VIRAL VECTOR FOR TREATING AUTOIMMUNE DISEASE AND DIABETES AND CONSTRUCTION METHOD AND APPLICATION THEREOF

Abstract

Provided are a viral vector for treating autoimmune disease and diabetes and a construction method and an application thereof. The viral vector is a lentiviral expression plasmid or an adeno-associated viral expression plasmid cloned with mfat-1 gene, and the mfat-1 gene is as shown in SEQ ID NO: 1.

Claims

1-15. (canceled)

16. A viral vector comprising a mammalianized fat-1 (mfat-1) gene having a polynucleotide sequence of SEQ ID NO: 1.

17. The viral vector according to claim 16, wherein the mfat-1 gene is cloned into a lentiviral expression plasmid or an adeno-associated viral expression plasmid.

18. The viral vector according to claim 17, wherein the lentiviral expression plasmid is pLJM1-CMV-hPGK-EGFP plasmid, pLJM1-CMV-hPGK-mkate2 plasmid, pLenti-CMV-MCS-GFP-SV-puro plasmid, FUGW, pLenti-puro, pLenti-MP2 or pLenti plasmid.

19. The viral vector according to claim 17, wherein the adeno-associated viral expression plasmid is pEMBL-AAV-D(+)-CMV-eGFP-SV40 plasmid, AAV GFP plasmid, AAV1 plasmid, AAV2 plasmid, rAAV2 plasmid, AAV5 plasmid, AAV8 plasmid, AAV9 plasmid or pAV-FH AAV plasmid.

20. A viral particle comprising the viral vector of claim 17.

21. A pharmaceutic composition comprising the viral particle of claim 20 and a therapeutically acceptable carrier.

22. A method for treating an inflammatory or autoimmune related disease in a subject comprising administering to the subject a therapeutically effective amount of the viral particle according of claim 20.

23. The method according to claim 22, wherein the inflammatory or autoimmune related disease is induced by imbalance of Th cell differentiation and imbalance of cytokines.

24. The method according to claim 22, wherein the inflammatory or autoimmune related disease is diabetes.

25. The method according to claim 24, wherein the diabetes is type 1 diabetes or type 2 diabetes.

26. The method according to claim 22, wherein the inflammatory or autoimmune related disease is selected from multiple sclerosis, rheumatoid arthritis and systemic lupus erythematosus.

27. The method according to claim 22, wherein gene product of the mfat-1 gene converts ω-6 polyunsaturated fatty acids (PUFAs) in the subject into ω-3 PUFAs.

28. The method according to claim 27, wherein a ratio of ω-6/ω-3 in the subject is balanced to about 1:1.

29. The method according to claim 22, wherein Th cell differentiation is rebalanced.

30. The method according to claim 22, wherein ratios of Th1/Th2 and T17/Treg are rebalanced.

31. The method according to claim 22, wherein the viral particle is administered to the subject by intravenous injection.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0055] FIG. 1 is a 1% agarose gel electrophoresis pattern of pLJM1-mfat-1-EGFP plasmid after digestion with NheI and EcoRI;

[0056] FIG. 2 is a graph showing results of differentiations of human peripheral blood CD4 T-cells and changes in cytokines thereof, after treating peripheral blood mononuclear cells of normal humans (FIG. 2A-E) and type 1 diabetic patients (FIG. 2F-J) in vitro for 24 h by using lentiviral particles in Example 2 of the present invention;

[0057] FIG. 3 is a graph showing results of changes in peripheral blood CD4 T-cell transcription factors after treating peripheral blood mononuclear cells of patients with type 1 diabetes in vitro for 24 h according to Example 2 of the present invention;

[0058] FIG. 4 is a graph showing results of changes in incidences of diabetes in NOD mice after administrating lentivirus for 9 weeks in Example 3 of the present invention;

[0059] FIG. 5 is a graph showing results of blood glucose changes in NOD mice after administrating lentivirus in Example 3 of the present invention;

[0060] FIG. 6 is a graph showing results of insulin secretion in NOD mice after administrating lentivirus for 9 weeks in Example 3 of the present invention;

[0061] FIG. 7 is a graph showing results of changes in the development of islet inflammatory infiltration in NOD mice after administrating lentivirus for 9 weeks of in Example 3 of the present invention;

[0062] FIG. 8 is a graph showing results of differentiation of CD4 cells in NOD mice after administrating lentivirus for 9 weeks in Example of the present invention.

DETAILED DESCRIPTION

[0063] The invention can be better understood in light of the following examples. However, it is to be easily understood by those skilled in the art that the description of examples is only intended to illustrate the invention and should not be construed as limiting the invention as described in claims. In the following examples, DH5a E. coli is supplied by TAKARA; LB medium is supplied by Sigma; 293FT cell line is supplied by ATCC; growth medium DMEM is supplied by GIBCO; FBS fetal bovine serum is supplied by GIBCO; OPTI-MEM is supplied by GIBCO; Lipofectamine 2000 transfection reagent is supplied by Invitrogen-Thermo Fisher Scientific; NOD mouse is supplied by The Jackson Laboratory. A centrifuge is FRESCO17 high-speed refrigerated centrifuge manufactured by American Thermo Company; an electrophoresis apparatus is PowerPac™ and Mini-Sub cell GT manufactured by American BIO-RAD company; a multiImager is ChemiDoc™ XRS+System manufactured by American BIO-RAD company.

Example 1: Construction of a Viral Vector Cloned with Mfat-1 Lentiviral Expression Plasmid

[0064] Firstly, a mfat-1 gene as shown in SEQ ID NO: 1, provided by Kingsray, was synthesized, and then was subcloned to pLJM1-CMV-hPGK-EGFP plasmid (addgene, Plasmid #19319) by NheI and EcoRI restriction sites, to obtain pLJM1-CMV-hPGK-EGFP-mfat-1 (PLJM1-mfat-1) expression plasmid. The detailed construction process is as follows:

1) PCR Amplification of Mfat-1 Region

[0065] Primers were designed by using the synthesized mfat-1 gene sequence as a template. PCR amplification of the mfat-1 gene sequence was carried out with the primers to obtain a PCR amplification product which is a mfat-1 gene sequence with NheI and EcoRI restriction sites at both ends, wherein the primers are as follows:

TABLE-US-00002 upstream primer mfat-1-F: 5′-TATTAAGCTAGCATGGTCGCCCACAGCA-3′; downstream primer mfat-1-R: 5′-CAACCGGAATTCTCATCACTTGGCCT-3′.

[0066] A system of PCR amplification reaction is shown in Table 1.

TABLE-US-00003 TABLE 1 System of PCR amplification reaction 5 × PrimeSTAR ® Buffer (Mg2+ plus) 10 μl dNTP Mixture (each dNTP 2.5 mM) 4 μl template DNA 100 ng upstream primer (10 μM) 1 μl downstream primer (10 μM) 1 μl PrimeSTAR ® HS DNA Polymerase (2.5 U/μl) 0.5 μl adding sterile ultrapure water to 50 μL

[0067] A procedure of the PCR amplification reaction is as follows: denaturation at 98° C. for 5 min; and holding at 98° C. for 10 s, and holding at 60° C. for 15 s, holding at 72° C. for 2 min, a total of 30 cycles; finally extension at 72° C. for 10 min.

2) Digestion

[0068] The PCR amplification product obtained in the above step was subjected to 1.5% agarose gel electrophoresis to recover a target DNA fragment. An Agarose Gel DNA Extraction Kit was used to obtain a purified target DNA fragment, which was digested with restriction enzymes NheI and EcoR to obtain a digested target DNA fragment. A pLJM1-EGFP empty lentiviral expression plasmid was digested with restriction enzymes NheI and EcoR to obtain a digested pLJM1-EGFP plasmid. A reaction system of digestion is shown in Table 2:

TABLE-US-00004 TABLE 2 System of digestion reaction plasmid or target DNA fragment 1 μg 10 × buffer 2 μl endonuclease NheI 1 μl endonuclease EcoRI 1 μl adding sterile ultrapure water to 20 μL

[0069] Conditions of digestion reaction: 37° C. water bath for 2 h. The above digested DNA fragment was subjected to 1% agarose gel electrophoresis to separate a target DNA fragment, and a purified plasmid or target DNA fragment was obtained using an Agarose Gel DNA Extraction Kit.

3) Ligation

[0070] The digested target DNA fragment was ligated to the digested pLJM1-EGFP plasmid.

TABLE-US-00005 TABLE 3 System of ligation reaction digested pLJM1-EGFP plasmid 45 ng digested target DNA fragment 60 ng 10 × buffer 2 μl ligase (350 units/μl) 1 μl adding sterile ultrapure water to 20 μL

[0071] Conditions of ligation reaction: ligating at room temperature (16° C.) overnight, i.e. 12 h, to obtain a PLJM1-mfat-1 vector, i.e. a ligated product.

[0072] The example also provides a viral particle, which was prepared by transforming the above viral vector into a cell. Detailed steps are as follows:

[0073] 1. A competent bacteria used for transformation is DH5α E. coli, prepared by CaCl.sub.2 method. The preparation method and subsequent transformation operations refer to the second edition of Molecular Cloning, as follows:

[0074] A. Preparation of a Competence:

[0075] i. 1 ml saturated bacterial solution was added to 100 ml LB, and then shaken for 2-3 h at 37° C., 280 rpm, until an optical density OD600 reflecting the bacterial density was between 0.4 and 0.6;

[0076] ii. the solution obtained in step i was transferred to ice-cold 50 ml polypropylene tube and then placed in an ice bath for 10 min;

[0077] iii. the solution obtained in step ii was centrifuged for 10 min at 4° C., 1000 g, to obtain a supernatant and a precipitate;

[0078] iv. the supernatant was discarded, the tube was inverted for 1 min to make the supernatant drained, and the precipitate was resuspended by adding 10 ml ice-cold 0.1 mol/1 CaCl.sub.2, and placed in an ice bath for 30 min;

[0079] v. the solution obtained in step iv was centrifuged for 10 min at 4° C., 1000 g, to obtain a supernatant and a precipitate;

[0080] vi. the supernatant was discarded, and the precipitate was resuspended by adding 2 ml ice-cold 0.1 mol/1 CaCl.sub.2, and then was well mixed with 0.5 ml 75% sterilized glycerol (prepared with 0.1 mol/1 CaCl.sub.2) to obtain a mixture. The mixture was dispensed into a 1.5 ml centrifuge tube at 100 μl/tube, and can be stored in a −80° C. refrigerator for half a year;

[0081] vii. a conversion efficiency was identified.

[0082] B. Conversion of the ligated product:

[0083] i. 1 tube of competent cells was thawed on ice, 3 μl ligated product was added thereto, mixed well, and then placed in ice bath for 30 min;

[0084] ii. the solution obtained in step i was left to stand in 42° C. water bath for 90 sec, and then transferred quickly to ice for ice bath for 1-2 min;

[0085] iii. the solution obtained in step ii was mixed with 900 μl LB medium, then placed in 37° C. water bath for 10 min;

[0086] iv. the bacteria were amplified for 45 min at 37° C., 210 rpm, to obtain transformed bacteria;

[0087] v. 100 μl of the above transformed bacteria were coated on an agar plate containing 100 μg/ml ampicillin;

[0088] vi. the plate was inverted and incubated at 37° C. for 16-20 h.

[0089] C. Positive clone screening (digestion identification, sequencing identification)

[0090] The positive clone was identified by 1% agarose gel electrophoresis, and the results are shown in FIG. 1, which indicates that the viral vector PLJM1-mfat-1 was successfully constructed.

[0091] 2. Packaging, amplification and purification of the lenti-mfat-1 lentiviral particle.

[0092] A packaging cell of lentivirus is 293FT cell strain, and its growth medium is DMEM (containing 10% FBS). Adherent cells were proliferated in the medium to form monolayer cells.

[0093] (1) Amplification of virus:

[0094] 1) 293FT cells in logarithmic growth phase were digested with trypsin, and adjusted to a cell density of 1×10.sup.5 cells/ml using DMEM high glucose medium (supplied by GIBCO) containing 10% FBS fetal bovine serum (provided by GIBCO), and then inoculated in a six-well plate, and cultured at 37° C. in a 5% CO.sub.2 incubator. After 24 h, the cell density can reach 0.8×10.sup.6 cells/ml and can be used for transfection. State of the cells is critical for packaging of virus, therefore the cells need to be in good state and have fewer passages.

[0095] 2) The cell culture medium was replaced with fresh complete medium 1 h before transfection.

[0096] 3) In one 1.5 ml EP tube, a plasmid reagent, including 1.5 μg PLJM1-mfat-1 vector, 1.125 μg psPAX2 vector Addgene (Plasmid #12260), 0.375 μg pMD2.G vector Addgene (Plasmid #12259), was added, and then mixed well with 100 μl OPTI-MEM and incubated for 5 min at room temperature (20° C.-30° C.).

[0097] 4) In another 1.5 ml EP tube, 100 μl OPTI-MEM was firstly added, and then 3 μl Lipofectamine2000 transfection reagent was added, mixed gently, and incubated for 5 min at room temperature (20° C.-30° C.).

[0098] 5) The incubated plasmid reagent (mixture of three kinds of plasmids PLJM1-mfat-1, psPAX2 and pMD2.G) obtained in step 3) was mixed gently with the diluted Lipofectamine 2000 transfection reagent obtained in step 4), incubated for 15 min at room temperature (20° C.-30° C.) to form a transfection complex of the plasmid reagent with the Lipofectamine 2000 transfection reagent.

[0099] 6) 200 μl reagent obtained in step 5) (i.e. the transfection complex of PLJM1-mfat-1, psPAX2 and pMD2.G and Lipofectamine2000 transfection reagent, obtained in step 5)) was dropped into the culture medium containing 293FT cells in obtained step 1), gently shaken and incubated at 37° C. in a 5% CO 2 cell incubator.

[0100] (2) Purification of virus:

[0101] 1) After the 293FT cell culture medium was cultured for 24 h in the above steps, the medium containing the transfection complex was discarded, and 2 ml fresh complete medium was added to continue the culture.

[0102] 2) After continuing the culture for 48 h and 72 h respectively, the 293FT cell culture mediums were centrifuged to collect supernatants respectively. The supernatants collected at 48 h and 72 h were mixed, centrifuged at 1250 rpm for 5 min to remove the 293FT cells, and filtered with 0.45 μm filter to obtain the Lenti-mfat-1 lentiviral particle. The viral particle may be stored at 4° C. for a short time (<3 d), and may be stored at −80° C. for a long time, and was separate packed to avoid repeated freezing and thawing.

[0103] (2) Determination of titer of virus

[0104] The titer of virus was determined by TCID50 method. The amplification, purification and determination of titer of control naked virus were carried out with the same methods described as above.

Example 2: Regulation of ω-3 PUFAs on Peripheral Blood CD4 T-Cells in Patients with Type 1 Diabetes

[0105] (1) 5 ml peripheral blood was extracted from patients and normal humans separately, diluted twice with PBS of pH 7.2, and then mixed with lymphocyte separation solution Lymphoprep (Axis-Shield, Norway), centrifuged at 3500 rpm for 20 min, then the lymphocytes were taken out and cultured in RPMI1640 medium (purchased from Gibco) to obtain the cultured lymphocytes. The above cultured lymphocytes were treated in vitro with DHA (docosahexaenoic acid) at a final concentration of 100 μM, EPA (eicosapentaenoic acid) at a final concentration of 100 μM, and AA (arachidonic acid) at a final concentration of 100 μM for 24 h, respectively. After treating, changes in intracellular cytokines of CD4 T-cells in the above lymphocytes were detected. (The above DHA, EPA and AA are all purchased from Sigma).

[0106] (2) labeling of intracellular cytokines of CD4 T-cells: The lymphocytes (number up to 1×10.sup.5) from patients and normal humans in the above steps were placed to different marked tubes. Labeled fluorescent monoclonal antibodies CD3 (0.1 μg) and CD8 (0.1 μg) were added into each tube. Then 1 ml paraformaldehyde having a concentration of 4% by weight was added into each tube, mixed well and incubated at room temperature (20° C.-30° C.) for 20 min. 1 ml intraprep permeabilization reagent, i.e. 0.5% saponin, was added, incubated at room temperature (20° C.-30° C.) for 30 min, then centrifuged for 5 min at 300 g, 4° C. to obtain a supernatant and a precipitate. The supernatant was removed, and 80 μl 0.5% saponin was added to re-suspend the precipitate to obtain suspensions. The obtained suspensions were labeled respectively with IFN-γ fluorescent monoclonal antibody (0.1 μg), IL-17 fluorescent monoclonal antibody (0.1 μg), IL-4 fluorescent monoclonal antibody (0.1 μg), and Foxp3 fluorescent monoclonal antibody (0.1 μg), and incubated in dark place at 4° C. for 1 h (for the suspension labeled with Foxp3 fluorescent monoclonal antibody, incubation and rupture took 8 h). The labeled cells were washed three times with 0.5% saponin, centrifuged for 5 min at 300 g, 4° C. to obtain a supernatant and a precipitate. The supernatant was removed, and the precipitate was fixed with paraformaldehyde having a mass concentration of 1%. After 24 h, percentages of Th1, Th2, Th17 and Treg cells were analyzed with a flow cytometer Accuri-C6. The antibodies and instruments used in the above steps were all purchased from BD Bioscience.

[0107] (3) Labeling of intracellular transcription factors of CD4 T cells: The lymphocytes (number up to 1×10.sup.5) from patients and normal humans in the steps 1) were placed to different marked tubes. Labeled fluorescent monoclonal antibodies CD3 (0.1 μg) and CD8 (0.1 μg) are added into each tube. 1 ml 4% paraformaldehyde was then added into each tube, mixed well and incubated at room temperature for 20 min. 1 ml intraprep permeabilization reagent, i.e. 0.5% saponin, was added, incubated at room temperature for 30 min, then centrifuged for 5 min at 300 g, 4° C. to obtain a supernatant and a precipitate. The supernatant was removed, and the precipitate was re-suspended by adding 80 μl 0.5% saponin to obtain suspensions. The obtained suspensions were labeled respectively with T-bet fluorescent monoclonal antibody (0.1 μg), GATA3 fluorescent monoclonal antibody (0.1 μg), RORγT fluorescent monoclonal antibody (0.1 μg), and Foxp3 fluorescent monoclonal antibody (0.1 μg), and incubated in dark place at 4° C. for 1 h (For Foxp3, intraprep permeabilization takes 8 h). The labeled cells were washed three times with 0.5% saponin, centrifuged for 5 min at 300 g, 4° C. to obtain a supernatant and a precipitate. The supernatant was removed, and the precipitate was fixed with 1% paraformaldehyde, and then analyzed with a flow cytometer Accuri-C6 to determine the percentages of the above transcription factors. The antibodies and instruments used in the above steps were all purchased from BD Bioscience.

[0108] (4) Results: The results are shown in FIG. 2 and FIG. 3. The CON in FIG. 2 represents a control group, i.e. the lymphocytes of peripheral blood from normal humans not treated with DHA, EPA or AA. The CON in FIG. 3 represents a control group, i.e. the lymphocytes of peripheral blood from patients not treated with DHA, EPA, or AA. In FIG. 2, figures A, B, C, D, and E show percentages of Th1, Th2, Th1/Th2, Th17 and Treg cells in peripheral blood CD4 T-cells of normal subjects, respectively, and figures F, G, H, I and J show percentages of Th1, Th2, Th1/Th2, Th17 and Treg cells in peripheral blood CD4 T-cells of patients, respectively. In FIG. 3, figures A, B, C, and D show percentages of transcription factors T-bet, GATA3, RORγT, and Foxp3 in peripheral blood CD4 T-cells of patients, respectively. As shown in FIGS. 2-3, in peripheral blood CD4 T-cells of patients with type 1 diabetes treated with DHA, EPA and AA respectively, the percentages of Th1 and Th17 cells decreased, and the percentages of Th2 and Treg cells increased, and the ratio of Th1/Th2 and the ratio of T17/Treg were rebalanced. The transcription factor T-bet that regulates the expression of Th1 cytokines and the transcription factor RORγT that regulates the expression of Th17 cytokines were inhibited significantly by ω-3PUFAs, while the transcription factors GATA3 and Foxp3 that regulate the expressions of cytokines Th2 and Treg were up-regulated under the action of ω-3PUFAs. These results fully indicate that ω-3 PUFAs have effects of re-balancing Th cell differentiation and significantly inhibiting inflammation caused by the imbalance of the ratios of Th1/Th2 and T17/Treg, and further indicate that the mfat-1 gene carried by the viral particles of the present invention can convert high levels of ω-6 PUFAs in the body into ω-3 PUFAs by using ω-6 PUFAs as a substrate, so that a ratio of ω-6/ω-3 is balanced and tends to 1:1, so as to inhibit autoimmune diseases caused by imbalance of the ratios of Th1/Th2 and T17/Treg, such as type 1 diabetes, multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus.

Example 3: Effect of Lenti-Mfat-1 Lentiviral Particle in the Treatment of Type 1 Diabetes

[0109] (1) Construction of a mouse model of type 1 diabetes:

[0110] Some NOD mice, each weighing 25 g were used. Blood were collected from the mice at a fixed time every day to detect random blood glucose in NOD mice. Mice with blood glucose of more than 11.1 mmol/L for two consecutive weeks were selected. The selected mice were randomly divided into two groups, a control group (Lenti-Con) and a lentiviral treatment group (Lenti-mfat-1), with 5 mice in each group.

[0111] (2) Administration method:

[0112] In the treatment group, Lenti-mfat-1 lentiviral particles in the form of a concentrate having a virus titer of 10.sup.8 TU/mL were injected through a tail vein of the mouse in an amount of 10.sup.9 TU/Kg mouse. In the control group, the pLJM1-CMV-hPGK-EGFP plasmid without mfat-1 gene were injected under the same conditions. Changes in random blood glucose in mice were detected.

[0113] (3) Results and discussions:

[0114] 1) General physiological conditions of NOD mouse after receiving treatment with Lenti-mfat-1 lentiviral particles

[0115] The mice after receiving treatment with Lenti-mfat-1 lentiviral particles in the treatment group had normal feeding and drinking situations compared with the mice in the control group. After 2 weeks of treatment, body weights of the mice in the treatment group were higher than that of the control group, but there was no statistical significance.

[0116] 2) After treatment with the viral particles of Example 1 for 9 weeks, a level of polyunsaturated fatty acid in the peripheral blood of the mice was detected. The detailed steps of detecting are as follows: after extracting fatty acids from peripheral blood of mice of the two groups by conventional organic chemical methods, the extracted samples (the extracted fatty acids) were dissolved in heptane, and then dropped into sample bottles for sample loading and testing. The sample was loaded through an automatic sampler and tested by Agilent 7890A. The running time of a single loading was about 1 h. Gas chromatographic conditions: column model: SP2380, 105 m*0.53 mm*0.20 μm (Agilent); operating procedures: holding at an initial temperature of 140° C. for 3 min, then rising to 220° C. at a rate of 8° C./min and holding at this temperature for 12 min; temperature of injector and detector: 260° C.; carrier gas was helium, speed was set to 12 psi. Finally, the type of fatty acid was determined according to peak times of the sample and a standard product (purchased from Sigma, USA), and the percentage of each fatty acid was obtained as the final result. The peak of interest was determined by comparing peak time, peak shape, and peak area percentage with those of the standard product; the sum of all the area percentages of ω-3 and ω-6 polyunsaturated fatty acids was calculated, and the ratio of ω-6/ω-3 was calculated. Column type: SP2380, 105 m*0.53 mm*0.20 μm). It was found that the proportion of EPA in mouse receiving Lenti-mfat-1 lentiviral particles increased, indicating the lentiviral particles with mfat-1 gene successfully worked (Table 5).

TABLE-US-00006 TABLE 5 Changes in polyunsaturated fatty acids in blood of mouse after treatment with viral particles. (%) Lenti-con Lenti-mfat-1 ω-3 PUFA species C18:3 (α-LA) 0.34 ± 0.12 0.36 ± 0.48 C20:5 (EPA) 0.53 ± 0.09  5.75 ± 2.96* C22:5 0.59 ± 0.04 1.82 ± 1.73 C22:6 (DHA) 6.26 ± 1.05 7.82 ± 5.22 ω-6 PUFA species C18:2 19.5 ± 1.67 16.7 ± 18.3 C18:3 (γ-LA) NP NP C20:4 (AA) 12.2 ± 3.18 8.01 ± 4.02 C22:4 0.94 ± 0.19 NP ω-6/ω-3 ratio 4.24 ± 0.31 1.77 ± 1.71

[0117] Each of ω-3 and ω-6 polyunsaturated fatty acid is expressed as a relative percentage, wherein the peak area of all fatty acids detected by gas chromatography is 100%, and the content of the target fatty acid is calculated by dividing the peak area by the total peak area the target fatty acid. Data of each group was repeated three times, and counted using oneway-ANOVA. Compared with the control group, *P<0.05, NP: not detected.

[0118] 3) Monitoring of random blood glucose levels, insulin levels, and islet inflammation in two groups of NOD mouse

[0119] Mouse having random blood glucose of below 11.1 mmol/L for two consecutive weeks was considered to have returned to normal blood glucose. Blood samples were taken from eye sockets of mouse in the Lenti-mfat-1 treatment group and the control group NOD, and then analyzed with an American Roche Accu-Chek blood glucose meter to determine the blood glucose levels. Results were shown in FIGS. 4-5. Compared with the control group, the randomized blood glucose of the NOD mouse in the treatment group had been significantly down-regulated at the second week, and had decreased to and maintained at the normal level by the sixth week, reaching 6.3 mmol/L, indicating Lenti-mfat-1 lentiviral particles had a significant therapeutic effect on NOD mouse. After 9 weeks of treatment, 2 ml of blood was taken from eyeballs of the two groups of mice, left to stand for 5 min, centrifuged at 3500 rpm for 15 min to obtain an upper serum. 200 μl of the upper serum was diluted 100 times with PBS having a pH of 7.4, and then analyzed with a kit purchased from Mercodia to determine the concentration of insulin in the serum. The result was shown in FIG. 6. After 9 weeks of treatment, the pancreas of the two groups of mice was taken under a stereoscopic microscope, and then routinely embedded in paraffin, sectioned, and further HE stained to observe an infiltration of islet lymphocytes of mouse. The result was shown in FIG. 7.

[0120] 4) Changes in CD4 T lymphocyte differentiation in NOD mouse after treatment with Lenti-mfat-1lentiviral particles

[0121] Spleen lymphocytes (number up to 1×10.sup.5) in the NOD mouse of the treatment group and the control group were collected and placed to different marked tubes. Labeled fluorescent monoclonal antibodies CD3 (0.1 μg) and CD8 (0.1 μg) are added into each tube. 1 ml paraformaldehyde having a concentration of 4% by weight was then added into each tube, mixed well and incubated at room temperature (20° C.-30° C.) for 20 min. 1 ml intraprep permeabilization reagent, i.e. 0.5% saponin, was added, incubated at room temperature (20° C.-30° C.) for 30 min, then centrifuged for 5 min at 300 g, 4° C. to obtain a supernatant and a precipitate. The supernatant was removed, and the precipitate was resuspended by adding 80 μl 0.5% saponin to obtain suspensions. The obtained suspensions were labeled respectively with IFN-γ fluorescent monoclonal antibody (0.1 μg), IL-17 fluorescent monoclonal antibody (0.1 μg), IL-4 fluorescent monoclonal antibody (0.1 μg), and Foxp3 fluorescent monoclonal antibody (0.1 μg), and incubated in dark place at 4° C. for 1 h (For the sample labeled with Foxp3 fluorescent monoclonal antibody, incubation and rupture takes 8 h). The labeled cells were washed three times with 0.5% saponin, centrifuged for 5 min at 300 g, 4° C. to obtain a supernatant and a precipitate. The supernatant was removed, and the precipitate was fixed with paraformaldehyde having a concentration of 1% by weight. After 24 h, percentages of Th1, Th2, Th17 and Treg cells were analyzed with a flow cytometer Accuri-C6. The antibodies and instruments used in the above steps were all purchased from BD Bioscience. Results were shown in FIG. 8. In CD4 T cells of NOD mouse treated with Lenti-mfat-1 lentiviral particles, the percentages of Th1 and Th17 cells decreased, and the percentages of Th2 and Treg cells increased, and the ratios of Th1/Th2 and T17/Treg were rebalanced, thereby significantly improving inflammatory environment caused by the imbalance of the ratios of Th1/Th2 and T17/Treg.

Example 4: Mfat-1 Transgenic Mice Completely Resist Inducers to Induce Occurrences of MS

[0122] (1) Establishment of model: 3 wide type mice (6-8 weeks, two females and one male, purchased from Model Animal Research Center of Nanjing University), and 3 maft-1 transgenic mice (6 weeks, two females and one male, provided by Model Animal Research Center of Nanjing University; the maft-1 transgenic mice had fat-1 gene expressed by pST181 prokaryotic expression vector, and named mfat-1 transgenic mouse because the muscle creatine kinase (MCK) enhancer was designed upstream of the fat-1 gene promoter CMV-β-actin to increase expression efficiency of the fat-1 gene in mammals (mammalianized)) were used. All of them were immunized with MOG by subcutaneous injection of MOG 35-55 (purchased from Sigma) into four points of dorsal areas of immunizing animals at 300 μg/body weight (kg). A second immunization was performed at the same dose six days after the first immunization. Animals survived for 24 days.

[0123] (2) Observation index of model:

[0124] Behavioral observation of clinical neuropathy: changes of animal behavioral were observed and recorded every day from the 12th day after establishment of model according to Kerlero scoring method. Scores were recorded according to the following behaviors: 1 point, weakness of tail; 2 points, paralysis of tail; 2.5 points, mild weakness of unilateral hind limbs; 3 points, significantly weakness of unilateral hind limbs; 4 points, paralysis of unilateral hind limbs; 4.5 points, paralysis of unilateral hind limbs accompanied with weakness of contralateral hind limbs or mild weakness of ipsilateral fore limbs; 5 points, paralysis of bilateral hind limbs; 6 points, paralysis of bilateral hind limbs accompanied with paralysis of unilateral fore limbs.

[0125] (3) Results: The results were shown in Table 6 below. The normal wild type mouse began to develop mild symptoms of weakness of tail from the 14th day after establishment of model, and by 23th day, all showed near-paralysis of unilateral hind limbs; while under the same induction conditions, mfat-1 transgenic mice showed no obvious symptoms during the whole process. It is indicated that the mfat-1 gene plays significant role in resisting or treating MS in the body.

TABLE-US-00007 TABLE 6 Behavior test scores of MS in mfat-1 transgenic mice and normal wildtype mice Behavior test scores of MS day day day day day day day day day day day day Groups 12 13 14 15 16 17 18 19 20 21 22 23 Wild type ♂ 0 0 1 1 1 1 3.5 1 3.5 3.5 3.5 3.5 Wild type ♀ 1 1 1 1 1 1 1 3.5 3.5 3.5 3.5 3.5 Wild type ♀ 0 0 1 1 1 1 1 3.5 1 3.5 3.5 3.5 mfat-1 0 0 0 0 0 0 0 0 0 0 0 0 transgenic mice ♀ mfat-1 0 0 0 0 0 0 0 0 0 0 0 1 transgenic mice ♀ mfat-1 0 0 0 0 0 0 0 0 0 0 0 1 transgenic mice ♂