USE OF NOCARDIA RUBRA CELL WALL SKELETON IN REGENERATIVE MEDICINE

Abstract

The present disclosure relates to a use of Nocardia rubra cell wall skeleton in regenerative medicine, and in particular, to a use of Nocardia rubra (especially the cell wall skeleton thereof) in the following aspects: promoting proliferation, growth, differentiation and migration of stem cells, and improving the survival rate of the stem cells, wherein the stem cells are selected from: adult stem cells, iPSCs, and mesenchymal stem cells.

Claims

1-10. (canceled)

11. A method of regulating stem cells comprising exposing the stem cells to a product derived from Nocardia rubra cell wall; wherein the ratio of the number of stem cells to the product derived from Nocardia rubra cell wall is 1 to 100 stem cells per 1 ng of the product derived from Nocardia rubra cell wall.

12. The method of claim 11, wherein the ratio is 5 to 50 stem cells per 1 ng of the product derived from Nocardia rubra cell wall.

13. The method of claim 11, wherein the regulation is selected from one of the following: promoting the proliferation of stem cells, promoting the growth of stem cells, promoting the differentiation of stem cells, promoting the migration of stem cells, improving the survival rate of stem cells, and a combination thereof.

14. The method of claim 11, wherein the stem cells are selected from the group consisting of: adult stem cells, iPSCs, mesenchymal stem cells, or combinations thereof.

15. The method of claim 14, wherein the stem cells are mesenchymal stem cells selected from the group consisting of: bone marrow mesenchymal stem cells, adipose-derived mesenchymal stem cells, synovial mesenchymal stem cells, umbilical cord mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, placental mesenchymal stem cells, amniotic mesenchymal stem cells, liver mesenchymal stem cells, muscle mesenchymal stem cells, lung mesenchymal stem cells, pancreatic mesenchymal stem cells and dental pulp mesenchymal stem cells.

16. The method of claim 14, wherein the mesenchymal stem cells are adipose-derived mesenchymal stem cells; and the adult stem cells are epithelial adult stem cells.

17. The method of claim 11, wherein the product derived from Nocardia rubra cell wall is Nocardia rubra cell wall or a component thereof.

18. The method of claim 11, wherein the product derived from Nocardia rubra cell wall is obtained by the following method comprising the following steps: 1) providing a Nocardia rubra; 2) disrupting the Nocardia rubra to obtain a disrupted product; 3.1) optionally, removing lipids from the disrupted product; 3.2) optionally, removing nucleic acids from the disrupted product; 3.3) optionally, removing proteins from the disrupted product; 3.4) obtaining a product derived from Nocardia rubra cell wall; 4) optionally, removing water from the product derived from Nocardia rubra cell wall, preferably lyophilizing the product derived from Nocardia rubra cell wall; 5) optionally, performing aliquoting; wherein, steps 3.1), 3.2) and 3.3) are interchangeable in order or performed in parallel; step 4) and step 5) are interchangeable in order.

19. A stem cell culture medium, comprising a product derived from Nocardia rubra cell wall, wherein the product derived from Nocardia rubra cell wall is Nocardia rubra cell wall or a component thereof.

20. The stem cell culture medium of claim 19, wherein the product derived from Nocardia rubra cell wall is obtained by the following method comprising the following steps: 1) providing a Nocardia rubra; 2) disrupting the Nocardia rubra to obtain a disrupted product; 3.1) optionally, removing lipids from the disrupted product; 3.2) optionally, removing nucleic acids from the disrupted product; 3.3) optionally, removing proteins from the disrupted product; 3.4) obtaining a product derived from Nocardia rubra cell wall; 4) optionally, removing water from the product derived from Nocardia rubra cell wall, preferably lyophilizing the product derived from Nocardia rubra cell wall; 5) optionally, performing aliquoting; wherein, steps 3.1), 3.2) and 3.3) are interchangeable in order or performed in parallel; step 4) and step 5) are interchangeable in order.

21. A method for promoting wound healing, comprising exposing a subject having a wound to a therapeutically effective amount of stem cells and a product derived from Nocardia rubra cell wall, wherein the stem cells are selected from the group consisting of adult stem cells, iPSCs, mesenchymal stem cells, or combinations thereof.

22. The method of claim 21, wherein the ratio of the number of stem cells/the product derived from Nocardia rubra cell wall is 1 to 100 stem cells per 1 ng of the product derived from Nocardia rubra cell wall.

23. The method of claim 21, wherein the ratio is 5 to 50 stem cells per 1 ng of the product derived from Nocardia rubra cell wall; the wound is a diabetes-related wound; and the regulation is selected from one of the following: promoting the proliferation of stem cells, promoting the growth of stem cells, promoting the differentiation of stem cells, promoting the migration of stem cells, improving the survival rate of stem cells, and a combination thereof.

24. The method of claim 21, wherein the stem cells are mesenchymal stem cells selected from the group consisting of: bone marrow mesenchymal stem cells, adipose-derived mesenchymal stem cells, synovial mesenchymal stem cells, umbilical cord mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, placental mesenchymal stem cells, amniotic mesenchymal stem cells, liver mesenchymal stem cells, muscle mesenchymal stem cells, lung mesenchymal stem cells, pancreatic mesenchymal stem cells and dental pulp mesenchymal stem cells.

25. The method of claim 21, wherein the mesenchymal stem cells are adipose-derived mesenchymal stem cells.

26. The method of claim 21, wherein stem cells are adult stem cells, and the adult stem cells are epithelial adult stem cells and the product derived from Nocardia rubra cell wall is Nocardia rubra cell wall or a component thereof.

27. A pharmaceutical composition comprising: (i) stem cells, and (ii) a product derived from Nocardia rubra cell wall, wherein the ratio of the number of stem cells to the product derived from Nocardia rubra cell wall is: 1 to 100 stem cells to 1 ng of the product derived from Nocardia rubra cell wall.

28. The pharmaceutical composition of claim 27, wherein the ratio is 5 to 50 stem cells per 1 ng of the product derived from Nocardia rubra cell wall; and the regulation is selected from one of the following: promoting the proliferation of stem cells, promoting the growth of stem cells, promoting the differentiation of stem cells, promoting the migration of stem cells, improving the survival rate of stem cells, and a combination thereof.

29. The pharmaceutical composition of claim 27, wherein the stem cells are selected from the group consisting of: adult stem cells, iPSCs, mesenchymal stem cells, or combinations thereof.

30. The pharmaceutical composition of claim 29, wherein the stem cells are mesenchymal stem cells selected from the group consisting of: bone marrow mesenchymal stem cells, adipose-derived mesenchymal stem cells, synovial mesenchymal stem cells, umbilical cord mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, placental mesenchymal stem cells, amniotic mesenchymal stem cells, liver mesenchymal stem cells, muscle mesenchymal stem cells, lung mesenchymal stem cells, pancreatic mesenchymal stem cells and dental pulp mesenchymal stem cells; and the product derived from Nocardia rubra cell wall is Nocardia rubra cell wall or a component thereof from Nocardia rubra.

Description

DESCRIPTION OF THE DRAWINGS

[0123] FIG. 1A: Ad-MSCs digested and isolated from human adipose tissue are spindle-shaped under light microscope.

[0124] FIG. 1B: Results of adipogenic induction show obvious presence of red lipid droplets.

[0125] FIG. 1C: Results of osteogenic induction show obvious presence of red calcium deposits.

[0126] FIG. 1D: Results of flow cytometry show: positive for CD105 (95.6%), CD90 (96.9%) and CD44 (78.6%); negative for CD31 (4.68%), CD34 (2.13%) and CD106 (9.58%).

[0127] FIG. 2A: 10 μg/ml of Composition 1 of the present application was applied to Ad-MSCs for 24 h, 48 h, 72 h and 96 h. Then, the absorbance (OD) of the Ad-MSCs was detected at a wavelength of 450 nm by an enzyme-linked immunometric meter using CCK-8.

[0128] FIG. 2B: Statistical analysis of cell viability in each group.

[0129] FIG. 2C: 10 μg/ml, 50 μl of Composition 1 of the present application was applied to Ad-MSCs for 72 h and 96 h. Then the EdU incorporation analysis was performed by using EDU kits, and positive cells were observed by fluorescence microscopy.

[0130] FIG. 2D: Statistical analysis of the cell proliferation rate in each group. The experiment was repeated for 3 times. The data are represented by bar graphs as mean±SD (*P<0.05, **P<0.01, vs. control).

[0131] FIG. 3A to FIG. 3B: The apoptosis rate of stem cells in blank control group, high glucose group, and the group treated with the composition of the present application, was detected by flow cytometry at 48 h and 72 h respectively.

[0132] FIG. 3C to FIG. 3D: Statistical analysis of the apoptosis rate in each group. The experiment was repeated for 3 times. The data are represented by bar graphs as mean±SD (**P<0.01, vs. high glucose).

[0133] FIG. 4A: Fluorescence results after TUNEL staining. The apoptosis rate of stem cells in blank control group, high glucose group, and the group treated with the composition of the present application, was detected at 48 h and 72 h respectively.

[0134] FIG. 4B: Quantitative analysis of the percentage of TUNEL positive staining. The experiment was repeated for 3 times. The data are represented by bar graphs as mean±SD (**P<0.01, ***P<0.001, vs. high glucose).

[0135] FIG. 5A and FIG. 5B: High glucose was added to the cells to induce apoptosis. Western blotting was performed to detect the protein expression levels of c-caspase-3 and Bax.

[0136] FIG. 5C and FIG. 5D: Grayscale analysis of protein expression. The experiment was repeated for 3 times. The data are represented by bar graphs as mean±SD (*P<0.05, **P<0.01, ***P<0.001, vs. high glucose).

[0137] FIG. 6A: Cells were inoculated into the wounds of diabetic mice. Then the survival rate of Ad-MSCs in each group was observed according to the cells labeled with the fluorescent dye CM-Dil on day 0, day 2 and day 7.

[0138] FIG. 6B: Results showing the statistical analysis of the fluorescence flux in CM-Dil-labeled cells.

[0139] FIG. 6C: The wound healing status in each group immediately after modeling and on day 7 and day 14. Compared with the blank control group and the Ad-MSC group, the group treated with the composition of the present application shows better wound healing.

[0140] FIG. 6D: Measurement and calculation of the wound healing rate in each group (*P<0.05, **<0.01, vs. blank).

[0141] FIG. 7A: HE staining shows the regeneration of diabetic wounds. The staining result represents collagen levels.

[0142] FIG. 7B: CD31-positive immunofluorescence staining of red vesicle-like structures in the figure suggests angiogenesis. Green positive fluorescence represents TNF-α level.

DETAILED DESCRIPTION OF THE INVENTION

[0143] “Isolation” refers to the separation of the Nocardia rubra of the present disclosure from its original growth environment.

[0144] The skilled person knows that the cell wall structures of gram-positive bacteria and gram-negative bacteria are different. Specifically, the cell wall of gram-positive bacteria is thicker (usually 20 nm to 80 nm), comprising about 90% peptidoglycan and about 10% teichoic acid (a polymer formed by alcohol and phosphoric acid molecules, usually existing in the form of sugar ester or amino acid ester). The peptidoglycan layer is dense, even as many as 20 layers. However, the cell wall of gram-negative bacteria is much thinner than that of gram-positive bacteria, and the structure is more complex, divided into outer membrane and peptidoglycan layer (usually 2 nm to 3 nm).

[0145] The peptidoglycan layer is a unique component of the bacterial cell wall and is a derivative of heteropolysaccharide. Each peptidoglycan monomer comprises 3 moieties: the sugar unit (for example, at least two sugar molecules are connected by glycosidic bonds to form the framework of peptidoglycan), the peptide tail (a short peptide chain formed by linking several amino acids, which is connected to a N-acetylmuramic acid molecule), and the peptide bridge (which crosslinks the adjacent “peptide tails” to form a high-strength network structure). Different bacteria have different peptide bridges, peptide tails and cross-linking manners.

[0146] Isolated Nocardia rubra Cell Wall

[0147] In the present disclosure, the “isolated Nocardia rubra cell wall” can be understood as either a complete cell wall or an incomplete cell wall (for example, disrupted or partially degraded). Under the teaching of the present disclosure, the skilled person will understand that the components exhibiting the desired activity are derived from Nocardia rubra cell wall (for example, the cell wall itself or components thereof). Therefore, complete cell wall, disrupted cell wall, incompletely degraded product of cell wall, cell wall components, cell wall extracts and other various forms are allowed to be used in clinical applications, which are all encompassed in the scope of the present disclosure.

[0148] Cell Wall Skeleton

[0149] A component that constitutes the main structure of the cell wall; however, it cannot be interpreted as merely representing the cross-linked network-like entity of the cell wall, and the skilled person understands that other cell wall components adsorbed by, bound to and carried by the cross-linked network-like entity are not excluded.

[0150] Identification of Nocardia rubra

[0151] According to known or future microbial identification techniques, the skilled person can perform taxonomic identification on a strain of bacteria. For example, the available identification techniques include morphology, physiological and biochemical characteristics, 16S rRNA, and the like. The skilled person understands that with the development of science and technology, identification techniques involve different methods. In the earlier period, morphological and biochemical identification methods were mainly used, but the reliability of these methods is not high. After the advent of sequencing technology, the skilled person can identify bacteria strains in a more reliable way. For example, when DNA sequences of 16S rRNA are identified as having more than 97% (inclusive) of identity, the two bacteria would be deemed as belonging to the same species. For Nocardia rubra, the known strains deposited in international (or national) collection authorities are used as model strains, and the strains to be identified are compared with the model strains.

[0152] Dosage Form

[0153] The medicament or pharmaceutical composition or active component or product of the present disclosure can be formulated into, but not limited to, the following forms: ointment, cream, plaster, gel, lotion, tincture, liniment, oil, paste, lyophilized powder, aerosol, suppository, patch, suspension, oral solution, buccal tablet and skin care product (cleanser, toning lotion, serum, lotion, cream and mask).

[0154] Formulation Unit

[0155] The medicament or pharmaceutical composition or active component or product of the present disclosure can be formulated into the form of a formulation unit.

[0156] In some embodiments, the unit dose of the medicament (or formulation, or therapeutic agent, or medical device) comprises: [0157] 1 μg to 1000 μg of the product derived from Nocardia rubra cell wall; or [0158] 1 μg to 1000 μg of the Nocardia rubra cell wall; or [0159] 1 μg to 1000 μg of the Nocardia rubra cell wall skeleton.

[0160] Particular examples of the unit dose are 1, 2, 5, 10, 15, 20, 25, 30, 40, 50, 55, 56, 57, 58, 59, 60, 61, 62, 63, 65, 66, 67, 68, 69, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 mg±10%, and the ranges between any two of the above values.

[0161] “Administering”, “giving”, “provided . . . with” and “treating”, when applied to animals, humans, cells, tissues, organs or biological samples, refer to the contact of the medicament or medical device with the animals, humans, cells, tissues, organs or biological samples.

[0162] “Treatment” means administrating an internal or external medicament (therapeutic agent, active component or composition) (such as the Nocardia rubra cell wall or pharmaceutical composition thereof according to the present disclosure) or a medical device to a subject (or population), in order to alleviate (relieve, delay, improve, cure) one or more disease symptoms to a clinically measurable degree in the subject (or population) to be treated, wherein the subject has, is suspected of having, or is susceptible to one or more diseases or symptoms thereof.

[0163] The amount of the medicament (therapeutic agent, active component or composition) that can effectively alleviate any disease symptoms is called the therapeutically effective amount. It can vary depending on a variety of factors, such as the disease state, age and body weight of the subject. It should be understood that the medicament (therapeutic agent, active component or composition) may be ineffective in alleviating the target disease or symptoms thereof in single subject, but is statistically effective for the target disease or symptoms thereof according to any statistical test method known in the art (such as Student t test, chi-square test and U test according to Mann and Whitney).

[0164] The term “optionally” means that the event following this term can happen, but not necessarily happen; it depends on the situation. For example, “optionally, performing aliquoting” means that the product is allowed to be aliquoted, but it is not necessary to be aliquoted; whether the product is aliquoted or not does not affect the realization of the technical effects.

[0165] “A”, “an”, “single” and “the”, if not explicitly stated, also involve plural forms.

[0166] The present disclosure is further described below with reference to the examples, preparation examples and test examples. However, these examples, preparation examples and test examples do not limit the scope of the present disclosure. When the particular conditions are not specified, operation should be performed in accordance with the normal conditions and the conditions recommended by the manufacturer. The reagents without giving particular sources are conventional reagents purchased on the market.

EXAMPLES

Example 1. Commercially Available Nocardia rubra Cell Wall Skeleton

[0167] The Nocardia rubra cell Wall Skeleton (Trade Name: NAKEJIA) was Purchased from Liaoning Greatest Bio-pharmaceutical Co., Ltd., with the medicine permission No. S20030009 (2 ml/ampule; lyophilized powder), which comprises 60 μg of active components and 15 mg of dextran 40.

PREPARATION EXAMPLES

Preparation Example 1. Culture Methods

[0168] In addition to commercially available product, the Nocardia rubra cell wall skeleton can also be prepared in-house. For example:

[0169] 1. Nocardia rubra can be cultured by conventional microbial production methods.

[0170] 2. The culture method can be solid culture or liquid culture.

[0171] 3. There are no special requirements on the nutrient sources in the culture medium. The culture medium can contain carbon sources, nitrogen sources and other nutrient sources that are commonly used for microbial culture. [0172] The carbon source can be any carbon source that can be consumed by Nocardia rubra, for example fructose, glucose, etc. [0173] The nitrogen source can be broth, peptone, ammonium salt, nitrate and other organic or inorganic nitrogen-containing compounds. [0174] For other nutrient sources, some inorganic salts can be added appropriately, for example NaCl and phosphates.

[0175] 4. There are no strict limitations on the culture conditions (temperature, time, etc.). Those skilled in the art can choose the conditions that maximize the yield based on the data collected from preliminary small-scale pilot test.

[0176] 5. As an example, the following culture conditions was used to ferment Nocardia rubra:

[0177] (1) The composition of medium comprising:

[0178] peptone, broth, sodium chloride, phosphate, glycerin (and, optionally, agar, when in solid culture).

[0179] (2) Parameters of the Culture Method:

[0180] After the working strain was recovered, it was transferred to a solid culture medium for 3-5 days, and then transferred to liquid culture (30-37° C., maintained for 3-5 days). The fed-batch semi-continuous mode or the batch mode can be used. The pH, bacterial density, dissolved oxygen and carbon source consumption were monitored during culture.

Preparation Example 2. Bacteria Disruption

[0181] The bacteria obtained in Preparation Example 1 were collected and the cells were disrupted (for example, but not limited to by sonication). Any appropriate well-known method in the art for disrupting the bacteria can be used, for example CN101250490A or CN101323865A.

[0182] The disruption state was checked under a microscope. There should be no more than 5 intact bacteria in each visual field. The disruption was deemed as being qualified, when several (10 to 30) visual fields checked met this standard.

Preparation Example 3. Removal of Nucleic Acids, Lipids, Proteins and Cell Membranes

[0183] 1. Removal of Nucleic Acids:

[0184] The supernatant after disruption was centrifuged. DNase and RNase were added to the obtained precipitate, and nucleic acids were removed according to the operation recommended by the supplier of the enzymes.

[0185] 2. Removal of Proteins:

[0186] Commonly used protease (such as trypsin) was added to the precipitate, and proteins were removed according to the operation recommended by the supplier of the enzyme.

[0187] 3. Removal of Lipids:

[0188] Organic reagents (for example, but not limited to, any one of acetone, ether and ethanol or a combination thereof) were added to the precipitate, and lipids were removed according to conventional operations in the art.

[0189] 4. Removal of Cell Membranes:

[0190] Triton X-100 was added to the precipitate, and the precipitate was collected by centrifugation according to conventional operations in the art, and rinsed with PBS.

[0191] It should be understood that among the above steps for removing impurities, those skilled in the art can adjust the order of the steps to make them compatible with each other.

[0192] After removing the non-cell wall components, the precipitate was re-dissolved in water for injection, and then stored for later use. Optionally, it could be sterilized at 115° C. for 20-30 minutes as the stock solution of the cell wall skeleton (mainly comprising the cell wall skeleton and components thereof).

Preparation Example 4. Preparation Methods of the Pharmaceutical Compositions

[0193] 1. Liquid Composition

[0194] An excipient (such as dextran 40, mannitol or trehalose) was added to the product obtained in Preparation Example 3. It was referred to as the pharmaceutical composition after aliquoting.

TABLE-US-00001 TABLE 1 The pharmaceutical composition can be formulated into various forms Composition Capacity of each vial Components and amount Composition 1 2 ml Active component 60 μg Dextran 40 15 mg Composition 2 2 ml Active component 60 μg Dextran 40 12 mg Composition 3 2 ml Active component 120 μg Dextran 40 36 mg Composition 4 2 ml Active component 60 μg Trehalose 12 mg Composition 5 2 ml Active component 120 μg Trehalose 36 mg Composition 6 2 ml Active component 120 μg Mannitol 36 mg Composition 7 2 ml Active component 60 μg Mannitol 12 mg

[0195] 2. Powder Composition

[0196] The pharmaceutical compositions of item 1 were lyophilized to prepare lyophilized powders (numbered as lyophilized composition 1 to lyophilized composition 7, respectively).

[0197] 3. Quality Inspection (Lyophilized Composition 1 was Taken as an Example)

TABLE-US-00002 TABLE 2 Quality inspection Appearance White unconsolidated solid or powder Water content ≤6% Solubility The product was deemed as qualified, if it could be dissolved within 1 min when 2.0 ml of NaCl injection was added Residual amount of proteins 0.4 μg/vial (criteria: ≤9.0 μg/vial) Residual amount of RNA 0.8% (criteria: not more than 5%) Residual amount of DNA 0.9% (criteria: not more than 5%) Residual amount of Triton Undetectable (criteria: not more X-100 than 5%) Residual amount of lipids 3.8% (criteria: not more than 5%) Phagocytosis rate 75% (criteria: ≥40%) Phagocytic index 1.05 (criteria: ≥0.50) Abnormal toxicity in mice All the mice should survive and have no abnormal reactions during the observation period. The composition was deemed as being qualified, if the body weight of each mouse increased at the due date Abnormal toxicity in All the guinea pigs should survive and guinea pigs have no abnormal reactions during the observation period. The composition was deemed as being qualified, if the body weight of each guinea pig increased at the due date

[0198] Materials and Methods

[0199] 1. Isolation, Culture and Passage of Human Ad-MSCs

[0200] Adipose tissue samples were obtained from liposuction aspirate of subjects (age range 30-45 years) and Ad-MSCs were isolated and cultured. Subjects were plastic surgery patients in the Affiliated Hospital of Xuzhou Medical University. The experiments were approved by the ethics committee, and informed consents of the patients were collected.

[0201] The obtained fresh adipose tissue extract was digested with 0.25% trypsin-EDTA, filtered, and centrifuged to retain the cell pellet. DMEM (Invitrogen) culture medium containing 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin was added. The cell culture dish was placed in a 37° C., 5% CO.sub.2 incubator to culture the cells. After that, the medium was changed every 2-3 days after washing the cells with PBS. The cells were passaged when growing to 80%.

[0202] 2. Determination of Cell Viability

[0203] Ad-MSCs in logarithmic growth phase were seeded in a 96-well plate at 4×10.sup.3 cells/well; DMEM+10% fetal bovine serum+1% penicillin/streptomycin was used for co-culture; the composition of the present application (Composition 1 or the commercially available composition of Example 1) was dissolved and diluted to 10 μg/ml in PBS buffer. Composition 1 was added after the Ad-MSCs adhered to the wall, and the experiment was divided into four groups: control, 25 μl, 50 μl and 75 μl. After 24 h, 48 h, 72 h and 96 h, 10 μL of CCK-8 reagent was added to each well, and incubated for further 2 h. Then the absorbance of each well at a wavelength of 450 nm was detected by an enzyme-linked immunometric meter. The cell growth curve was plotted with the culture time as the horizontal axis and the cell number (absorbance) as the vertical axis.

[0204] 3. EdU Incorporation Experiment

[0205] EdU incorporation assay was performed by using Cell-Light EdU 567 in vitro imaging kit (RiboBio). Ad-MSCs in the logarithmic growth phase were seeded in a 96-well plate at 4×10.sup.3 cells/well. 10 μg/ml, 50 μl of the composition of the present application was applied onto the Ad-MSCs. 100 μl of EDU medium was added to each well after 72 h and 96 h and incubated for two hours, and the wells were washed for 1-2 times with PBS. 4% paraformaldehyde fixative was added to each well and the plate was incubated at room temperature for 30 min. Then 2 mg/ml glycine solution was added and the plate was shaken on a shaker for 5 minutes. After washing with PBS, osmotic agent was added, the plate was shaken on a shaker for 10 min and washed with PBS. Apollo staining reaction solution was added and the plate was incubated at room temperature in the dark for 30 minutes. The staining reaction solution was discarded. Penetrant (0.5% TritonX-100 in PBS) was added and the plate was shaken on a shaker 2-3 times, 10 minutes each time, and the penetrant was discarded. The plate was washed with PBS again and Hoechest 33342 reaction solution was added. The plate was incubated at room temperature in the dark for 30 minutes, and the reaction solution was discarded. After washing 1-3 times with PBS, positive cells were observed by fluorescence microscopy.

[0206] 4. Detection of Apoptosis

[0207] 4.1 Flow Cytometry:

[0208] When Ad-MSCs reached 80% confluence, they were seeded at a density of 1×10.sup.4 cells/well in a 6-well plate. The experiment was divided into four groups. After the cells adhered to the wall, 100 μl of 50% sucrose was added to each experimental group to induce apoptosis of Ad-MSCs, and 100 μl or 250 μl of the composition of the present application at 10 μg/ml was respectively added to two experimental groups. After 48 h and 72 h, the supernatant of each group of cells was collected in flow tubes, the adherent cells were digested with EDTA-free trypsin and collected into flow tubes of the same group. The tubes were centrifuged at 2000 rpm for 5 min, washed twice with PBS buffer, shaken and mixed well. 500 μl of binding buffer (Annexin V-FITC apoptosis detection kit, Shanghai Beyotime Biotechnology Co., Ltd.), 5 μl of FITC and 5 μl of PI were successively added to each tube. The tubes were shaken and mixed well and incubated in the dark at 4° C. for 5-15 min. The results were detected and analyzed by flow cytometry (BD Biosciences).

[0209] 4.2 TUNEL Method:

[0210] Several 18 mm×18 mm coverslips were sterilized by soaking in 75% ethanol. The coverslips were placed in a 6-well plate and rinsed with PBS buffer several times until the ethanol residue was completely removed. Ad-MSCs were cultured in a 6-well plate at a cell density of 1×10.sup.4 cells per well. The experimental grouping was the same as that of flow cytometry. The cells were washed 3 times with PBS buffer and fixed in 4% paraformaldehyde at room temperature for 30 min; washed 3 times with PBS buffer and incubated with 0.1% TritionX-100 at 2° C.-8° C. for 10 min; washed 3 times with PBS buffer. 500 μl of TUNEL reaction solution (TUNEL apoptosis kit, Roche) was prepared. 50 μl of enzyme solution and 450 μl of labeling solution were mixed to prepare reagent A. 50 μl of reagent A was added to the negative control group, which was placed in a 37° C. incubator in the dark for 60 min. DNase I was added to the positive control group, which was incubated at room temperature for 10 min. The cells were washed 3 times with PBS buffer and 50 μl/well of TUNEL reaction mixture was added. The cells were incubated in a 37° C. incubator in the dark for 60 min and washed 3 times with PBS buffer. 50 μl/well of DAPI staining solution was added and the cells were incubated at room temperature for 3 min. The samples were photographed under a fluorescence microscope for analysis, and the wavelength range of detection light was 570-620 nm (maximum wavelength 580 nm).

[0211] 5. Western Blot Analysis

[0212] The treated cells were collected. 300 μl of cell lysis mixture (PIPA cell lysis solution was dissolved on ice, PMSF was added at a ratio of 100:1) was added and then placed on ice. The cells were fully lysed and adherent cells were scraped. The mixture was centrifuged in a Centrifuge-5810R refrigerated high-speed centrifuge at 4° C., 12,000 rpm for 20 min and the supernatant was collected.

[0213] The cell extracts were resolved on SDS-polyacrylamide gels. Then the proteins were transferred to nitrocellulose membranes and incubated with the following antibodies: rabbit-anti-human caspase-3 monoclonal antibody (1:400; CST, USA), rabbit-anti-human Bax monoclonal antibody (1:400; CST, USA). After incubation with the primary antibody, the membranes were washed for 5 min×3 times and incubated with diluted secondary antibody (1:10000) at room temperature in the dark for 2 h. The secondary antibody was discarded and the membranes were washed for 5 min×3 times. Equal amounts of ECL luminescent solution A and B were pipetted respectively and mixed well to prepare the ECL working solution. The ECL working solution was evenly dropped on the membranes. The membranes were placed in a TANON gel imager for exposure and development, photographed, and analyzed by ImageJ software.

[0214] 6. Establishment of Diabetic Wound Animal Model

[0215] The experimental animals were 4-week-old BALB/c athymic nude mice, SPF grade. All animal studies were approved by the Animal Care and Use Committee. The experimental mice were kept in SPF grade animal facility. Mice were fasted for 12 h before model establishment, weighed and recorded. Diabetic mice were induced by intraperitoneal injection of 2% STZ (Sigma) at a dose of 150 mg/kg.

[0216] Blood glucose was measured on day 7 after injection. Blood was collected through tail vein, and the blood glucose concentration of the mice was measured with a blood glucose monitor and recorded. From day 7, the blood glucose concentration of the mice was greater than 16.7 mmol/L and the typical symptoms of diabetes “polydipsia, polyphagia, polyuria and weight loss” could be observed, which was regarded as successful model establishment.

[0217] The experimental animals were divided into 3 groups (5 animals in each group): [0218] blank control group, [0219] Ad-MSC group, [0220] The composition of the present application+Ad-MSC group.

[0221] Once the diabetic mouse model was successfully established, the mice were anesthetized; and wounds with a diameter of 1.5 cm were prepared. The pretreated Ad-MSCs were injected into the skin of mice in each group by multi-point intradermal injection, with six points in each wound; and 0.1 ml of cells injected at each point. The wound surface was covered with sterile gauze, and feeding were continued for further observation. The survival of cells in the wound and the skin healing of mice were observed by using LB983 in vivo imaging system.

[0222] 7. Staining with CM-Dil Viable Cell Stain

[0223] Ad-MSCs were labeled with CM-Dil before injection. According to the method recommended by the supplier, CM-Dil viable cell stain was added to the cells and incubated for 30 minutes. The supernatant was discarded after centrifugation and the cells were washed 3 times with PBS buffer. Finally, an appropriate amount of PBS was added and mixed well, and the labeled Ad-MSCs were placed in an ice box for later use.

[0224] 8. HE Staining

[0225] On day 14 after operation, the mice in each group were sacrificed by cervical dislocation. The whole skin layer was excised 3 mm beyond the boundary of the wound, reaching the muscle layer; and the samples were stored in formaldehyde fixative. After 48 h, the steps of dehydrating-transparentizing-immersing in paraffin-embedding-sectioning were performed. After being deparaffinized, the sections were stained with hematoxylin for 10 min, rinsed with running water, treated with 1% hydrochloric acid for a few seconds, the sections turned blue with 0.2% ammonia and rinsed. Then the sections were stained with eosin for 5 min, rinsed and dehydrated with ethanol, and finally transparentized with xylene, sealed with neutral gum. The thickness of newly formed tissue, the arrangement of collagen and connective tissue and the infiltration of inflammatory cells in the wounds of each group were observed under a microscope, and photographed for analysis.

[0226] 9. Masson Staining

[0227] The sections were deparaffinized, and rinsed with water: The sections were washed with distilled water and stained with hematoxylin for 10 min to stain the nuclei, thoroughly washed with distilled water and then treated with hydrochloric acid-alcohol. Masson Ponceau acid fuchsin solution was applied for 5-10 min. The sections were washed with 2% glacial acetic acid aqueous solution, treated with 1% phosphomolybdic acid aqueous solution for 3-5 min, and then directly stained with aniline blue or light green solution for 5 min and washed with 0.2% glacial acetic acid aqueous solution. Finally, the sections were dehydrated with 95% alcohol and anhydrous alcohol, soaked in xylene till fully transparent; the sections were sealed with neutral gum, dried and then observed under a microscope.

[0228] 10. Histo-Immunofluorescence Staining

[0229] The tissue paraffin sections were deparaffinized and incubated with 3% H.sub.2O.sub.2 at room temperature for 5-10 min to eliminate endogenous peroxidase activity. The sections were rinsed with distilled water, soaked twice in PBS for 5 min each time, and blocked with 10% normal goat serum (diluted in PBS) at room temperature for 10 min. The blocking solution was discarded without washing the sections. CD31 primary antibody (dilution ratio 1:300) working solution was added dropwise and incubated overnight. The sections were rinsed 3 times with PBS for 5 min each time. An appropriate amount of biotin-labeled fluorescent secondary antibody working solution (dilution ratio 1:400) was added dropwise in the dark. The sections were incubated at 37° C. in the dark for 1 h and rinsed 3 times with PBS for 5 min each time. An appropriate amount of DAPI staining solution was added dropwise. The sections were incubated at room temperature in the dark for 3 min and rinsed 3 times with PBS for 5 min each time. Finally, anti-quenching sealing solution was added dropwise to each section. The sections were covered with coverslips, fixed, and stored in the dark.

[0230] 11. Statistical Analysis

[0231] Statistical analysis was performed by using SPSS software (SPSS 16.0). The experimental results were represented as mean±SD. The comparison between two groups was performed by independent samples t-test, and the comparison of means among multiple groups was performed by one-way ANOVA. α=0.05 was the level of significance, and the difference with P<0.05 was considered to be statistically significant.

Test Example 1. Identification of Morphology, Surface Markers and Induced Differentiation Ability of Human Ad-MSCs

[0232] The collagenase digestion method was used. Primary Ad-MSCs extracted from adipose tissue extracts were seeded in cell culture dishes and passaged when they reached 80% confluence. The proliferation rate of the cells increased significantly after passage. The cells were uniform in terms of morphology and spindle-shaped (FIG. 1A). Well-grown Ad-MSCs of the P3 generation were used for adipogenic differentiation and osteogenic differentiation, respectively. Observation of the cells stained with oil red after 2 weeks under a microscope showed obvious presence of red lipid droplets of different sizes in the cells (FIG. 1B); observation of the cells stained with alizarin S stain showed obvious deposition of red calcium deposits (FIG. 1C). This indicated that the isolated and extracted cells had the characteristics of stem cell differentiation potential.

[0233] Six different cell surface markers were detected by flow cytometry. The results were as shown: positive for CD105 (95.6%), CD90 (96.9%) and CD44 (78.6%); negative for CD31 (4.68%), CD34 (2.13%) and CD106 (9.58%) (FIG. 1D). These results were consistent with the characteristics of the Ad-MSC immunophenotype.

Test Example 2. Effects of the Composition of the Present Application on the Activity and Proliferation of Ad-MSCs

[0234] In order to detect the effect of the composition of the present application on the activity of Ad-MSCs, Composition 1 of the present application was applied to Ad-MSCs for 24 h, 48 h, 72 h and 96 h. Then the absorbance of the stem cells at a wavelength of 450 nm was detected by an enzyme-linked immunometric meter using CCK-8 reagent. The results showed that the viability of Ad-MSCs was significantly enhanced, at 72 h and 96 h after exposing the stem cells to 50 μl of the composition of the present application at a concentration of 10 μg/ml (FIG. 2A and FIG. 2B).

[0235] In order to verify the effect of the composition of the present application on the proliferation of Ad-MSCs, 10 μg/ml, 50 μl of the composition of the present application was applied to stem cells. The incorporation of EdU was analyzed by using the EDU kit 72 h and 96 h later, respectively. The positive cells were observed by fluorescence microscopy. The results showed that the composition of the present application could increase the proliferation rate of Ad-MSCs (FIG. 2C and FIG. 2D).

Test Example 3. The Composition of the Present Application Inhibits the High Glucose-Induced Apoptosis of Ad-MSCs

[0236] High glucose was added to Ad-MSCs to induce apoptosis. After treating the cells with different concentrations of Composition 1 of the present application for 48 h and 72 h, respectively, apoptosis was detected by using FITC-PI flow cytometry apoptosis detection kit.

[0237] The results showed that the composition of the present application could inhibit the high glucose-induced apoptosis of stem cells (FIG. 3A, FIG. 3B, FIG. 3C and FIG. 3D). In order to confirm the results, apoptosis was detected by using the TUNEL method. The TUNEL fluorescence images were quantitatively analyzed, and the results were consistent with the results of flow cytometry (FIG. 4A and FIG. 4B).

Test Example 4. Detection of Expression Levels of Apoptosis Marker Proteins

[0238] High glucose was added to the cells to induce apoptosis. The cells were treated with different concentrations of the composition of the present application for 48 h and 72 h, and proteins were extracted. The protein expression levels of c-caspase-3 and Bax (two major apoptotic markers) were detected by Western blotting, and β-actin was used as an internal reference. The results showed that the expression levels of c-caspase-3 and Bax proteins were decreased after treatment with the composition of the present application (FIG. 5A, FIG. 5B, FIG. 5C and FIG. 5D).

Test Example 5. The Composition of the Present Application Improves the Survival Rate of Ad-MSCs and Accelerates Wound Healing in Nude Mice

[0239] In order to further explore the effect of the composition of the present application on Ad-MSCs in diabetic wound healing, a wound model simulating the mechanism of wound repair in human skin was established in diabetic nude mice.

[0240] After the successful establishment of diabetic mouse model, the mice were anesthetized and wounds with a diameter of 1.5 cm were prepared. The pretreated cells labeled with fluorescent dye CM-Dil were injected into the wound of mice in treatment group by intradermal injection. The survival rate of cells was observed by using LB983 in vivo imaging system.

[0241] The results showed that the Ad-MSCs in the group treated with the composition of the present application had a higher survival rate than the group using Ad-MSCs only (FIG. 6A and FIG. 6B). The wound healing process of diabetic mice treated with Ad-MSCs was also evaluated for 14 days. Compared with the blank group, the wounds of the mice treated with Ad-MSCs healed well, while the group treated with the composition of the present application healed better, with accelerated wound healing (FIG. 6C and FIG. 6D) and an increased wound healing rate (Table 3).

TABLE-US-00003 TABLE 3 Wound healing on day 14 after operation (mean ± SD, n = 3 ) Wound Group healing rate (%) Blank 60.5 ± 2.8  Ad-MSC  80.0 ± 1.8** Ad-MSC + the composition of the  92.5-0.8** present application **P < 0.01, vs. blank

[0242] The skin samples from the wounds of nude mice in each group were collected after 14 days of healing, with a diameter of about 1 cm and a thickness of the whole skin layer. The samples of each group were subjected to HE staining and Masson staining (FIG. 7A). The results showed that both the Ad-MSC group and the group treated with Ad-MSC+ the composition of the present application could promote the regeneration of diabetic wounds, while in the group treated with the composition of the present application, the collagen fibers grew more neatly and the stripes were clearer. The microvascular regeneration and inflammatory factors in the skin were detected by histo-immunofluorescence of CD31 and TNF-α (FIG. 7B). The results showed that the group treated with the composition of the present application can better promote the skin regeneration and revascularization of the diabetic wound than the Ad-MSC group.

[0243] Discussions:

[0244] There are many reasons for diabetic refractory wounds, for example, the absence of cellular and molecular signals required for normal wound healing process. In addition, peripheral neuropathy, peripheral circulation damage and disturbance of protease balance are important factors for difficult healing of diabetic wounds [11]. On the other hand, the abnormal vascular microenvironment under high glucose conditions can lead to abnormal cell growth environment, and ultimately damage the vascular reconstruction in the trauma area [12]. In addition, the reduced number of fibroblasts, increased glycosylation of proteins, abnormal expression of growth factor, delayed inflammatory process, and accumulation of glycosylation end products in damaged tissues under diabetic high glucose conditions affect the migration and function of myeloid cells [13]. Studies have shown that Nr-CWS can enhance the activity of T cells, macrophages and natural killer cells in vivo and promote cytokine production. It can promote the killing activity of macrophages, killer T cells, LAK cells and NK cells by increasing the levels of a variety of cytokines, including IL-1, IL-2, IL-6, TNF and IFN [14].

[0245] Ad-MSCs are a kind of stem cells extracted from adipose tissue and they have multi-directional differentiation potential [15-17]. Ad-MSCs can migrate to damaged sites through differentiation potential, repair damaged skin with differentiated cells, and secrete a variety of growth factors [18]. They accelerate angiogenesis of wound and promote wound healing [19]. Some scholars have found that Ad-MSCs can differentiate into fibroblasts, showing not only morphological similarity, but also the ability to express fibroblast surface proteins, including vimentin and fibronectin [20]. Meanwhile, Ad-MSCs can also be directly transformed into fibroblasts and keratinocytes for wound repair [21]. However, previous studies have found that most Ad-MSCs undergo apoptosis when injected into wounds of diabetic mice, resulting in delayed wound healing. Ad-MSCs cultured under high glucose conditions undergo apoptosis in a time-dependent manner [22,23].

[0246] In the test examples, the effect of the composition of the present application on in vitro activity and proliferation ability of Ad-MSCs was detected by CCK-8 and EdU methods. The results showed that the composition of the present application could improve the activity and proliferation ability of Ad-MSCs. Apoptosis of cells was detected by flow cytometry and TUNEL method, and it was found that the composition of the present application could inhibit the high glucose-induced apoptosis of Ad-MSCs in a time- and concentration-dependent manner.

[0247] The effect of the composition of the present application on the survival rate of Ad-MSCs was tested in vivo in animals, and the results showed that the survival rate of Ad-MSCs treated with the composition of the present application was higher than that of Ad-MSCs only, and the wound healing rate was higher. According to the results of histo-immunofluorescence, the endothelial cell adhesion factor CD31 (which represents angiogenesis) was increased, and the TNF-α (which represents the inflammatory level) was decreased.

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