctc agtgttcaga agttgaatgc 540 atgacctgca atggggagag ttatcgaggt ctcatggatc atacagaatc aggcaagatt 600 tgtcagcgct gggatcatca gacaccacac cggcacaaat tcttgcctga aagatatccc 660 gacaagggct ttgatgataa ttattgccgc aatcccgatg gccagccgag gccatggtgc 720 tatactcttg accctcacac ccgctgggag tactgtgcaa ttaaaacatg cgctgacaat 780 actatgaatg acactgatgt tcctttggaa acaactgaat gcatccaagg tcaaggagaa 840 ggctacaggg gcactgtcaa taccatttgg aatggaattc catgtcagcg ttgggattct 900 cagtatcctc acgagcatga catgactcct gaaaatttca agtgcaagga cctacgagaa 960 aattactgcc gaaatccaga tgggtctgaa tcaccctggt gttttaccac tgatccaaac 1020 atccgagttg gctactgctc ccaaattcca aactgtgata tgtcacatgg acaagattgt 1080 tatcgtggga atggcaaaaa ttatatgggc aacttatccc aaacaagatc tggactaaca 1140 tgttcaatgt gggacaagaa catggaagac ttacatcgtc atatcttctg ggaaccagat 1200 gcaagtaagc tgaatgagaa ttactgccga aatccagatg atgatcttca tggaccctgg 1260 tgctacacgg gaaatccact cattccttgg gattattgcc ctatttctcg ttgtgaaggt 1320 gataccacac ctacaatagt caatttagac catcccgtaa tatcttgtgc caaaacgaaa 1380 caattgcgag ttgtaaatgg gattccaaca cgaacaaaca taggatggat ggttagtttg 1440 agatacagaa ataaacatat ctgcggagga tcattgataa aggagagttg ggttcttact 1500 gcacgacagt gtttcccttc tcgagacttg aaagattatg aagcttggct tggaattcat 1560 gatgtccacg gaagaggaga tgagaaatgc aaacaggttc tcaatgtttc ccagaggta 1620 tatggccctg aaggatcaga tctggtttta atgaagcttg ccaggcctgc tgtcctggat 1680 gattttgtta gtacgattga tttacctaat tatggatgca caattcctga aaagaccagt 1740 tgcagtgttt atggctgggg ctacactgga ttgatcaact atgatggcct attacgagtg 1800 gcacatact atataatggg aaatgagaaa tgcagccagc atcatcgagg gaaggtgact 1860 ctgaatgagt ctgaaatatg tgctggggct gaaaagattg gatcaggacc atgtgagggg 1920 gattatggtg gcccacttgt ttgtgagcaa cataaaatga gaatggttct tggtgtcatt 1980 gttcctggtc gtggatgtgc cattccaaat cgtcctggta tttttgtccg agtagcatat 2040 tatgcaaaat ggatacacaa aattatttta acatataagg taccacagtc atag 2094 (nucleotide sequence encoding 130Arg-hHGF) SEQ ID NO: 6 caaaggaaaa gaagaaatac aattcatgaa ttcaaaaaat cagcaaagac taccctaatc 60 aaaatagatc cagcactgaa gataaaaacc aaaaaagtga atactgcaga ccaatgtgct 120 aatagatgta ctaggaataa aggacttcca ttcacttgca aggattttgt ttttgataaa 180 gcaagaaaac aatgcctctg gttccccttc aatagcatgt caagtggagt gaaaaaagaa 240 tttggccatg aatttgacct ctatgaaaac aaagactaca ttagaaactg catcattggt 300 aaaggacgca gctacaaggg aacagtatct atcactaaga gtggcatcaa atgtcagccc 360 tggagttcca tgataccaca cgaacacaga tttttgcctt cgagctatcg gggtaaagac 420 ctacaggaaa actactgtcg aaatcctcga ggggaagaag ggggaccctg gtgtttcaca 480 agcaatccag aggtacgcta cgaagtctgt gacattcctc agtgttcaga agttgaatgc 540 atgacctgca atggggagag ttatcgaggt ctcatggatc atacagaatc aggcaagatt 600 tgtcagcgct gggatcatca gacaccacac cggcacaaat tcttgcctga aagatatccc 660 gacaagggct ttgatgataa ttattgccgc aatcccgatg gccagccgag gccatggtgc 720 tatactcttg accctcacac ccgctgggag tactgtgcaa ttaaaacatg cgctgacaat 780 actatgaatg acactgatgt tcctttggaa acaactgaat gcatccaagg tcaaggagaa 840 ggctacaggg gcactgtcaa taccatttgg aatggaattc catgtcagcg ttgggattct 900 cagtatcctc acgagcatga catgactcct gaaaatttca agtgcaagga cctacgagaa 960 aattactgcc gaaatccaga tgggtctgaa tcaccctggt gttttaccac tgatccaaac 1020 atccgagttg gctactgctc ccaaattcca aactgtgata tgtcacatgg acaagattgt 1080 tatcgtggga atggcaaaaa ttatatgggc aacttatccc aaacaagatc tggactaaca 1140 tgttcaatgt gggacaagaa catggaagac ttacatcgtc atatcttctg ggaaccagat 1200 gcaagtaagc tgaatgagaa ttactgccga aatccagatg atgatgctca tggaccctgg 1260 tgctacacgg gaaatccact cattccttgg gattattgcc ctatttctcg ttgtgaaggt 1320 gataccacac ctacaatagt caatttagac catcccgtaa tatcttgtgc caaaacgaaa 1380 caattgcgag ttgtaaatgg gattccaaca cgaacaaaca taggatggat ggttagtttg 1440 agatacagaa ataaacatat ctgcggagga tcattgataa aggagagttg ggttcttact 1500 gcacgacagt gtttcccttc tcgagacttg aaagattatg aagcttggct tggaattcat 1560 gatgtccacg gaagaggaga tgagaaatgc aaacaggttc tcaatgtttc ccagctggta 1620 tatggccctg aaggatcaga tctggtttta atgaagcttg ccaggcctgc tgtcctggat 1680 gattttgtta gtacgattga tttacctaat tatggatgca caattcctga aaagaccagt 1740 tgcagtgttt atggctgggg ctacactgga ttgatcaact atgatggcct attacgagtg 1800 gcacatctct atataatggg aaatgagaaa tgcagccagc atcatcgagg gaaggtgact 1860 ctgaatgagt ctgaaatatg tgctggggct gaaaagattg gatcaggacc atgtgagggg 1920 gattatggtg gcccacttgt ttgtgagcaa cataaaatga gaatggttct tggtgtcatt 1980 gttcctggtc gtggatgtgc cattccaaat cgtcctggta tttttgtccg agtagcatat 2040 tatgcaaaat ggatacacaa aattatttta acatataagg taccacagtc atag 2094 (nucleotide sequence encoding 130His-hHGF) SEQ ID NO: 7 caaaggaaaa gaagaaatac aattcatgaa ttcaaaaaat cagcaaagac taccctaatc 60 aaaatagatc cagcactgaa gataaaaacc aaaaaagtga atactgcaga ccaatgtgct 120 aatagatgta ctaggaataa aggacttcca ttcacttgca aggattttgt ttttgataaa 180 gcaagaaaac aatgcctctg gttccccttc aatagcatgt caagtggagt gaaaaaagaa 240 tttggccatg aatttgacct ctatgaaaac aaagactaca ttagaaactg catcattggt 300 aaaggacgca gctacaaggg aacagtatct atcactaaga gtggcatcaa atgtcagccc 360 tggagttcca tgataccaca cgaacaccac tttttgcctt cgagctatcg gggtaaagac 420 ctacaggaaa actactgtcg aaatcctcga ggggaagaag ggggaccctg gtgtttcaca 480 agcaatccag aggtacgcta cgaagtctgt gacattcctc agtgttcaga agttgaatgc 540 atgacctgca atggggagag ttatcgaggt ctcatggatc atacagaatc aggcaagatt 600 tgtcagcgct gggatcatca gacaccacac cggcacaaat tcttgcctga aagatatccc 660 gacaagggct ttgatgataa ttattgccgc aatcccgatg gccagccgag gccatggtgc 720 tatactcttg accctcacac ccgctgggag tactgtgcaa ttaaaacatg cgctgacaat 780 actatgaatg acactgatgt tcctttggaa acaactgaat gcatccaagg tcaaggagaa 840 ggctacaggg gcactgtcaa taccatttgg aatggaattc catgtcagcg ttgggattct 900 cagtatcctc acgagcatga catgactcct gaaaatttca agtgcaagga cctacgagaa 960 aattactgcc gaaatccaga tgggtctgaa tcaccctggt gttttaccac tgatccaaac 1020 atccgagttg gctactgctc ccaaattcca aactgtgata tgtcacatgg acaagattgt 1080 tatcgtggga atggcaaaaa ttatatgggc aacttatccc aaacaagatc tggactaaca 1140 tgttcaatgt gggacaagaa catggaagac ttacatcgtc atatcttctg ggaaccagat 1200 gcaagtaagc tgaatgagaa ttactgccga aatccagatg atgatgctca tggaccctgg 1260 tgctacacgg gaaatccact cattccttgg gattattgcc ctatttctcg ttgtgaaggt 1320 gataccacac ctacaatagt caatttagac catcccgtaa tatcttgtgc caaaacgaaa 1380 caattgcgag ttgtaaatgg gattccaaca cgaacaaaca taggatggat ggttagtttg 1440 agatacagaa ataaacatat ctgcggagga tcattgataa aggagagttg ggttcttact 1500 gcacgacagt gtttcccttc tcgagacttg aaagattatg aagcttggct tggaattcat 1560 gatgtccacg gaagaggaga tgagaaatgc aaacaggttc tcaatgtttc ccagctggta 1620 tatggccctg aaggatcaga tctggtttta atgaagcttg ccaggcctgc tgtcctggat 1680 gattttgtta gtacgattga tttacctaat tatggatgca caattcctga aaagaccagt 1740 tgcagtgttt atggctgggg ctacactgga ttgatcaact atgatggcct attacgagtg 1800 gcacatctct atataatggg aaatgagaaa tgcagccagc atcatcgagg gaaggtgact 1860 ctgaatgagt ctgaaatatg tgctggggct gaaaagattg gatcaggacc atgtgagggg 1920 gattatggtg gcccacttgt ttgtgagcaa cataaaatga gaatggttct tggtgtcatt 1980 gttcctggtc gtggatgtgc cattccaaat cgtcctggta tttttgtccg agtagcatat 2040 tatgcaaaat ggatacacaa aattatttta acatataagg taccacagtc atag 2094 (nucleotide sequence encoding 130Lys-hHGF) SEQ ID NO: 8 caaaggaaaa gaagaaatac aattcatgaa ttcaaaaaat cagcaaagac taccctaatc 60 aaaatagatc cagcactgaa gataaaaacc aaaaaagtga atactgcaga ccaatgtgct 120 aatagatgta ctaggaataa aggacttcca ttcacttgca aggcttttgt ttttgataaa 180 gcaagaaaac aatgcctctg gttccccttc aatagcatgt caagtggagt gaaaaaagaa 240 tttggccatg aatttgacct ctatgaaaac aaagactaca ttagaaactg catcattggt 300 aaaggacgca gctacaaggg aacagtatct atcactaaga gtggcatcaa atgtcagccc 360 tggagttcca tgataccaca cgaacacaag tttttgcctt cgagctatcg gggtaaagac 420 ctacaggaaa actactgtcg aaatcctcga ggggaagaag ggggaccctg gtgtttcaca 480 agcaatccag aggtacgcta cgaagtctgt gacattcctc agtgttcaga agttgaatgc 540 atgacctgca atggggagag ttatcgaggt ctcatggatc atacagaatc aggcaagatt 600 tgtcagcgct gggatcatca gacaccacac cggcacaaat tcttgcctga aagatatccc 660 gacaagggct ttgatgataa ttattgccgc aatcccgatg gccagccgag gccatggtgc 720 tatactcttg accctcacac ccgctgggag tactgtgcaa ttaaaacatg cgctgacaat 780 actatgaatg acactgatgt tcctttggaa acaactgaat gcatccaagg tcaaggagaa 840 ggctacaggg gcactgtcaa taccatttgg aatggaattc catgtcagcg ttgggattct 900 cagtatcctc acgagcatga catgactcct gaaaatttca agtgcaagga cctacgagaa 960 aattactgcc gaaatccaga tgggtctgaa tcaccctggt gttttaccac tgatccaaac 1020 atccgagttg gctactgctc ccaaattcca aactgtgata tgtcacatgg acaagattgt 1080 tatcgtggga atggcaaaaa ttatatgggc aacttatccc aaacaagatc tggactaaca 1140 tgttcaatgt gggacaagaa catggaagac ttacatcgtc atatcttctg ggaaccagat 1200 gcaagtaagc tgaatgagaa ttactgccga aatccagatg atgatgctca tggaccctgg 1260 tgctacacgg gaaatccact cattccttgg gattattgcc ctatttctcg ttgtgaaggt 1320 gataccacac ctacaatagt caatttagac catcccgtaa tatcttgtgc caaaacgaaa 1380 caattgcgag ttgtaaatgg gattccaaca cgaacaaaca taggatggat ggttagtttg 1440 agatacagaa ataaacatat ctgcggagga tcattgataa aggagagttg ggttcttact 1500 gcacgacagt gtttcccttc tcgagacttg aaagattatg aagcttggct tggaattcat 1560 gatgtccacg gaagaggaga tgagaaatgc aaacaggttc tcaatgtttc ccagctggta 1620 tatggccctg aaggatcaga tctggtttta atgaagcttg ccaggcctgc tgtcctggat 1680 gattttgtta gtacgattga tttacctaat tatggatgca caattcctga aaagaccagt 1740 tgcagtgttt atggctgggg ctacactgga ttgatcaact atgatggcct attacgagtg 1800 gcacatctct atataatggg aaatgagaaa tgcagccagc atcatcgagg gaaggtgact 1860 ctgaatgagt ctgaaatatg tgctggggct gaaaagattg gatcaggacc atgtgagggg 1920 gattatggtg gcccacttgt ttgtgagcaa cataaaatga gaatggttct tggtgtcatt 1980 gttcctggtc gtggatgtgc cattccaaat cgtcctggta tttttgtccg agtagcatat 2040 tatgcaaaat ggatacacaa aattatttta acatataagg taccacagtc atag 2094 (nucleotide sequence of pSN vector) SEQ ID NO: 9 gcgcagcacc atggcctgaa ataacctctg aaagaggaac ttggttaggt accttctgag 60 gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc 120 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt 180 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca 240 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc 300 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg 360 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagcttg 420 ctagccaccg cggccgcaac ttgtttattg cagcttataa tggttacaaa taaagcaata 480 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca 540 aactcatcaa tgtatcttat catgtctgga tccaggataa tatatggtag ggttcatagc 600 cagagtaacc ttttttttta atttttattt tattttattt tgagctgcag gcatgcaagc 660 tggcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta 720 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg 780 atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgcctgatg cggtattttc 840 tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct 900 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac 960 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca 1020 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac 1080 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt 1140 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 1200 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta 1260 tgctggggag tcgaaattca gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc 1320 gaatcgggag cggcgatacc gtaaagcacg aggaagcggt cagcccattc gccgccaagc 1380 tcttcagcaa tatcacgggt agccaacgct atgtcctgat agcggtccgc cacacccagc 1440 cggccacagt cgatgaatcc agaaaagcgg ccattttcca ccatgatatt cggcaagcag 1500 gcatcgccat gggtcacgac gagatcctcg ccgtcgggca tgctcgcctt gagcctggcg 1560 aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg atcgacaaga 1620 ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg 1680 caggtagccg gatcaagcgt atgcagccgc cgcattgcat cagccatgat ggatactttc 1740 tcggcaggag caaggtgaga tgacaggaga tcctgccccg gcacttcgcc caatagcagc 1800 cagtcccttc ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg 1860 gccagccacg atagccgcgc tgcctcgtct tgcagttcat tcagggcacc ggacaggtcg 1920 gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc ggcatcagag 1980 cagccgattg tctgttgtgc ccagtcatag ccgaatagcc tctccaccca agcggccgga 2040 gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg atcctcatcc tgtctcttga 2100 tcagatcttg atccctgtca gaccaagttt actcatatat actttagatt gatttaaaac 2160 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa 2220 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 2280 cttcttgaga tccttifitt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 2340 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 2400 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc 2460 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 2520 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 2580 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 2640 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg 2700 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 2760 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 2820 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 2880 gcaaccgg 2889
Specific Models for Carrying Out the Present Invention
[0094] The present application will be described with reference to the following examples which are intended to illustrate (not limit the present application) the present application.
[0095] Unless otherwise specified, the molecular biology experimental methods and immunoassay methods used in the present application basically refer to J. Sambrook et al., Molecular Cloning: Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., Compiled Molecular Biology Experiment Guide, 3rd edition, John Wiley & Sons, Inc., 1995; the restriction enzymes were used in accordance with the conditions recommended by the product manufacturer. Those skilled in the art know that the examples describe the present application by way of example, and are not intended to limit the scope sought to be protected by the present application.
Example 1: Preparation of hHGF and its Mutants
[0096] The amino acid sequence of nature hHGF (SEQ ID NO: 1) could be found in NCBI accession number NP_000592.3. Using SEQ ID NO:1 as a template, the following three hHGF mutants were designed:
[0097] (1) hHGF mutant 130Arg-hHGF, that was obtained by mutating Ser at position 130 of SEQ ID NO: 1 to Arg, and its amino acid sequence was shown in SEQ ID NO: 2;
[0098] (2) hHGF mutant 130His-hHGF, that was obtained by mutating Ser at position 130 of SEQ ID NO: 1 to His, and its amino acid sequence was shown in SEQ ID NO: 3;
[0099] (2) hHGF mutant 130Lys-hHGF, that was obtained by mutating Ser at position 130 of SEQ ID NO: 1 to Lys, and its amino acid sequence was shown in SEQ ID NO: 4.
[0100] Full-gene synthesis was performed to obtain the polynucleotides encoding natural hHGF (SEQ ID NO: 1) and the above three hHGF mutants (SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4), respectively, restriction enzyme cleavage sites and initiation codons were introduced at their 5′ ends, and restriction enzyme cleavage sites and termination codons were introduced at their 3′ ends, so as to obtain nucleic acid molecules encoding natural hHGF and various mutants.
[0101] The nucleic acid molecules prepared as described above were cloned into expression vectors, and transformed into CHO host cells, respectively. Under conditions that allowed the expression of foreign proteins, the transformed CHO host cells were cultured, and then the cultures were collected and centrifuged to obtain supernatants containing the target proteins (natural hHGF, 130Arg-hHGF, 130His-hHGF, or 130Lys-hHGF). According to the manufacturer's instructions, an anion exchange chromatography medium (DEAE Sepharose Fast Flow, GE healthcare, 17-0709-10) was used to separate the target proteins in the supernatants, and a heparin affinity chromatography medium (Heparin Sepharose 6 Fast Flow, GE healthcare, 17-0998-01) was used to further purify the target proteins.
[0102] The purified target proteins were detected by non-reduced polyacrylamide gel electrophoresis (non-reduced SDS-PAGE, Molecular Cloning Experiment Guide, 4.sup.th Edition), and the results were shown in
Example 2: Preparation of Recombinant Plasmids Encoding hHGF and its Mutants
[0103] Full-gene synthesis was performed to obtain polynucleotides respectively encoding natural hHGF (SEQ ID NO: 1) and the above three hHGF mutants (SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4), which respectively had the nucleotide sequences as shown in SEQ ID NOs: 5-8. The polynucleotide molecules prepared as above were respectively cloned into pSN vector (see, for example, Chinese Patent CN 108611367 B; its nucleotide sequence was shown in SEQ ID NO: 9), and then transformed into E. coli. After screening and sequencing verification, engineered strains containing the target recombinant plasmids were obtained. The constructed engineered strains were subjected to fermentation, and the plasmids were extracted to obtain stock solutions containing the target recombinant plasmids. The target recombinant plasmids were specifically as follows:
[0104] (1) pSN-hHGF, which carried the polynucleotide (SEQ ID NO: 5) encoding natural hHGF (SEQ ID NO: 1);
[0105] (2) pSN-130Arg-hHGF, which carried the polynucleotide (SEQ ID NO: 6) encoding 130Arg-hHGF (SEQ ID NO: 2);
[0106] (3) pSN-130His-hHGF, which carried the polynucleotide (SEQ ID NO: 7) encoding 130His-hHGF (SEQ ID NO: 3); and
[0107] (4) pSN-130Lys-hHGF, which carried the polynucleotide (SEQ ID NO: 8) encoding 130Lys-hHGF (SEQ ID NO: 4).
[0108] The contents of plasmids in the prepared stock solutions were determined by using ultraviolet spectrophotometer. The results showed that the contents of plasmids in the various prepared stock solutions were in the range of 2.0 to 2.2 mg/mL. Specifically, the contents of recombinant plasmids in the four stock solutions were: 2.12 mg/mL (pSN-hHGF), 2.05 mg/mL (pSN-130Arg-hHGF), 2.15 mg/mL (pSN-130His-hHGF) and 2.10 mg/mL (pSN-130Lys-hHGF), respectively.
[0109] The stock solutions containing the target recombinant plasmids were taken, diluted with water for injection to plasmid concentration of about 30 μg/ml, and then subjected to purity detection by HPLC. The detection conditions used were as follows:
[0110] The chromatographic column used was an anion exchange HPLC analytical column DNA-NPR, which was equilibrated with a buffer of 20 mM Tris-HCl, 0.5M NaCl, pH8.8. After equilibration, the samples were loaded and detected. The loading volume was 100 μl, the flow rate was 0.5 ml/min, and the detection wavelength was 260 nm. After loading the samples, the equilibration was performed by using a buffer of 20 mM Tris-HCl, 0.5M NaCl, pH8.8 (5 min), and then linear gradient elution was carried out under the conditions as follows: (1) by linearly transiting from 100% of solution A (the solution A was 20 mM Tris-HCl, 0.5M NaCl, pH8.8) to 100% of solution B (the solution B was 20 mM Tris-HCl, 0.8M NaCl), the elution was performed for 30 min; and (2) then by using a buffer of 20 mM Tris-HCl, 0.8M NaCl, pH8.8, the elution was performed for 5 min. The results showed that in the various samples of the prepared stock solutions, the plasmids all had HPLC purity of greater than 95.0%. Specifically, the HPLC purity values of the recombinant plasmids in the four stock solutions were: 97.5% (pSN-hHGF), 98.2% (pSN-130Arg-hHGF), 98.0% (pSN-130His-hHGF), 97.8% (pSN-130Lys-hHGF), respectively.
Example 3: Evaluation of In Vitro Biological Activity of hHGF Mutants and Natural hHGF
[0111] In this example, an in vitro endothelial cell migration experiment was used to evaluate the effects of hHGF mutants and natural hHGF on endothelial cell migration, so as to evaluate the in vitro biological activities of hHGF mutants and natural hHGF.
[0112] 1. Materials and Methods
[0113] 1.1 Protein Samples
[0114] The hHGF mutants prepared as above (130Arg-hHGF, 130His-hHGF and 130Lys-hHGF) and natural hHGF were formulated to have required concentrations with normal saline before use.
[0115] 1.2 Cell Line
[0116] ECV304 cell line (umbilical vein endothelial cells) was used to test the biological activity of HGF.
[0117] 1.3 Reagents
[0118] DMEM medium: Provided by Hyclone. The preparation method was as follows: 1 bag of DMEM medium powder (specification was 1 L) was taken, added with water to dissolve and diluted to 1000 ml, and then added with 2.1 g of sodium bicarbonate. Then, the prepared medium was sterilized and filtered, and stored at 4° C.
[0119] Complete medium: 100 ml of fetal bovine serum was taken and added with the DMEM medium to 1000 ml.
[0120] Transwells: Provided by Costar.
[0121] 1.4 Instruments
[0122] Carbon dioxide cell incubator: Provided by Medical Equipment Factory of Shanghai Boxun Industrial Co., Ltd., model: HH. CP.
[0123] Inverted microscope: Provided by Chongqing Optoelectronic Instrument Corporation, model: XDS-1B.
[0124] Ultra-clean workbench: Provided by Suzhou Purification Equipment Co., Ltd., model: SW-CJ-1F.
[0125] Bench-top cell washing centrifuge: Provided by Hunan Xingke Scientific Instrument Co., Ltd., model TDL-50B.
[0126] Optical microscope: Provided by Chongqing Optoelectronic Instrument Corporation, model: BP104.
[0127] 1.5 Experimental Method
[0128] According to the method of “in vitro HGF activity detection test” (The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes. J. Exp. Med., 1962, 115: 453-466), the cell migration test was performed. In short, 600 μl of DMEM medium was added into each well of the lower tank of the migration plate so as to submerge the Transwells therein. The ECV304 cells digested with 0.1% trypsin were prepared with 1640 medium containing 10% fetal bovine serum to form a cell suspension containing 1×10.sup.6 cells per 1 ml. To each well, 200 μl of cell suspension was added and incubated at 37° C. for 1 h. Then, the original DMEM medium in the lower tank of the migration plate was replaced with 600 μl of medium containing 2 μg of natural hHGF or hHGF mutant, and the incubation was continued for 2 hours. After the incubation, the Transwells were transferred to another well with 20% paraformaldehyde, and the cells were fixed for 10 minutes. The non-migrated cells on the membrane were gently wiped off using cotton swab, and then stained with crystal violet for 5 min. The membrane was carefully removed using scalpel blade, placed on a glass slide (the side with cells was up), and observed with an optical microscope. In addition, a blank control that did not use natural hHGF or hHGF mutant is also provided.
[0129] In this test, the count of migrating cells was used to evaluate the biological activity of the protein to be tested (natural hHGF or hHGF mutant). The method of quantitatively evaluating cell migration with optical microscope was as follows: firstly, an area with uniform cell distribution was selected under a low-power (4× objective) optical microscope, then a medium-power (20× objective) microscope with grid attached to the eyepiece was used to select randomly and continuously 5 fields of view, and the count of migrating cells was performed. The measurement results were analyzed and evaluated by statistical t test method.
[0130] 1.6 Statistical Analysis
[0131] The data were expressed as mean±standard deviation (
[0132] 2. Experimental Results
[0133] As described above, in the in vitro test, the effects of hHGF mutants and natural hHGF on endothelial cell migration was evaluated by performing an endothelial cell migration test with hHGF mutants and natural hHGF. The experimental results were shown in Table 1.
TABLE-US-00002 TABLE 1 Effects of hHGF mutants and natural hHGF on endothelial cell migration Number of Number of migrating cells Group times (cells/field of view) Blank control group 3 30.7 ± 8.1 Natural hHGF 3 98.1 ± 11.5*.sup. 130Arg-hHGF 3 415.7 ± 33.1**.sup.## 130His-hHGF 3 355.3 ± 27.3**.sup.## 130Lys-hHGF 3 388.9 ± 35.6**.sup.## wherein, “*” means p < 0.05 compared with the blank control group; “**” means p < 0.01 compared with the blank control group; “#” means p < 0.05 compared with the natural hHGF test group; “.sup.##” means p < 0.01 compared with the natural hHGF test group.
[0134] As shown in Table 1, in the blank control wells, few endothelial cells passed through the migration membrane; while in the test wells containing hHGF mutants or natural hHGF, the number of migrating cells was significantly increased. Compared with the blank control wells, there were significant differences (test wells containing natural hHGF: p<0.05; test wells containing hHGF mutants: p<0.01). Furthermore, in the test wells containing hHGF mutants, the numbers of migrating cells were significantly higher than that of the test wells containing natural hHGF (p<0.01). These results indicated that both hHGF mutants and natural hHGF could induce/stimulate the migration of endothelial cells, and that the hHGF mutants had stronger ability to induce endothelial cell migration as compared with the natural hHGF. The three hHGF mutants (130Arg-hHGF, 130His-hHGF and 130Lys-hHGF) of the present application could better promote the cell migration.
Example 4: Evaluation of Therapeutic Effect of hHGF Mutants and Natural hHGF on Rabbit Lower Extremity Artery Ischemia Model
[0135] In this example, a rabbit lower extremity artery ischemia model was used to evaluate the effects of hHGF mutants and natural hHGF on the re-formation of blood vessels and collateral circulation in the rabbit lower extremity ischemia model, thereby evaluating the therapeutic effects of hHGF mutants and natural hHGF.
[0136] 1. Materials and Methods
[0137] 1.1 Protein Samples
[0138] The hHGF mutants prepared as above (130Arg-hHGF, 130His-hHGF and 130Lys-hHGF) and natural hHGF were formulated to have required concentrations with normal saline before use.
[0139] 1.2 Animal Model
[0140] New Zealand male white rabbits, 12-14 months old, body weight 3.5 to 4.0 kg, provided by Beijing Weitong Lihua Company. According to the method described by Takeshita et al. (Therapeutics the formation of a blood vessel. A single intraarterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hind limb model. J. Clin. Invest., 1994, 93: 662-670), the rabbit lower extremity artery ischemia model was established. After intramuscular injection of Xylazine at a dose of 5 mg/kg, the rabbits were anesthetized with Ketamine at a dose of 50 mg/kg. The inner skin of the left thigh was disinfected with alcohol and iodine. Under aseptic conditions, the skin of the thigh from the midpoint of the groin to the knee joint on the left side was cut, the myofascial membrane was cut, the muscles were separated to fully expose the femoral artery, its main trunk and branches were ligated, and the artery from the femoral artery root to the popliteal artery and the artery at the bifurcation of the great saphenous artery were excised. After ensuring that there was no bleeding, the myofascial membrane and skin were sutured. After the operation, continuous intramuscular injection of gentamicin (3 mg/kg/d) was carried out for 3 days to prevent infection, and intramuscular injection of morphine (0.3 mg/kg/d) was carried out for 10 days for analgesia. On the 10.sup.th day after the operation, an arterial cannula was inserted into the right carotid artery, a 3F catheter (Terumo, Japan) was inserted into the entrance of the left internal iliac artery, 5 ml of contrast agent was infused at a rate of 1 ml per second, and selective internal iliac angiography was performed to confirm the establishment of sick animal model.
[0141] 1.3 Animal Grouping
[0142] On the 10.sup.th day after the establishment of the animal model, the animals were randomly divided into model control group (6), hHGF test group (8), 130Arg-hHGF test group (8), 130His-hHGF test group (8) and 130Lys-hHGF test group (8).
[0143] 1.4 Method of Administration
[0144] Four points at the ischemic site of the left inner thigh of each animal (1 point for adductor muscle, and 3 points for semimembranosus muscle) were taken, and 250 μg/250 μl test drug was administrated to each point by intramuscular injection (that was, 1 mg/1 ml of the test drug was administrated once per animal), once a day. The model control group was given an equal volume of saline. The administration was continued for 15 days, a total of 15 administrations.
[0145] 1.5 Evaluation of Effect of Drugs on the Formation of a Blood Vessel by Selective Internal Iliac Angiography
[0146] Since the collateral circulation of the lower extremity artery ischemia model animals originated from the branches of the internal iliac artery, selective internal iliac angiography was performed on the 10.sup.th and 40.sup.th day (the 30.sup.th day after the first administration) after the operation to observe the formation of collateral circulation before and after the administration. A 3F catheter (Terumo, Japan) was inserted into the right carotid artery, passed through the abdominal aorta, and placed at the left internal iliac artery entrance. A total of 5 ml of contrast agent was injected at a speed of 1 ml/sec, and Cine film photography was performed. On the 4.sup.th second angiogram, three straight lines perpendicular to the femur and dividing the femur into 4 parts were drawn on the femur, the number of blood vessels crossing the straight lines were counted, the counting was repeated for 3 times, and the average thereof was taken.
[0147] 1.6 Histological Determination of Capillary Density
[0148] On the 40.sup.th day after the operation, the ischemic muscle tissues of the lower limbs (adductor muscles and semimembranous muscles) were taken, placed into O.C.T. compound (Miles Inc., Elkhart, USA) solution, and quickly frozen with liquid nitrogen, and then the tissues were frozen and sectioned. According to the Indoxyl-tetrazolium method, the capillary endothelial cells were stained with alkaline phosphatase. Under the microscope (×200), the number of capillary endothelial cells in the tissue was counted, which was then convert into the number of capillaries per 1,000 muscle cells so as to quantify the density of capillaries.
[0149] 1.7 Statistical Analysis
[0150] The data were expressed as mean±standard deviation (
[0151] 2. Experimental Results
[0152] The detection results of the number of collateral vessels at the ischemic site and the number of new collateral vessels at the ischemic site before and after the administration of each group of experimental animals were shown in Table 2.
TABLE-US-00003 TABLE 2 Counting results of collateral vessels of each group of animals Number Before Day 30 after of administration administration Increment Group animals (number) (number) (number) Model control group 6 39.87 ± 3.10 43.52 ± 2.16 3.65 ± 2.25 hHGF 8 38.73 ± 3.22 51.39 ± 3.82* 12.66 ± 2.96**.sup. 130Arg-hHGF 8 40.47 ± 2.76 62.37 ± 2.27 21.90 ± 2.47**.sup.## 130His-hHGF 8 42.29 ± 1.87 60.73 ± 3.25 18.44 ± 2.48**.sup.# 130Lys-hHGF 8 45.07 ± 3.61 64.35 ± 1.97 19.28 ± 2.65**.sup.## wherein, “*” means p < 0.05 compared with the model control group; “**” means p < 0.01 compared with the model control group; “.sup.#” means p < 0.05 compared with the hHGF test group; “.sup.##” means p < 0.01 compared with the hHGF test group.
[0153] As shown in Table 2, there was no significant difference (between the five groups) in the number of collateral vessels of experimental animals in each group before administration (p>0.05). After administration, the number of collateral vessels in the model control group was not statistically different from that before administration (p>0.05); while the numbers of collateral vessels in the four test groups were significantly increased than before administration. When comparing between the groups, the increments of vessels of the three hHGF mutant test groups were all significantly greater than that of the hHGF test group (p<0.05).
[0154] The experimental results in Table 2 showed that the three hHGF mutants and natural hHGF had good therapeutic effects on lower extremity artery ischemia. Compared with the model control group, each test group could significantly promote the formation of collateral vessels, as shown by angiography. On the 30.sup.th day after treatment (calculated from the first day of administration), the density of arterioles in the left hind limb of the rabbits of experimental group was significantly higher than that of the model control group. Furthermore, the treatment results of the three hHGF mutants were significantly different from that of the natural hHGF test group: the densities of left hind limb arterioles of the rabbits receiving the hHGF mutants were higher than that of the rabbits receiving the natural hHGF; in which the 130Arg-hHGF test group (p<0.01), 130His-hHGF test group (p<0.05) and 130Lys-hHGF test group (p<0.01) were significantly better than the natural hHGF test group in promoting the formation of collateral vessels. These results indicated that the three hHGF mutants of the present application were unexpectedly superior to natural hHGF in the treatment of lower extremity artery ischemia.
Example 5: Evaluation of Therapeutic Effect of Recombinant Plasmids Encoding hHGF or its Mutants on Rabbit Lower Extremity Artery Ischemia Model
[0155] In this example, a rabbit lower extremity artery ischemia model was used to evaluate the effects of recombinant plasmids encoding hHGF or its mutants in promoting the formation of collateral vessels, so as to evaluate the therapeutic effects of recombinant plasmids encoding hHGF or its mutants.
[0156] 1. Materials and Methods
[0157] 1.1 Plasmid Samples
[0158] The four recombinant plasmids prepared as above (pSN-hHGF, pSN-130Arg-hHGF, pSN-130His-hHGF and pSN-130Lys-hHGF) were formulated to have the required concentration with physiological saline before use.
[0159] 1.2 Animal Model
[0160] New Zealand male white rabbits, 12-14 months old, body weight 3.5 to 4.0 kg, provided by Beijing Weitong Lihua Company. According to the method described by Takeshita et al. (Therapeutics the formation of a blood vessel. A single intraarterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hind limb model. J. Clin. Invest., 1994, 93: 662-670), the rabbit lower extremity artery ischemia model was established. After intramuscular injection of Xylazine at a dose of 5 mg/kg, the rabbits were anesthetized with Ketamine at a dose of 50 mg/kg. The inner skin of the left thigh was disinfected with alcohol and iodine. Under aseptic conditions, the skin of the thigh from the midpoint of the groin to the knee joint on the left side was cut, the myofascial membrane was cut, the muscles were separated to fully expose the femoral artery, its main trunk and branches were ligated, and the artery from the femoral artery root to the popliteal artery and the artery at the bifurcation of the great saphenous artery were excised. After ensuring that there was no bleeding, the myofascial membrane and skin were sutured. After the operation, continuous intramuscular injection of gentamicin (3 mg/kg/d) was carried out for 3 days to prevent infection, and intramuscular injection of morphine (0.3 mg/kg/d) was carried out for 10 days for analgesia. On the 10.sup.th day after the operation, an arterial cannula was inserted into the right carotid artery, a 3F catheter (Terumo, Japan) was inserted into the entrance of the left internal iliac artery, 5 ml of contrast agent was infused at a rate of 1 ml per second, and selective internal iliac angiography was performed to confirm the establishment of sick animal model.
[0161] 1.3 Animal Grouping
[0162] On the 10.sup.th day after the establishment of the animal model, the animals were randomly divided into model control group (8), pSN-hHGF test group (8), pSN-130Arg-hHGF test group (8), pSN-130His-hHGF test group (8) and pSN-130Lys-hHGF test group (8).
[0163] 1.4 Method of Administration
[0164] Four points at the ischemic site of the left inner thigh of each animal (1 point for adductor muscle, and 3 points for semimembranosus muscle) were taken, and 250 μg/250 μl test drug was administrated to each point by intramuscular injection (that was, 1 mg/1 ml of the test drug was administrated once per animal) for a total of 1 administration. The model control group was given an equal volume of saline.
[0165] 1.5 Evaluation of Effect of Drugs on the Formation of a Blood Vessel by Selective Internal Iliac Angiography
[0166] Since the collateral circulation of the lower extremity artery ischemia model animals originated from the branches of the internal iliac artery, selective internal iliac angiography was performed on the 10.sup.th and 40.sup.th day (the 30.sup.th day after the first administration) after the operation to observe the formation of collateral circulation before and after the administration. A 3F catheter (Terumo, Japan) was inserted into the right carotid artery, passed through the abdominal aorta, and placed at the left internal iliac artery entrance. A total of 5 ml of contrast agent was injected at a speed of 1 ml/sec, and Cine film photography was performed. On the 4.sup.th second angiogram, three straight lines perpendicular to the femur and dividing the femur into 4 parts were drawn on the femur, the number of blood vessels crossing the straight lines were counted, the counting was repeated for 3 times, and the average thereof was taken.
[0167] 1.6 Statistical Analysis
[0168] The data were expressed as mean±standard deviation (
[0169] 2. Experimental Results
[0170] The detection results of the number of collateral vessels at the ischemic site and the number of new collateral vessels at the ischemic site before and after the administration of each group of experimental animals were shown in Table 3.
TABLE-US-00004 TABLE 3 Counting results of collateral vessels of each group of animals Number Before Day 30 after of administration administration Increment Group animals (number) (number) (number) Model control group 8 45.62 ± 2.15 48.92 ± 3.75 3.30 ± 2.82 pSN-hHGF 8 42.51 ± 4.30 55.78 ± 4.57* 13.27 ± 4.32** pSN-130Arg-hHGF 8 46.73 ± 5.22 78.37 ± 3.32* 31.64 ± 4.80**## pSN-130His-hHGF 8 48.32 ± 2.84 80.54 ± 6.28* 32.22 ± 4.91**## pSN-130Lys-hHGF 8 46.72 ± 5.14 78.41 ± 4.20* 31.69 ± 4.18**## wherein, “*” means p < 0.05 compared with the model control group; “**” means p < 0.01 compared with the model control group; “#” means p < 0.05 compared with the pSN-hHGF test group; “##” means p < 0.01 compared with the pSN-hHGF test group.
[0171] As shown in Table 3, there was no significant difference (between the five groups) in the number of collateral vessels of experimental animals in each group before the administration (p>0.05). After administration, the number of collateral vessels in the model control group was not statistically different from that before administration (p>0.05); while the numbers of collateral vessels in the four test groups were significantly increased than before administration. When comparing between the groups, the increments of vessels of the 3 test groups of recombinant plasmids encoding hHGF mutants (pSN-130Arg-hHGF, pSN-130His-hHGF and pSN-130Lys-hHGF) were all significantly greater than that of the test group of recombinant plasmid encoding natural hHGF (pSN-hHGF) (p<0.01).
[0172] The experimental results in Table 3 showed that the recombinant plasmids encoding hHGF or its mutants all had good therapeutic effects on lower extremity artery ischemia. Compared with the model control group, each test group could significantly promote the formation of collateral vessels. Furthermore, the experimental results in Table 3 also showed that the therapeutic results of the three recombinant plasmids encoding hHGF mutants were significantly different from that of the recombinant plasmid encoding natural hHGF, that was, the pSN-130Arg-hHGF test group (p<0.01), pSN-130His-hHGF test group (p<0.01) and pSN-130Lys-hHGF test group (p<0.01) were all significantly better than the pSN-hHGF test group in promoting collateral vessel formation. These results indicated that the three recombinant plasmids encoding hHGF mutants (pSN-130Arg-hHGF, pSN-130His-hHGF, and pSN-130Lys-hHGF) were unexpectedly superior to the recombinant plasmid encoding natural hHGF (pSN-hHGF) in aspect of treatment effect on lower extremity artery ischemia.
Example 6: Evaluation of Therapeutic Effect of hHGF Mutants and Natural hHGF on Rat Diabetic Peripheral Neuropathy Model
[0173] In this example, a rat diabetic peripheral neuropathy model was used to evaluate the effects of hHGF mutants and natural hHGF in the treatment of diabetic peripheral neuropathy.
[0174] 1. Materials and Methods
[0175] 1.1 Protein Samples
[0176] The hHGF mutants prepared as above (130Arg-hHGF, 130His-hHGF and 130Lys-hHGF) and natural hHGF were formulated to have the required concentration with normal saline before use.
[0177] 1.2 Instruments
[0178] ONE-TOUCH Select Simple blood glucose meter: Provided by Johnson & Johnson;
[0179] Neuromatic-2000 electromyography instrument: Provided by Dandi;
[0180] EG1160 paraffin embedding machine: Provided by Leica, Germany;
[0181] RM2255 slicer: Provided by Leica, Germany;
[0182] DM6000B optical microscope: Provided by Leica, Germany.
[0183] 1.3 Animal Model
[0184] Wistar rats (SPF grade, male, body weight 180-200 g, 2.5 to 3 months old) were provided by Beijing Weitong Lihua Company. After the rats were bought back, they were fed adaptively for 5 days, and the animals were confirmed to be in good condition. 10 rats were randomly selected as the normal control group, and the remaining 60 rats were modeled as follows. The rats were fasted for 12 hours without fasting water, and then weighed, measured to determine blood glucose, and numbered. Streptozotocin (STZ) was added into the pre-prepared 0.1 mol/L citric acid/sodium citrate buffer (pH 4.4) under ice bath environment to prepare 2% STZ solution. The STZ was administrated once to the modeled animals by intraperitoneal injection on the left side at a dose of 65 mg/kg; the animals in the normal control group received the same dose of the same buffer by left intraperitoneal injection. After 72 hours, the blood glucose values of the rats were measured. A total of 52 rats with blood glucose >16.7 mmol/L and urine glucose from +++ to ++++ were selected as model animals. After 10 weeks of feeding, the model rats with diabetic peripheral neuropathy were obtained.
[0185] 1.4 Animal Grouping
[0186] The 52 model rats were randomly divided into model control group (10), hHGF test group (10), 130Arg-hHGF test group (10), 130His-hHGF test group (11), 130Lys-hHGF test group (11 only).
[0187] 1.5 Method of Administration
[0188] The animals in the test groups began to receive the drugs after 10 weeks of successful modeling. Four points on the left inner thigh of each animal (1 point for adductor muscle and 3 points for semimembranous muscle) were taken, and 250 μg/250 μl test drug was administrated to each point by intramuscular injection (that was, a total of 1 mg/1 ml test drug was administrated to each animal once), 1 administration per day. The model group was given an equal volume of normal saline for 20 days for a total of 20 injections.
[0189] 1.6 Measurement of Motor Nerve Conduction Velocity (MNCV) and Sensory Nerve Conduction Velocity (SNCV)
[0190] The measurement was performed at the 10.sup.th week after the first administration. After the rats were anesthetized, the left sciatic nerve was surgically separated, and the MNCV and SNCV of the rats were measured with Neuromatic-2000 electromyography instrument. The MNCV measurement method was as follows: the recording electrode was vertically pierced into the middle of the abdominal part of the tibialis anterior muscle, the stimulating electrode was used to stimulate the proximal end of the sciatic nerve with a stimulation current of 20 mA, the electromyography instrument displayed and recorded the action potential on the oscilloscope, and then the MNCV was calculated according to the distance between the two electrodes. The SNCV measurement method was as follows: the recording electrode was placed at the proximal end of the sciatic nerve, the stimulating electrode was used to stimulate the proximal end of the sural nerve with a stimulation current of 30 mA, the electromyography instrument recorded the obtained waveform, and then the SNCV was calculated according to the distance between the two electrodes.
[0191] 1.7 Quantitative Analysis of Myelinated Nerve Fibers of Sural Nerve
[0192] The measurement was performed at the 10.sup.th week after the first administration. The distal end of the right sural nerve was fixed in 3% glutaraldehyde/0.1 mol/L phosphate buffer and kept at 4° C. overnight; rinsed with PBS buffer, fixed with 1% osmium acid, then rinsed, dehydrated, and embedded with epoxy resin. 1 μm semi-thin cross-sectional sections were prepared, stained with 1% toluidine blue solution for 30 minutes, then washed with 85% alcohol, decolorized until the background was light blue, and then the slide was mounted with gum. The images of cross sections of sural nerve were collected at 200 times magnification, the myelinated nerve fibers were counted by using a multifunctional true color pathological image analysis system, the total cross-sectional area of sural nerve fibers, the nerve fiber density and the average cross-sectional area of nerve fibers were measured so as to observe the pathological changes of the sural nerve.
[0193] 1.8 Statistical Processing
[0194] The data were expressed as mean±standard deviation (
[0195] 2. Experimental Results
[0196] 2.1 Determination of MNCV and SNCV of Experimental Animals in Each Group
[0197] Table 4 showed the measurement results of MNCV and SNCV of experimental animals in each group.
TABLE-US-00005 TABLE 4 MNCV and SNCV of animals in each group at the 10.sup.th week after the first administration Group MNCV SNCV Normal control group 43.5 ± 4.7 46.7 ± 5.3 Model control group 31.6 ± 3.5** 33.2 ± 5.7** hHGF group 35.2 ± 3.2* 38.9 ± 4.5* 130Arg-hHGF group 42.8 ± 4.7 44.8 ± 5.6 130His-hHGF group 40.3 ± 4.3 39.7 ± 3.8 130Lys-hHGF group 39.5 ± 3.8 42.6 ± 4.5 wherein, “*” means p < 0.05 compared with the normal control group; “**” means p < 0.01 compared with the normal control group.
[0198] As shown in Table 4, at the 10.sup.th week after the first administration, the MNCV and SNCV of the model control group were the lowest (significantly slower than those of the normal control group, p<0.01); the hHGF test group followed (significantly slower than those of the normal control group, p<0.05). The MNCV and SNCV of the hHGF mutant groups were slightly lower than those of the normal control group, but the difference was not statistically significant (p>0.05). These results indicated that natural hHGF and hHGF mutants could promote the repair of diabetic peripheral neuropathy, restore the damaged MNCV and SNCV, and that the therapeutic effects of hHGF mutants were better than that of natural hHGF.
[0199] 2.2 Quantitative Analysis of Myelinated Nerve Fibers of Sural Nerve
[0200] The results of quantitative analysis of the myelinated nerve fibers of sural nerve of experimental animals in each group were shown in Table 5.
TABLE-US-00006 TABLE 5 Quantitative analysis of myelinated nerve fibers of sural nerve in each group of animals at the 10.sup.th week after the first administration Total nerve Average nerve cross-sectional area fiber area Group (μm.sup.2) (μm.sup.2) Normal control group 31771 ± 1265.sup. 23.5 ± 2.1.sup. Model control group .sup. 16449 ± 1082** .sup. 13.7 ± 1.4** hHGF group .sup. 20454 ± 1571* .sup. 17.3 ± 2.5* 130Arg-hHGF group 27539 ± 1852.sup.# 22.9 ± 1.7.sup.# 130His-hHGF group 25991 ± 1451.sup.# 20.3 ± 1.1.sup.# 130Lys-hHGF group 26193 ± 1615.sup.# 19.9 ± 2.1.sup.# wherein, “*” means p < 0.05 compared with the normal control group; “**” means p < 0.01 compared with the normal control group. “.sup.#” means p < 0.05 compared with the hHGF test group; “##” means p < 0.01 compared with the hHGF test group.
[0201] As shown in Table 5, at the 10th week after the first administration, compared with the normal control group, the total nerve cross-sectional area and the average nerve fiber area of the model control group were significantly reduced, and there was a significant difference (p<0.01); the total nerve cross-sectional area and average nerve fiber area of the animals in the hHGF test group also decreased significantly (p<0.05), but the degree of reduction decreased; the total nerve cross-sectional area and average nerve fiber area of the animals in the hHGF mutant test groups showed non-obvious reduction, and there was no significant difference. Furthermore, compared with the hHGF test group, the total nerve cross-sectional area and the average nerve fiber area of the hHGF mutant test groups were significantly increased (p<0.05). These results indicated that the natural hHGF and hHGF mutants could promote the repair of diabetic peripheral neuropathy, restore the total cross-sectional area of myelinated nerve fibers and the average area of nerve fibers of the sural nerve, and that the therapeutic effects of hHGF mutants were better than that of natural hHGF.
[0202] The results in Table 4-5 showed that both hHGF mutants and natural hHGF had a good therapeutic effects on diabetic peripheral neuropathy, could significantly increase MNCV and SNCV in diabetic rats, and significantly improve the total cross-sectional area of sural nerve fibers in diabetic rats and the average area of nerve fibers. In addition, the treatment results of the three hHGF mutants were significantly better than those of the natural hHGF. The three hHGF mutants of the present application were unexpectedly superior to the natural hHGF in the treatment of diabetic peripheral neuropathy in rats.
Example 7: Evaluation of Therapeutic Effects Recombinant Plasmids Encoding hHGF or its Mutants on Rat Diabetic Peripheral Neuropathy Model
[0203] In this example, a rat diabetic peripheral neuropathy model was used to evaluate the effects of recombinant plasmids encoding hHGF or its mutants on diabetic peripheral neuropathy.
[0204] 1. Materials and Methods
[0205] 1.1 Plasmid Samples
[0206] The four recombinant plasmids prepared as above (pSN-hHGF, pSN-130Arg-hHGF, pSN-130His-hHGF and pSN-130Lys-hHGF) were formulated to have the required concentration with physiological saline before use.
[0207] 1.2 Instruments
[0208] ONE-TOUCH Select Simple blood glucose meter: Provided by Johnson & Johnson;
[0209] Neuromatic-2000 electromyography instrument: Provided by Dandi;
[0210] EG1160 paraffin embedding machine: Provided by Leica, Germany;
[0211] RM2255 slicer: Provided by Leica, Germany;
[0212] DM6000B optical microscope: Provided by Leica, Germany.
[0213] 1.3 Animal Model
[0214] Wistar rats (SPF grade, male, body weight 180-200 g, 2.5 to 3 months old) were provided by Beijing Weitong Lihua Company. After the rats were bought back, they were fed adaptively for 5 days, and the animals were confirmed to be in good condition. 10 rats were randomly selected as the normal control group, and the remaining 60 rats were modeled as follows. The rats were fasted for 12 hours without fasting water, and then weighed, measured to determine blood glucose, and numbered. Streptozotocin (STZ) was added into the pre-prepared 0.1 mol/L citric acid/sodium citrate buffer (pH 4.4) under ice bath environment to prepare 2% STZ solution. The STZ was administrated once to the modeled animals by intraperitoneal injection on the left side at a dose of 65 mg/kg; the animals in the normal control group received the same dose of the same buffer by left intraperitoneal injection. After 72 hours, the blood glucose values of the rats were measured. A total of 52 rats with blood glucose >16.7 mmol/L and urine glucose from +++ to ++++ were selected as model animals. After 10 weeks of feeding, the model rats with diabetic peripheral neuropathy were obtained.
[0215] 1.4 Animal Grouping
[0216] 52 model rats were randomly divided into model control group (10), pSN-hHGF test group (10), pSN-130Arg-hHGF test group (10), pSN-130His-hHGF test group (11), and PSN-130Lys-hHGF test group (11 animals).
[0217] 1.5 Method of Administration
[0218] The animals in the test groups began to receive the drugs after 10 weeks of successful modeling. Four points on the left inner thigh of each animal (1 point for adductor muscle and 3 points for semimembranous muscle) were taken, and 250 μg/250 μl test drug was administrated to each point by intramuscular injection (that was, a total of 1 mg/1 ml test drug was administrated to each animal once), in total of 1 administration. The model group was given an equal volume of normal saline.
[0219] 1.6 Measurement of Motor Nerve Conduction Velocity (MNCV) and Sensory Nerve Conduction Velocity (SNCV)
[0220] The measurement was performed at the 10.sup.th week after the administration. After the rats were anesthetized, the left sciatic nerve was surgically separated, and the MNCV and SNCV of the rats were measured with Neuromatic-2000 electromyography instrument. The MNCV measurement method was as follows: the recording electrode was vertically pierced into the middle of the abdominal part of the tibialis anterior muscle, the stimulating electrode was used to stimulate the proximal end of the sciatic nerve with a stimulation current of 20 mA, the electromyography instrument displayed and recorded the action potential on the oscilloscope, and then the MNCV was calculated according to the distance between the two electrodes. The SNCV measurement method was as follows: the recording electrode was placed at the proximal end of the sciatic nerve, the stimulating electrode was used to stimulate the proximal end of the sural nerve with a stimulation current of 30 mA, the electromyography instrument recorded the obtained waveform, and then the SNCV was calculated according to the distance between the two electrodes.
[0221] 1.7 Quantitative Analysis of Myelinated Nerve Fibers of Sural Nerve
[0222] The measurement was performed at the 10.sup.th week after the administration. The distal end of the right sural nerve was fixed in 3% glutaraldehyde/0.1 mol/L phosphate buffer and kept at 4° C. overnight; rinsed with PBS buffer, fixed with 1% osmium acid, then rinsed, dehydrated, and embedded with epoxy resin. 1 μm semi-thin cross-sectional sections were prepared, stained with 1% toluidine blue solution for 30 minutes, then washed with 85% alcohol, decolorized until the background was light blue, and then the slide was mounted with gum. The images of cross sections of sural nerve were collected at 200 times magnification, the myelinated nerve fibers were counted by using a multifunctional true color pathological image analysis system, the total cross-sectional area of sural nerve fibers, the nerve fiber density and the average cross-sectional area of nerve fibers were measured so as to observe the pathological changes of the sural nerve.
[0223] 1.8 Statistical Processing
[0224] The data were expressed as mean±standard deviation (
[0225] 2. Experimental Results
[0226] 2.1 Determination of MNCV and SNCV of Experimental Animals in Each Group
[0227] Table 6 showed the measurement results of MNCV and SNCV of experimental animals in each group.
TABLE-US-00007 TABLE 6 MNCV and SNCV of animals in each group at the 10.sup.th week after administration Group MNCV SNCV Normal control group 51.2 ± 3.6 48.5 ± 4.8 Model control group 33.6 ± 3.0** 31.5 ± 3.7** pSN-hHGF test group 36.2 ± 2.8* 37.6 ± 3.1* pSN-130Arg-hHGF test group 45.4 ± 4.6 48.3 ± 5.1 pSN-130His-hHGF test group 48.8 ± 4.0 47.9 ± 3.1 pSN-130Lys-hHGF test group 47.5 ± 4.6 46.3 ± 3.5 wherein, “*” means p < 0.05 compared with the normal control group; “**” means p < 0.01 compared with the normal control group.
[0228] As shown in Table 6, at the 10.sup.th week after administration, the MNCV and SNCV of the model control group were the lowest (significantly slower than those of the normal control group, p<0.01); the pSN-hHGF test group followed (significantly slower than those of the normal control group, p<0.05). The MNCV and SNCV of the test groups of recombinant plasmids encoding hHGF mutants were slightly lower than those of the normal control group, but the difference was not statistically significant (p>0.05). These results indicated that the recombinant plasmids encoding natural hHGF and its mutants could promote the repair of diabetic peripheral neuropathy, restore damaged MNCV and SNCV, and the therapeutic effects of the recombinant plasmids encoding hHGF mutants (pSN-130Arg-hHGF, pSN-130His-hHGF and pSN-130Lys-hHGF) were better than those of the recombinant plasmid encoding natural hHGF (pSN-hHGF).
[0229] (2) Quantitative Analysis of Myelinated Nerve Fibers of Sural Nerve
[0230] The results of quantitative analysis of the myelinated nerve fibers of sural nerve of experimental animals in each group were shown in Table 7.
TABLE-US-00008 TABLE 7 Quantitative analysis of myelinated nerve fibers of sural nerve of animals in each group at the 10.sup.th week after administration Total nerve Average nerve cross-sectional area fiber area Group (μm.sup.2) (μm.sup.2) Normal control group 36054 ± 2122.sup. 25.7 ± 2.0.sup. Model control group .sup. 15398 ± 971** .sup. 10.6 ± 1.2** pSN-hHGF test group .sup. 18123 ± 1025* .sup. 15.2 ± 1.8* pSN-130Arg-hHGF test group 31352 ± 1659.sup.# 23.9 ± 1.4.sup.# pSN-130His-hHGF test group 28232 ± 1321.sup.# 21.5 ± 0.9.sup.# pSN-130Lys-hHGF test group 29269 ± 1217.sup.# 24.1 ± 1.2.sup.# wherein, “*” means p < 0.05 compared with the normal control group; “**” means p < 0.01 compared with the normal control group. “.sup.#” means p < 0.05 compared with the pSN-hHGF test group; “##” means p < 0.01 compared with the pSN-hHGF test group.
[0231] As shown in Table 7, at the 10.sup.th week after administration, compared with the normal control group, the total nerve cross-sectional area and the average nerve fiber area of the model control group were significantly reduced, and there was a significant difference (p<0.01); the total nerve cross-sectional area and the average nerve fiber area of the animals in the pSN-hHGF test group were also significantly reduced (p<0.05), but the degree of reduction decreased; the total nerve cross-sectional area and the average nerve fiber area of the animals in the test groups of recombinant plasmids encoding hHGF mutants showed non-obvious reduction, and there was no significant difference. Furthermore, compared with the pSN-hHGF test group, the total nerve cross-sectional area and the average nerve fiber area of the animals in the test groups of recombinant plasmids encoding hHGF mutants were significantly increased (p<0.05). These results indicated that the recombinant plasmids encoding natural hHGF and its mutants could promote the repair of diabetic peripheral neuropathy, restore the total cross-sectional area of myelinated nerve fibers and the average area of nerve fibers of sural nerve, and the therapeutic effects of the three recombinant plasmids encoding hHGF mutants (PSN-130Arg-hHGF, pSN-130His-hHGF and pSN-130Lys-hHGF) were better than those of the recombinant plasmid encoding natural hHGF (pSN-hHGF).
[0232] The results in Table 6-7 showed that the recombinant plasmids encoding hHGF mutants and the recombinant plasmid encoding natural hHGF had good therapeutic effects on diabetic peripheral neuropathy, could significantly increase MNCV and SNCV in diabetic rats, and significantly improve the total cross-sectional area of sural nerve fibers and the average area of nerve fibers in diabetic rats. In addition, the treatment results of the three recombinant plasmids encoding hHGF mutants (pSN-130Arg-hHGF, pSN-130His-hHGF and pSN-130Lys-hHGF) were significantly better than those of the recombinant plasmid encoding natural hHGF (pSN-hHGF).
[0233] 3. Conclusion
[0234] The results of Examples 3, 4 and 6 showed that the hHGF mutants of the present application had significantly higher activity than natural hHGF in terms of promoting the migration of umbilical vein endothelial cells, promoting the growth of lower extremity arterioles, and promoting the repair of diabetic peripheral neuropathy.
[0235] The results of Examples 5 and 7 showed that the recombinant plasmids encoding the hHGF mutants could be used as gene therapy drugs to promote the growth of lower extremity arterioles and promote the repair of diabetic peripheral neuropathy in the subjects, and their therapeutic effects were significantly higher than those of the recombinant plasmid encoding natural hHGF.
[0236] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details according to all the teachings that have been disclosed, and these changes are within the scope of protection of the present application. The full scope of the present application is given by the appended claims and any equivalents thereof.