Use of the cell wall skeleton of Rhodococcus rhodochrous in regenerative medicine

Abstract

The present invention provides uses of the cell wall skeleton of Rhodococcus ruber in promoting stem cell proliferation, promoting stem cell growth, promoting stem cell differentiation, promoting stem cell migration, and improving stem cell survival, wherein the stem cells are selected from adult stem cells, iPSCs, and mesenchymal stem cells.

Description

Technical Field

[0001] This application claims the benefit of priority of a Chinese patent application (Application No. CN202010068249.1) filed on January 21, 2020.

[0002] This application relates to regenerative medicine. Specifically, it relates to the use of Rhodococcus rhodochrous (especially cell wall skeleton) in promoting the proliferation and differentiation of stem cells.

Background Art

[0003] Stem cells are primitive cells with self-renewal ability and multi-lineage differentiation ability. Under specific conditions, they can proliferate and differentiate directionally into different functional cells. Therefore, the research on stem cells plays a very important role in the renewal and damage repair of each tissue and organ of the living body, and offers hope for the treatment of many incurable diseases, especially diseases caused by cell and tissue loss or damage.

[0004] Stem cells include embryonic stem cells and adult stem cells. Due to ethical issues, the application of embryonic stem cells is greatly limited. Adult stem cells can differentiate into functional cells and tissues, thus providing a basis for the wide application of stem cells and a new cell source for cell replacement therapy for many diseases. Therefore, adult stem cells have become the focus of research.

[0005] However, normal adult mammalian stem cells are few in number, and their differentiation is affected by various intrinsic mechanisms and microenvironmental factors. It is difficult to perform long-term large-scale culture in vitro, especially serum-free growth culture, and they cannot be applied to actual treatment.

[0006] Many diseases can be caused by the loss or damage of functional cells, and cell replacement therapy is an effective method for treating these diseases. Stem cell medicine can prevent and treat diseases caused by cell loss or damage by regulating the proliferation and differentiation of stem cells in vivo. By using stem cell medicine to regulate the proliferation and directed differentiation ability of stem cells, damaged functional cells can be regenerated and their biological functions can be restored.

[0007] Adipose mesenchymal stem cells (Ad-MSCs) are adult stem cells derived from the adipose tissue matrix, have very high self-renewal ability, and can differentiate into various types of functional cells. There is evidence proving that Ad-MSCs have great potential in stem cell-based chronic wound treatment. However, one major obstacle to the successful application of Ad-MSCs in potential cell therapy is the survival rate of cells after transplantation. When cells are transplanted into damaged skin tissue, since they experience oxygen deficiency, inflammation, oxidative stress or other adverse conditions, the survival rate of the seeded cells after transplantation inevitably decreases, interfering with the therapeutic effect of Ad-MSCs.

[0008] Rhodococcus ruber is a Gram-positive bacterium. Generally, the colonies are circular, showing yellowish orange or reddish orange, the size of the colonies is about 1 mm - 2 mm, the cell morphology is spherical or short rod-shaped, can form primary branched mycelia, and has no flagella. Rhodococcus ruber is aerobic and auxotrophic.

[0009] Currently, researchers are performing whole-genome sequencing of Rhodococcus ruber. For example, Fan Xin et al. sequenced the whole genome of Rhodococcus ruber strain SD3 and performed bioinformatics analysis. The SD3 strain has a whole-genome length of about 5.37 Mb, a GC content of about 70.63%, and a GenBank accession number of CP029146 (Fan Xin, Whole-genome sequencing of Rhodococcus ruber SD3 and analysis of the expression of its heat shock protein DnaK, Genomics and Applied Biology, January 2019).

[0010] Since the genus Rhodococcus has very strong organic matter tolerance and a wide degradation spectrum by itself, it can adapt to various living environments. Therefore, the genus Rhodococcus is widely applied in fields such as pollution restoration, decomposition of organic compounds, and sewage treatment. Currently, Rhodococcus rhodochrous is mainly applied in the field of environmental improvement. Please refer to CN108862590A, CN107151635A, CN102250796A, CN1519312A, CN103627653A, CN101033454A, CN108130288A, CN104830738A, CN101619299A, CN103509833A, CN106434466A, CN101580808A, CN102604875A, CN103160491A, CN106591168A, CN106591172A, CN105820982A.

[0011] CN109576180A discloses a bacterium RDC-01 screened from red soil in the suburbs near Panyu District, Guangzhou. Through 16S rRNA gene sequence analysis and identification of culture characteristics, the strain was identified as Rhodococcus rhodochrous. After inactivating the bacterium and adding it to an inactivated vaccine for animals as an immune adjuvant, it was shown that the production of antibodies in animals could be promoted. However, there is still no report on applying Rhodococcus rhodochrous in the field of human medicine.

[0012] Therefore, finding active ingredients that specifically regulate the proliferation and differentiation of adult stem cells is a research hotspot in stem cell medicine.

Prior Art Documents

Patent Documents

[0013]

Patent Document 1

Patent Document 2

Patent Document 3

[0014]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Summary of the Invention

[0015] This application provides an active ingredient for regulating stem cells and its applications.

[0016] In some embodiments of the present disclosure, isolated Rhodococcus ruber is provided.

[0017] In some specific embodiments of the present disclosure, Rhodococcus rhodochrous with deposit number CGMCC No. 17431, which was deposited on March 22, 2019 at the China General Microbiological Culture Collection Center (No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101), is provided. This deposit complies with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

[0018] In some embodiments of the present disclosure, Rhodococcus rhodochrous and its derivative products are provided. The derivative products are derived from Rhodococcus rhodochrous and include the components of Rhodococcus rhodochrous (for example, proteins, nucleic acids, lipids, cell walls and their components, carbohydrates, metabolites).

[0019] In a specific embodiment, an isolated cell wall of Rhodococcus rhodochrous is provided.

[0020] In a specific embodiment, an isolated cell wall of Rhodococcus rhodochrous is provided, and the Rhodococcus rhodochrous refers to the strain with deposit number CGMCC No. 17431.

[0021] In a specific embodiment, a product derived from the cell wall of Rhodococcus rhodochrous is provided.

[0022] In a specific embodiment, an isolated cell wall skeleton of Rhodococcus rhodochrous is provided.

[0023] In a specific embodiment, an isolated cell wall skeleton of Rhodococcus rhodochrous is provided, and the Rhodococcus rhodochrous refers to the strain with deposit number CGMCC No. 17431.

[0024] In some embodiments of the present disclosure, there is provided a pharmaceutical composition comprising the cell wall of Rhodococcus rhodochrous or the cell wall skeleton of Rhodococcus rhodochrous.

[0025] In some embodiments of the present disclosure, there is provided a product derived from the cell wall of Rhodococcus rhodochrous, comprising a product obtained by grinding Rhodococcus rhodochrous.

[0026] In some other embodiments of the present disclosure, there is provided a product derived from the cell wall of Rhodococcus rhodochrous, comprising a product obtained by grinding Rhodococcus rhodochrous and then purifying (removing lipids, removing nucleic acids, removing proteins).

[0027] In some other embodiments of the present disclosure, there is provided a product derived from the cell wall of Rhodococcus rhodochrous, comprising the cell wall of Rhodococcus rhodochrous.

[0028] In some other embodiments of the present disclosure, there is provided a product derived from the cell wall of Rhodococcus rhodochrous, comprising the cell wall skeleton of Rhodococcus rhodochrous.

[0029] In some embodiments of the present disclosure, there is provided a pharmaceutical composition or a medical device comprising a product derived from the cell wall of Rhodococcus rhodochrous.

[0030] In some other embodiments of the present disclosure, there is provided a pharmaceutical composition or a medical device comprising a product obtained by grinding Rhodococcus rhodochrous and then purifying (removing lipids, and / or removing nucleic acids, and / or removing proteins).

[0031] In some other embodiments of the present disclosure, there is provided a pharmaceutical composition or a medical device comprising the cell wall of Rhodococcus rhodochrous.

[0032] In some other embodiments of the present disclosure, there is provided a pharmaceutical composition or a medical device comprising the cell wall skeleton of Rhodococcus rhodochrous.

[0033] In some other embodiments of the present disclosure, there is provided a pharmaceutical composition or a medical device comprising a product derived from the cell wall of the above-mentioned Rhodococcus rhodochrous.

[0034] In a specific embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0035] In some embodiments, the product derived from the cell wall of Rhodococcus rhodochrous in the pharmaceutical composition is 1 part by weight, and the pharmaceutically acceptable excipient is 50 to 5000 parts by weight (for example, 50, 100, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 500, 600, 700, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 and any value between any two numerical values).

[0036] In some other embodiments, the cell wall of Rhodococcus rhodochrous in the pharmaceutical composition is 1 part by weight, and the pharmaceutically acceptable excipient is 50 to 5000 parts by weight (for example, 50, 100, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 500, 600, 700, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 and any value between any two numerical values).

[0037] In still some other embodiments, the cell wall skeleton of Rhodococcus rhodochrous in the pharmaceutical composition is 1 part by weight, and the pharmaceutically acceptable excipient is 50 to 5000 parts by weight (for example, 50, 100, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 500, 600, 700, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 and any value between any two numerical values).

[0038] In some embodiments, the pharmaceutical composition may be manufactured as a liquid (liquid formulation).

[0039] In some other embodiments, the pharmaceutical composition may be manufactured as a solid (dry powder formulation or lyophilized powder formulation).

[0040] One skilled in the art can understand that for the pharmaceutical compositions of the present disclosure, both the liquid formulation and the dry powder formulation (or lyophilized powder formulation) can be converted into each other and differ only in the water content. Most or all of the water in the liquid formulation is removed to obtain the dry powder formulation (or lyophilized powder formulation). After dissolving (or redissolving) the dry powder formulation (or lyophilized powder formulation), the liquid formulation is obtained.

[0041] In some embodiments, the medicine or pharmaceutical composition is manufactured in a dosage form selected from ointments, creams, emulsions, suspensions, pastes, gels, detergents, tinctures, oils, tablets, aerosols, sprays, liniments, powders, and the ointments are selected from ointments, plasters, creams.

[0042] In some embodiments, pharmaceutically acceptable excipients include, but are not limited to, fillers, stabilizers, flavoring agents, disintegrants, adhesives, and lubricants.

[0043] In some embodiments, the pharmaceutically acceptable excipients include, for example, dextran, lactose, microcrystalline cellulose, trehalose, glycine, xylitol, sodium carboxymethyl cellulose, erythritol, gelatin, magnesium stearate, propellants, humectants, solvents, solubilizers, emulsifiers, antioxidants, pH adjusters, preservatives, but are not limited thereto. Specifically, non-limiting examples are white petrolatum, carbomer, hypromellose, methylcellulose, sodium carboxymethyl cellulose, chitosan, succinoyl chitosan, polyvinylpyrrolidone, polyvinyl alcohol, sodium hyaluronate, dimethyl ether, tetrafluoroethane, hydrofluoroalkane, glycerol, propylene glycol, deionized water, water for injection, distilled water, ethanol, cetyl alcohol, stearyl alcohol, p-aminobenzoic acid, acetamide, isopropanol, Tween, polyoxyethylene hydrogenated castor oil, stearic acid, glyceryl monostearate, triglycerol monostearate, sucrose fatty acid ester, sucrose ester, sucrose acetate isobutyrate, sorbitan tristearate, isopropyl myristate, cholesterol, squalene, squalane, n-butanol, ethylene glycol, ethanol, propylene glycol, polyglycerol ester, sulfite, cysteic acid, di-tert-butylhydroxytoluene, potassium sorbate, phosphate buffer, triethanolamine, sodium hydroxide, ethylenediamine, laurylamine, sodium bicarbonate, hydrochloric acid, parabens, thimerosal, chlorocresol, chlorobutanol, benzoic acid and its sodium salt.

[0044] In some embodiments of the present disclosure, 1) a step of preparing Rhodococcus ruber; 2) a step of culturing the Rhodococcus ruber as needed; 3) a step of collecting the cultured Rhodococcus ruber as needed; 4) a step of pulverizing the cultured Rhodococcus ruber to obtain a pulverized product; 5.1) If necessary, performing a lipid removal operation on the pulverized product; 5.2) If necessary, performing a nucleic acid removal operation on the pulverized product; 5.3) If necessary, performing a protein removal operation on the pulverized product; 5.4) Obtaining a purified product; 6) If necessary, removing water in the purified product, preferably removing the water in the purified product by lyophilization; 7) If necessary, performing individual packaging; 8) Obtaining a product derived from the cell wall of Rhodococcus rhodochrous, and providing a method for producing a product derived from the cell wall of Rhodococcus rhodochrous, which includes or consists of these steps.

[0045] Steps 5.1), 5.2), and 5.3) may be performed in any order or simultaneously, and steps 6) and 7) may be performed in any order.

[0046] If necessary, step 5) may include removing the cell membrane (e.g., with a non-ionic surfactant).

[0047] The culture of Rhodococcus rhodochrous is not limited to specific media and culture parameters. Those skilled in the art can culture it in a known and appropriate manner and can use petri dishes, culture flasks, or fermentation tanks according to the production scale.

[0048] Since the purpose of pulverizing Rhodococcus rhodochrous is to remove substances inside the cells, techniques such as ultrasonic disruption and lysozyme can be used. Those skilled in the art can understand that any known or future method applicable to disrupting Gram-positive bacteria can be applied to the technical solution of the present disclosure.

[0049] Those skilled in the art can adjust the specific parameters and equipment for culture, crushing, isolation, collection, impurity removal, and individual packaging according to the subsequent administration (such as oral administration, injection, topical application, etc.) of the active ingredients (cell wall and its components), so as to ensure that the factors affecting the subsequent administration are not introduced into the manufacturing steps.

[0050] In some embodiments, lipids in the crushed product are removed with an organic solvent. In some embodiments, DNA and RNA in the crushed product are removed with nuclease. In some embodiments, proteins in the crushed product are decomposed with hydrolase. In some embodiments, cell membranes in the crushed product are removed with a surfactant.

[0051] In some embodiments, the average particle size after grinding is 10 nm to 1000 nm, and may be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 nm ± 10 nm, or within the range between any two of the above numerical values. There are numerous methods for measuring particle size (Hu Songqing et al., Modern Particle Size Measurement Technology, Modern Chemical Industry, 2002, 22:1).

[0052] In some specific embodiments, the average particle size after grinding is 10 nm to 800 nm.

[0053] In some other specific embodiments, the average particle size after grinding is 10 nm to 500 nm.

[0054] In some embodiments, the individual packaging refers to packaging individually in a container or on a solid support. The container is selected from bottles, tubes, packs, bags, plates, ampoules, injection devices, aluminum foil packages, dressings, and capsules.

[0055] For example, in a specific embodiment, the individual packaging refers to packaging individually in a bottle / ampoule. Before administration, a solvent is added to the bottle / ampoule.

[0056] In some specific embodiments of the present disclosure, there is provided a product derived from the cell wall of Rhodococcus rhodochrous, which is produced by the method in the present disclosure.

[0057] In some embodiments of the present disclosure, there is provided a pharmaceutical composition or a medical device comprising a product derived from the cell wall of Rhodococcus rhodochrous, which is produced by the method in the present disclosure.

[0058] In some embodiments of the present disclosure, there is provided an isolated cell wall of Rhodococcus rhodochrous for regulating adult stem cells. The regulation refers to one or a combination selected from promoting the proliferation of stem cells, promoting the growth of stem cells, promoting the differentiation of stem cells, promoting the migration of stem cells, and improving the survival rate of stem cells. The stem cells are selected from adult stem cells, iPSCs, and mesenchymal stem cells.

[0059] In some embodiments, the mesenchymal stem cells are selected from bone marrow mesenchymal stem cells, adipose 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.

[0060] In some embodiments of the present disclosure, there is provided a product derived from the cell wall of Rhodococcus rhodochrous for regulating adult stem cells.

[0061] In some embodiments of the present disclosure, there is provided a pharmaceutical composition or a medical device for regulating adult stem cells.

[0062] In some embodiments of the present disclosure, there is provided the use of the cell wall of Rhodococcus rhodochrous in the present disclosure in the regulation of adult stem cells.

[0063] There is further provided the use of the cell wall of Rhodococcus rhodochrous in the present disclosure in the manufacture of a pharmaceutical / medical device for regulating adult stem cells.

[0064] In some embodiments of the present disclosure, provided is the use of a product derived from the cell wall of Rhodococcus rhodochrous in the present disclosure in the regulation of adult stem cells, and further provided is the use of a product derived from the cell wall of Rhodococcus rhodochrous in the present disclosure in the manufacture of a medicament / medical device for regulating adult stem cells.

[0065] In some embodiments of the present disclosure, provided is the use of a pharmaceutical composition in the present disclosure in the regulation of adult stem cells, and further provided is the use of a pharmaceutical composition in the present disclosure in the manufacture of a medicament / medical device for regulating adult stem cells.

[0066] In some embodiments of the present disclosure, - Rhodococcus rhodochrous in the present disclosure, - the isolated cell wall of Rhodococcus rhodochrous in the present disclosure, - a product derived from the cell wall of Rhodococcus rhodochrous in the present disclosure, - a pharmaceutical composition in the present disclosure, any one selected therefrom is provided for use in the manufacture of a medicament (or medical device).

[0067] In some specific embodiments, the medicament is used for regulating adult stem cells.

[0068] In some specific embodiments, the medical device (for example, dressing material, patch, bandage, film, patch, etc.) is used for regulating adult stem cells.

[0069] In some embodiments of the present disclosure, a subject is - the isolated cell wall of Rhodococcus rhodochrous in the present disclosure, - a product derived from the cell wall of Rhodococcus rhodochrous in the present disclosure, - a pharmaceutical composition in the present disclosure, - a therapeutically effective amount (or prophylactically effective amount) selected from a medical device in the present disclosure, contacting with any one of them, further provides a method for regulating adult stem cells.

[0070] In some specific embodiments, provided is a method for regulating stem cells, comprising the step of contacting the stem cells with a product derived from the cell wall of Rhodococcus rhodochrous.

[0071] In some specific embodiments, the ratio of the number of stem cells to the product derived from the cell wall of Rhodococcus rhodochrous is 1 to 1000 stem cells / 1 ng of the product derived from the cell wall of Rhodococcus rhodochrous, preferably 5 to 50 stem cells / 1 ng of the product derived from the cell wall of Rhodococcus rhodochrous. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 stem cells / 1 ng of the product derived from the cell wall of Rhodococcus rhodochrous, and any range therebetween, but not limited thereto.

[0072] In some embodiments, provided is a method for promoting wound healing, comprising the step of contacting a wound of a subject with a therapeutically effective amount of mesenchymal stem cells and a product derived from the cell wall of Rhodococcus rhodochrous.

[0073] In some embodiments, the ratio of the number of mesenchymal stem cells to the product derived from the cell wall of Rhodococcus rhodochrous is 1 to 100 stem cells / 1 ng of the product derived from the cell wall of Rhodococcus rhodochrous, preferably 5 to 50 stem cells / 1 ng of the product derived from the cell wall of Rhodococcus rhodochrous. In some specific embodiments, the wound is a wound associated with diabetes.

[0074] In some specific embodiments, depending on the differences in the area and depth of the lesion, a medicament (or medical device) is administered to the lesion. For example, - applying a medicament containing the cell wall skeleton of Rhodococcus rhodochrous, or - covering the lesion with a patch (film or gauze) impregnated with the cell wall skeleton of Rhodococcus rhodochrous, or - Administer the lyophilized powder containing the cell wall skeleton of Rhodococcus rhodochrous directly to the lesion, or - Administer, but not limited to, a paste / cream containing the cell wall skeleton of Rhodococcus rhodochrous to the lesion.

[0075] In some specific embodiments, the contact cycle is 2 days to 2 months or more. Specifically, for example, it is 2, 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 days, and also for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks. In a specific embodiment, the active ingredient is administered to the subject over 3 to 4 weeks.

[0076] In some embodiments, it is administered at a frequency of 1 to 3 times a day, 1 to 6 times every 2 days, 1 to 9 times every 3 days, 1 to 14 times a week, 1 to 60 times a month. In some embodiments, it is administered 2 times a day, or 1 time a day, or 1 time every 2 days.

[0077] The dosage per administration varies depending on the specific situation of the subject, and generally it is administered at 1 μg to 1000 μg / unit dosage / once. Specifically, for example, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 μg / unit dosage / once and within the range between any two of the aforementioned numerical values.

[0078] In some specific embodiments, the contact is achieved, but not limited to, by means such as oral administration, mucosal administration, transdermal administration, intraperitoneal administration, puncture, nasal drop, eye drop, suppository, sublingual administration.

[0079] In some specific embodiments, the subject is an animal other than a human, such as a farm animal, a pet, a draft animal, an ornamental animal, or an industrial animal.

[0080] In a specific embodiment, the subject is a human.

[0081] In some specific embodiments, the subject is a subject suspected of having, diagnosed as having, already having, or being susceptible to having a target disease or its symptoms.

[0082] In some embodiments, - Rhodococcus rhodochrous in the present disclosure, - the cell wall of the isolated Rhodococcus rhodochrous in the present disclosure, - a product derived from the cell wall of Rhodococcus rhodochrous in the present disclosure, - a pharmaceutical composition in the present disclosure, to provide a cell culture medium comprising one or a combination thereof selected from the group consisting of.

[0083] In some specific embodiments, a cell culture medium is provided that further comprises other components known in the art applicable to culturing stem cells (particularly mesenchymal stem cells). When applied to humans, in order to provide safer cells, those that do not contain xenogeneic animal components during the culturing process, such as serum-free culture media, are recommended.

[0084] In some specific embodiments, those skilled in the art can add one or a combination of cytokines, such as FGF, PDGF, TGF-β, HGF, EGF, CTGF, VEGF, insulin, insulin-like growth factor, to the cell culture medium as needed (e.g., to maintain dryness or promote differentiation).

[0085] In some specific embodiments, the content of FGF (calculated at the final concentration) is preferably 0.1 to 100 ng / ml. FGF refers to a growth factor belonging to the fibroblast growth factor family, preferably FGF-1 or FGF-2 (bFGF).

[0086] In some specific embodiments, the content of PDGF (calculated at the final concentration) is preferably 0.5 to 100 ng / ml. PDGF refers to a growth factor belonging to the platelet-derived growth factor family, preferably PDGF-BB or PDGF-AB.

[0087] In some specific embodiments, the content of TGF-β (calculated at the final concentration) is preferably 0.5 to 100 ng / ml. TGF-β refers to a growth factor belonging to the transforming growth factor-β family, preferably TGF-β3.

[0088] In some specific embodiments, the content of HGF (calculated at the final concentration) is preferably 0.1 to 50 ng / ml.

[0089] In some specific embodiments, the content of EGF (calculated at the final concentration) is preferably 0.5 to 200 ng / ml.

[0090] In some specific embodiments, a cell culture medium further containing at least one phospholipid and / or at least one fatty acid is provided.

[0091] Examples of phospholipids include phosphatidic acid, lysophosphatidic acid, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol. The total content of phospholipids (calculated at the final concentration) is preferably 0.1 to 30 μg / ml.

[0092] Examples of fatty acids include linoleic acid, oleic acid, linolenic acid, arachidonic acid, myristic acid, palmitoleic acid, palmitic acid, and stearic acid. The total content of fatty acids is preferably 1 / 1000 to 1 / 10 of the medium.

[0093] In some specific embodiments, a cell culture medium further containing cholesterol is provided.

[0094] In some specific embodiments, a cell culture medium further containing ascorbic acid is provided.

[0095] In some specific embodiments, a cell culture medium further containing an antioxidant is provided. Examples of the antioxidant include DL-α-tocopherol acetate (vitamin E).

[0096] In some specific embodiments, a cell culture medium further containing transferrin is provided.

[0097] In some specific embodiments, a cell culture medium further containing selenate is provided.

[0098] In some specific embodiments, a cell culture medium further containing amino acids, nucleotides, and trace elements necessary for maintaining cells is provided.

[0099] In a specific example, the composition of the present application is added to a known commercially available mesenchymal stem cell culture medium. Examples of the commercially available mesenchymal stem cell culture medium include MesenPRO RS TM , StemPro® MSC SFM, StemPro® MSC SFM XenoFee, and StemPro® Human Adipose-Derived Stem Cell Medium.

[0100] In some embodiments, - mesenchymal stem cells, - Provide a therapeutic composition comprising a product derived from the cell wall of Rhodococcus rhodochrous.

[0101] In some embodiments of the therapeutic composition, the ratio of the number of mesenchymal stem cells to the product derived from the cell wall of Rhodococcus rhodochrous is 1 to 100 stem cells / 1 ng of the product derived from the cell wall of Rhodococcus rhodochrous, preferably 5 to 50 stem cells / 1 ng of the product derived from the cell wall of Rhodococcus rhodochrous.

[0102] In some embodiments of the therapeutic composition, the mesenchymal stem cells are selected from bone marrow mesenchymal stem cells, adipose 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, dental pulp mesenchymal stem cells.

[0103] In some embodiments, provide a method for improving apoptosis of stem cells, comprising the step of contacting the stem cells with an effective amount of the above-mentioned product derived from the cell wall of Rhodococcus rhodochrous.

[0104] In some embodiments, provide a method for improving the survival rate of stem cells, comprising the step of contacting the stem cells with an effective amount of the above-mentioned product derived from the cell wall of Rhodococcus rhodochrous.

[0105] In the context of the present application, the only therapeutic (or preventive) active ingredient in a pharmaceutical or medical device is a product derived from the cell wall of Rhodococcus rhodochrous, in particular a product containing components of Rhodococcus rhodochrous (e.g., proteins, nucleic acids, lipids, cell walls and their components, carbohydrates, metabolites), specifically a product containing the cell wall of Rhodococcus rhodochrous (more preferably the cell wall skeleton of Rhodococcus rhodochrous or its composition).

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0107] "Isolation" refers to detaching the Rhodococcus rhodochrous of the present disclosure from its original growth environment.

[0108] It is known to those skilled in the art that the cell wall structures of Gram-positive bacteria and Gram-negative bacteria are different. Specifically, Gram-positive bacteria have a thicker cell wall (generally 20 nm to 80 nm), containing about 90% peptidoglycan and about 10% teichoic acid (a polymer formed by alcohol molecules and phosphate molecules, generally present in the form of sugar esters or amino acid esters). The peptidoglycan layer is dense and can reach 20 layers in many cases. However, the cell wall of Gram-negative bacteria is much thinner than that of Gram-positive bacteria, with a complex structure, and is divided into an outer membrane and a peptidoglycan layer (generally 2 nm to 3 nm).

[0109] The peptidoglycan layer is a unique component in the bacterial cell wall and is a derivative of heteropolysaccharide. Each monomer of peptidoglycan includes three parts: a sugar unit (for example, at least two sugar molecules are connected by glycosidic bonds to form the backbone structure of peptidoglycan), a peptide terminus (a short peptide chain composed of multiple amino acids connected and connected to the N-acetylmuramic acid molecule), and a peptide bridge (which crosslinks adjacent "peptide termini" to form a high-strength network structure). Different bacteria have different peptide bridges, peptide termini, and crosslinking methods.

[0110] Cell wall of isolated Rhodococcus rhodochrous In the present disclosure, the "isolated cell wall of Rhodococcus ruber" may be understood as a complete cell wall or an incomplete cell wall (e.g., disrupted or partially degraded). Based on the teachings of the present disclosure, those skilled in the art can understand that the component showing the desired activity is derived from the cell wall of Rhodococcus ruber (e.g., the cell wall itself or its composition). Therefore, various forms such as a complete cell wall, a disrupted cell wall, an incomplete degradation product of the cell wall, a cell wall component, and a cell wall extract can be used in clinical applications, and all of these are included within the scope of the present disclosure.

[0111] Cell wall skeleton It is a composition component that constitutes the main structure of the cell wall, but it should not be understood as only representing the substantial cross-linked network in the cell wall. Those skilled in the art can understand that other cell wall components adsorbed, bound, and retained by the substantial cross-linked network are not excluded.

[0112] Rhodococcus ruber In the embodiments of the present disclosure, Rhodococcus ruber used refers to the Rhodococcus ruber species of the genus Rhodococcus and is not limited to a specific cell strain.

[0113] Non-limiting examples include TOY7 strain (Nanjing Agricultural University Agricultural Environmental Microbial Strain Preservation Center), CGMCC No. 4795, DSM43338, CCTCC No. 2012035, CGMCC No. 16640, CGMCC No. 17431.

[0114] Identification of Rhodococcus ruber With known or unknown microbial identification techniques, a person skilled in the art can make a taxonomic identification of a bacterial strain. For example, available identification techniques include morphological characteristics, physiological and biochemical characteristics, 16S rRNA, etc. A person skilled in the art can understand that with the development of science and technology, identification techniques involve various means, and in the early stages, morphological identification and biochemical identification methods were mainly used, but these methods are less reliable. After the advent of sequencing technology, a person skilled in the art can identify strains in a more reliable manner. For example, when the DNA sequence of 16S rRNA is identified to have a homology of 97% or more, it is determined that two bacteria belong to the same species (Hua Xu et al., Progress in the classification and application research of the genus Rhodococcus, Microbiology Bulletin, 2003:30(4)). For Rhodococcus ruber, a known strain deposited at the International (or National) Culture Collection Center is used as a model strain for comparison.

[0115] Dosage form The medicaments or pharmaceutical compositions or active ingredients or products of the present disclosure may be in the form of, but are not limited to, an ointment, cream, plaster, gel, cleanser, tincture, liniment, oil, paste, lyophilized powder, aerosol, suppository, patch, suspension, oral liquid, lozenge, skin care product (facial cleanser, lotion, serum, emulsion, face cream, face mask).

[0116] Formulation Unit The medicament or pharmaceutical composition or active ingredient or product of the disclosure may be manufactured in the form of a unit dosage form.

[0117] In some embodiments, the unit dosage of the medicament (or formulation, or therapeutic agent, or medical device) is -1μg to 1000μg of the above-mentioned cell wall-derived product of Rhodococcus ruber, or -1μg to 1000μg of the cell wall of the Rhodococcus ruber, or -Containing 1μg to 1000μg of the cell wall skeleton of Rhodococcus ruber.

[0118] Specific examples of the unit dosage 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 ranges between any two of the above numerical values.

[0119] When the terms "administer", "give", "provide", "treat" are used with respect to an animal, a human, a cell, a tissue, an organ or a biological sample, they refer to the contact of a pharmaceutical or a medical device with the animal, the human, the cell, the tissue, the organ or the biological sample.

[0120] "Treatment" means administering to a subject to be tested a pharmaceutical (a therapeutic agent, an active ingredient or a composition) (for example, the cell wall of Rhodococcus rhodochrous or its pharmaceutical composition in the present disclosure) or a medical device, either orally or topically, so as to relieve (alleviate, delay, improve, cure) one or more disease symptoms of the subject to be tested (or a population) to a clinically measurable extent, and the above subject to be tested is a subject who already has, is suspected of having, or is likely to suffer from one or more diseases or their symptoms.

[0121] The amount of a pharmaceutical (a therapeutic agent, an active ingredient or a composition) that effectively relieves the symptoms of any disease is called a therapeutically effective amount. It can vary depending on various factors, such as the disease state, age and weight of the subject to be tested. When relieving the target disease or its symptoms of a single subject to be tested, the pharmaceutical (a therapeutic agent, an active ingredient or a composition) may not be effective, but it should be understood that it can be determined to be statistically effective against the target disease or its symptoms by any known statistical test method in this field (for example, Student's T test, chi-square test, Mann-Whitney U test).

[0122] "As needed" means that the matters described in the following text may occur, but not necessarily, and it is necessary to set according to the situation. For example, "individually package as needed" means that it is allowed to individually package the product, but it is not necessarily necessary to individually package, and whether to individually package or not does not affect the achievement of the technical effect.

[0123] "One", "a", "single", "the" include plural forms without clear explanation.

[0124] The following examples, production examples and test examples are combined to further illustrate the present disclosure. However, these examples, production examples and test examples do not limit the scope of the present disclosure. When specific conditions are not specified, operate according to general conditions and the conditions proposed by raw material suppliers. Reagents without specific supply sources specified are general reagents purchased on the market.

[0125] Examples Example 1. Origin of the Strain Rhodococcus ruber used in the following examples is CGMCC No. 17431, which was deposited on March 22, 2019 at China General Microbiological Culture Collection Center (CGMCC), No. 3, Yard 1, West Beichen Road, Chaoyang District, Beijing, China (Institute of Microbiology Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing China).

[0126] As will be particularly understood by those skilled in the art, the following specific examples use specific cell lines, but the realization of the technical effect is not limited to the specific cell lines, and any species belonging to the Rhodococcus ruber species can be applied.

[0127] Example 2. Identification of the Strain 1. Visual observation of colony morphological characteristics When cultured on glycerol agar medium at 30-37°C (specifically 32-35°C) for 12-72 (specifically 36-60, e.g. 40-50) hours, the following was found:

[0128] -The colony rises, - Reddish orange color (there may be slight differences due to the influence of light, medium color, etc.) - The surface is dry and cracked, with a slight sheen (there may be slight differences depending on the culture conditions), - It crumbles easily when touched lightly, -Colony size is approximately 1mm to 2mm (there may be slight differences depending on the culture conditions).

[0129] 2. Microscopic Observation - The fungus is branched, has a diaphragm, and forms a mycelium (there may be slight differences depending on the culture conditions). -The mycelium divides to form short, thick, regular cells (there may be slight differences depending on the culture conditions). -After 4 to 5 days of cultivation, the cells became short rod-shaped or spherical (there may be slight differences depending on the cultivation conditions).

[0130] 3.Stainability Gram staining was positive.

[0131] 4. Biochemical reactions A glycerol agar slant medium was prepared and cultured at 30 to 37° C. (specifically, 32 to 35° C.) for 12 to 72 hours (specifically, 36 to 60 hours, for example, 40 to 50 hours). Next, the culture was subjected to the following tests.

[0132] 4.1 Acid production from carbohydrates Table 1. Acid production test [Table 1]

[0133] 4.2 Measurement of Enzyme Activity (API ZYM) Table 2. Measurement of Enzyme Activity

Table 2

[0134] 4.3 Positive for nitrate reduction reaction, positive for catalase, positive for tyrosinase, negative for amylase, negative for oxidase, and negative for gelatin liquefaction were exhibited.

[0135] 4.4 Sole Carbon Source Table 3. Carbon Source

Table 3

[0136] 4.5. 16S rRNA Identification Genomic extraction, 16S rRNA amplification, and sequencing were performed on 15 isolated strains in the working seed tube and 10 different strains isolated in the original seed tube. The 16S rRNA gene homology of a total of 25 strains was 100%.

[0137] Moreover, according to the neighbor-joining strain phylogenetic tree constructed by the Kimura 2-parameter algorithm, the result showed that the strain belongs to Rhodococcus ruber.

[0138] Production Example Production Example 1. Culture Method 1. Rhodococcus ruber can be cultured by a general microbial production method.

[0139] 2. The culture method may be solid culture or liquid culture.

[0140] 3. There is no particular regulation for the nutrient sources in the medium, and carbon sources, nitrogen sources, and other nutrient sources generally used for microbial culture may be included in the medium.

[0141] - The carbon source is any carbon source that Rhodococcus ruber can utilize, such as fructose, glucose, etc.

[0142] - The nitrogen source may be broth, peptone, ammonium salts, nitrates and other organic or inorganic nitrogen-containing compounds.

[0143] - As other nutrient sources, some inorganic salts, such as NaCl, phosphates, can be appropriately added.

[0144] 4. There is no strict limitation on the culture conditions (temperature, time, etc.). Those skilled in the art can select the conditions that maximize the production volume based on the preliminary small-scale pilot test data by themselves.

[0145] 5. As an example, Rhodococcus ruber was fermented under the following culture conditions.

[0146] (1) The medium composition is containing peptone, beef broth, sodium chloride, phosphate, glycerol (and agar if necessary for solid culture).

[0147] (2) Culture method parameters: After regenerating the working bacterial strain, transfer it to a solid culture medium and maintain it for 3 to 5 days, then transfer it to a liquid for culture (maintain at 30 - 37 °C for 3 to 5 days). The auxiliary material semi-continuous batch feeding mode or the batch feeding mode can be used. During the culture period, pH, bacterial density, dissolved oxygen, and consumption of the carbon source were monitored.

[0148] Production Example 2. Disruption of Bacterial Cells Collect the bacteria obtained in Production Example 1 and disrupt the cells (for example, by ultrasonic disruption, but not limited thereto). Any appropriate known method in this field, such as the methods in CN101250490A or CN101323865A, can be used to disrupt the bacterial cells.

[0149] Check the fragmentation status under a microscope. The number of bacteria maintaining their shape per field of view should not exceed 5. If multiple (10 - 30) fields of view are checked and all meet this criterion, it is considered qualified.

[0150] Production Example 3. Nucleic acid removal, lipid removal, impurity protein removal, cell membrane removal 1. Nucleic acid removal: The supernatant after fragmentation was centrifuged, and DNA degrading enzyme and RNA degrading enzyme were added to the obtained precipitate, and nucleic acids were removed according to the operations proposed by the enzyme supplier.

[0151] 2. Protein removal: A general protease (for example, trypsin) was added to the precipitate, and proteins were removed according to the operations proposed by the enzyme supplier.

[0152] 3. Lipid removal: An organic reagent (for example, one or a combination of acetone, ether, ethanol, but not limited thereto) was added to the precipitate, and lipids were removed according to general operations in this field.

[0153] 4. Cell membrane removal: Triton X - 100 was added to the precipitate, and according to general operations in this field, it was centrifuged to collect the precipitate, which was then washed with PBS.

[0154] During the above steps of removing impurities, those skilled in the art should understand that the order can be adjusted to make the steps compatible with each other.

[0155] After removing components other than the cell wall, the precipitate was redissolved in water for injection and prepared for use. If necessary, it can be sterilized at 115°C for 20 - 30 minutes to obtain a stock solution of cell wall skeleton (mainly containing cell wall skeleton and its composition components).

[0156] Production Example 4. Method for manufacturing a pharmaceutical composition 1. Liquid composition An excipient (e.g., dextran 40, mannitol, or trehalose) was added to the product obtained in Production Example 3. After filling the container, a pharmaceutical composition was obtained.

[0157] Table 4. Multiple manufacturable forms of the pharmaceutical composition

Table 4

[0158] 2. Lyophilized powder composition The pharmaceutical composition of claim 1 was lyophilized to obtain lyophilized powders (each numbered lyophilized powder compositions 1 to 7).

[0159] 3. Quality inspection (taking lyophilized powder composition 1 as an example) Table 5. Quality inspection items

Table 5

[0160] Test example Materials and methods 1. Isolation, culture, and subculture of human Ad-MSCs Adipose tissue samples were obtained from the liposuction aspirates of the subjects (age range 25 - 35 years), and Ad-MSCs were isolated and cultured. The subjects were patients who underwent plastic surgery at the Affiliated Hospital of Xuzhou Medical University, approved by the ethics committee, and patient informed consent was obtained.

[0161] The obtained fresh adipose tissue extract was digested with 0.25% pancreatin - EDTA, filtered, centrifuged to retain the cell pellet layer, and added to DMEM (Invitrogen) medium containing 10% FBS fetal bovine serum (Gibco) and 1% penicillin / streptomycin. The cell culture dish was placed in an incubator at 37°C with 5% CO2 for culture. Thereafter, until the cells grew to 80% and were subcultured, they were washed with PBS every 2 - 3 days and then the medium was changed.

[0162] 2. Measurement of cell viability Ad-MSCs in the logarithmic growth phase were seeded into a 96-well plate at 4×10 3 / well and co-cultured with DMEM + 10% fetal bovine serum + 1% penicillin / streptomycin. The composition of the present application (Composition 1) was dissolved in PBS solution and diluted to 10 μg / ml. After the Ad-MSCs adhered, each measurement object was divided into four experimental groups: control, 25 μl, 50 μl, and 75 μl. 10 μL of CCK-8 reagent was added to each well at 24 h, 48 h, 72 h, and 96 h respectively, followed by incubation for 2 h. Then, the absorbance of each well was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay detector. A cell growth curve was drawn with the culture time on the x-axis and the number of cells (absorbance) on the y-axis.

[0163] 3. EdU Incorporation Experiment EdU incorporation analysis was performed using the Cell-Light EdU 567 In Vitro Imaging Kit (RiboBio). Ad-MSCs in the logarithmic growth phase were seeded into a 96-well plate at 4×10 3 / well. The composition of the present application at 10 μg / ml and 50 μl was allowed to act on the Ad-MSCs. 100 μl of EDU medium was added to each well at 72 h and 96 h, followed by incubation for 2 h and washing 1 - 2 times with PBS. 4% paraformaldehyde fixative was added to each well and incubated at room temperature for 30 min. Next, 2 mg / ml glycine solution was added and shaken for 5 min. After washing with PBS, a permeabilizing agent was added and shaken for 10 min, followed by washing with PBS. Apollo staining reaction solution was added and incubated in the dark at room temperature for 30 min. The staining reaction solution was removed, and a permeabilizing agent (0.5% TritonX-100 in PBS) was added and shaken 2 - 3 times, 10 min each time, and then the permeabilizing agent was removed. After washing again with PBS, Hoechest 33342 reaction solution was added and incubated in the dark at room temperature for 30 min, and then the reaction solution was removed. After washing 1 - 3 times with PBS, positive cells were observed under a fluorescence microscope.

[0164] 4. Cell Apoptosis Detection 4.1 Flow Cytometry: When the Ad-MSCs grew to 80% confluence, 1×10 4Inoculated into a 6-well plate at the density of wells. The experiment was divided into 4 groups. After the cells adhered, 100 μl of 50% sucrose was added to each experimental group to induce cell apoptosis of Ad-MSC. Then, 100 μl and 250 μl of the composition of the present application at 10 μg / ml were added to two of the experimental groups respectively. After 48 h and 72 h, the cell supernatants of each group were collected into flow cytometry tubes. The adherent cells were digested with pancreatin without EDTA and collected into the flow cytometry tubes of the same group, centrifuged at 2000 rpm for 5 min, washed twice with PBS buffer, shaken to mix uniformly, 500 μl of binding buffer (Annexin V-FITC Cell Apoptosis Detection Kit, Shanghai Biyuntian Biotechnology Co., Ltd.), 5 μl of FITC, and 5 μl of PI were sequentially added to each tube, shaken to make it uniform, incubated in the dark at 4 °C for 5 - 15 min, detected with a flow cytometer (BD Biosciences), and the results were analyzed.

[0165] 4.2 TUNEL method: Several 18 mm × 18 mm cover glasses were immersed in 75% ethanol for disinfection. When in use, the cover glasses were placed in a 6-well plate and washed repeatedly several times with PBS buffer until the residual ethanol solution was completely removed. Ad-MSC was cultured in a 6-well plate, and the cell density of each well was 1×10 4There were [number] of them. Grouping of the experiments was carried out in the same way as in flow cytometry. The cells were washed three times with PBS buffer, fixed with 4% paraformaldehyde at room temperature for 30 min, washed three times with PBS buffer, incubated with 0.1% Triton X-100 at 2 °C to 8 °C for 10 min, washed three 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 make reagent A, 50 μl of reagent A was added to the negative control group, incubated in an incubator at 37 °C in the dark for 60 min, DNase I was added to the positive control group, incubated at room temperature for 10 min, the cells were washed three times with PBS buffer, the TUNEL reaction mixture was added at 50 μl / well, incubated in the incubator at 37 °C in the dark for 60 min, the cells were washed three times with PBS buffer, 50 μl / well of DAPI staining solution was added, incubated at room temperature for 3 min, the samples were placed under a fluorescence microscope for photography and analysis, and the detection light wavelength range was set to 570 - 620 nm (maximum wavelength 580 nm).

[0166] 5. Western blot analysis The treated cells were taken out, 300 μl of cell lysis mixture (PIPA cell lysis buffer was dissolved on ice and PMSF was added at a ratio of 100:1) was added to each petri dish, then placed on ice and dissolved sufficiently, the adherent cells were scraped off, centrifuged at 12,000 rpm for 20 min at 4 °C using a Centrifuge-5810R freezing high-speed centrifuge, and the supernatant was collected.

[0167] The cell extracts were separated by SDS-polyacrylamide gel, and then the proteins were transferred to nitrocellulose film, and incubated with rabbit anti-human caspase-3 monoclonal antibody (1:400, CST, USA), rabbit anti-human Bax monoclonal antibody (1:400, CST, USA) as primary antibodies. After incubation, it was washed 3 times for 5 min each. Then, diluted secondary antibody (1:10000) was added and incubated at room temperature in the dark for 2 h. The secondary antibody was removed and washed 3 times for 5 min each. Equal amounts of solution A and solution B of the ECL luminescent solution were uniformly mixed to prepare the ECL working solution. The ECL working solution was uniformly dropped onto the film, placed in a TANON gel imaging device for exposure and development, photographed, and analyzed with ImageJ software.

[0168] 6. Establishment of an animal model with diabetic wounds The experimental animals were 4-week-old BALB / c athymic nude mice at the SPF level. All animal studies were approved by the Animal Protection and Use Committee. The experimental mice were housed in an animal breeding room at the SPF level. Before model establishment, the mice were fasted for 12 h, weighed and recorded. Then, 2% STZ (Sigma) was intraperitoneally injected at a dose of 150 mg / kg to induce diabetic mice.

[0169] On the 7th day after injection, blood glucose was measured by collecting blood from the tail vein, and the blood glucose concentration of the mice was measured and recorded with a glucometer. From the 7th day, if the blood glucose concentration of the mice was greater than 16.7 mmol / L and typical symptoms of diabetes such as "polydipsia, polyphagia, polyuria, and weight loss" appeared, it was considered that the model establishment was successful.

[0170] The experimental animals were - a blank control group, - an Ad-MSC group, - a composition of the present application + Ad-MSC group, divided into 3 groups (5 mice in each group).

[0171] After successfully establishing a model of diabetic mice, the mice were anesthetized, a wound with a diameter of 1.5 cm was created, and the pre-treated Ad-MSCs were injected into the skin of the mice in each group by intradermal injection at multiple sites. Six sites were selected for each wound, and 0.1 ml of cells was injected into each site. The surface was covered with sterile gauze and continued to be bred and observed. The LB983 in vivo imaging system was used to observe the survival status of wound cells and the healing status of mouse skin.

[0172] 7. Staining with CM-Dil live cell stain Before injection, Ad-MSCs were labeled with CM-Dil. According to the method proposed by the supplier, CM-Dil live cell stain was added, incubated for 30 minutes, centrifuged, the supernatant was removed, washed 3 times with PBS buffer, and finally an appropriate amount of PBS was added, mixed uniformly, and the labeled Ad-MSCs were placed in an ice box for use.

[0173] 8. Immunofluorescence staining of tissues The tissue paraffin sections were deparaffinized, incubated with 3% H2O2 at room temperature for 5 - 10 minutes to remove the activity of endogenous peroxidase. The sections were washed with distilled water, immersed in PBS twice at 5 minutes per time, sealed with 10% normal goat serum (diluted with PBS) at room temperature for 10 minutes, the sealing solution was removed without washing. The working solution of CD31 primary antibody (dilution ratio 1:300) was dropped and left overnight. The sections were washed 3 times with PBS at 5 minutes per time. An appropriate amount of the working solution of the fluorescent secondary antibody labeled with biotin (dilution ratio 1:400) (in the dark) was dropped, incubated at 37°C in the dark for 1 hour, then washed 3 times with PBS at 5 minutes per time, an appropriate amount of DAPI staining solution was dropped, incubated at room temperature in the dark for 3 minutes, and washed 3 times with PBS at 5 minutes per time. Finally, an anti-fading mounting solution was dropped on each section, covered with a cover glass, fixed, and stored in the dark.

[0174] 9. Statistical analysis Statistical analysis was performed using SPSS software (SPSS 16.0), and the experimental results were shown as mean ± SD. For comparison between two groups, an independent samples t-test was used, and for comparison of the means of multiple groups, one-way analysis of variance (One-way ANOVA) was used. α = 0.05 was set as the significance level, and P < 0.05 was considered to indicate a statistically significant difference.

[0175] Test Example 1. Identification of the morphology, surface markers, and differentiation induction ability of human Ad-MSCs Using the collagenase digestion method, the primary Ad-MSCs extracted from the adipose tissue extract were inoculated into cell culture dishes. When the cells grew to 80% confluence, they were passaged. After passage, the growth rate of the cells became significantly faster, the cell morphology was uniform and consistent, presenting a spindle shape. The well-grown Ad-MSCs of the P3 generation were used for adipogenic induction differentiation and osteogenic induction differentiation respectively. After 2 weeks, they were stained with Oil Red and observed under a microscope, showing that there were red lipid droplets with obviously inconsistent sizes in the cells. When stained with Alizarin Red S staining solution, obvious red calcium nodule deposits could be seen. This indicated that the isolated and extracted cells had the characteristics of stem cell differentiation ability.

[0176] Six different cell surface markers were detected using a flow cytometer. The results showed that CD105, CD90, and CD44 were positive, and CD31, CD34, and CD106 were negative, which was consistent with the characteristics of the Ad-MSC immunophenotype.

[0177] Test Example 2. Effects of the composition of the present application on the activity and proliferation of Ad-MSCs To detect the effect of the composition of the present application on the activity of Ad-MSCs, at 24 h, 48 h, 72 h, and 96 h after the composition 1 of the present application acted on Ad-MSCs respectively, stem cells were detected using the CCK-8 reagent, and the absorbance value was measured using an enzyme-linked immunosorbent assay device under a wavelength of 450 nm. The results showed that when the composition of the present application with a concentration of 10 μg / ml and a volume of 50 μl acted on stem cells, the viability of Ad-MSCs was significantly enhanced at 72 h and 96 h (Figure 1).

[0178] To verify the effect of the composition of the present application on the proliferation of Ad-MSCs, 10 μg / ml of 50 μl of the composition of the present application was allowed to act on the stem cells. After 72 h and 96 h respectively, EdU incorporation analysis was performed using an EDU kit, and positive cells were observed under a fluorescence microscope. The results showed that the composition of the present application could improve the proliferation rate of Ad-MSCs (Figure 2).

[0179] Test Example 3. Inhibition of the apoptosis of Ad-MSC cells induced by high glucose by the composition of the present application High glucose was added to Ad-MSC cells to induce cell apoptosis, and they were treated with the composition 1 of the present application at different concentrations. After 48 h and 72 h, the cell apoptosis situation was detected using a FITC-PI flow cytometry apoptosis detection kit.

[0180] The results showed that the composition of the present application could inhibit the apoptosis of stem cells induced by high glucose (Figure 3A, Figure 3B).

[0181] Test Example 4. Detection of the protein expression levels of apoptosis markers High glucose was added to the cells to induce cell apoptosis, and they were treated with the composition of the present application at different concentrations for 48 h and 72 h respectively. Cell proteins were extracted and Western blot experiments were performed to detect the protein expression levels of c-caspase-3 and Bax (two main apoptosis markers), with β-actin as an internal standard. The results showed that the protein expression levels of c-caspase-3 and Bax decreased after treatment with the composition of the present application (Figure 4A, Figure 4B).

[0182] Test Example 5. Improvement of the survival rate of Ad-MSCs and acceleration of wound healing in nude mice by the composition of the present application To further study the effect of the composition of the present application on the repair and healing of diabetic wounds by Ad-MSCs, a wound model simulating the wound repair mechanism of human skin was established on the body of diabetic nude mice.

[0183] After successfully establishing a diabetic mouse model, the mice were anesthetized, a wound with a diameter of 1.5 cm was created, and pre-treated cells labeled with the fluorescent dye CM-Dil were injected intradermally into the wounded skin of the mice in the treatment group. The LB983 in vivo imaging system was used to detect the cell viability.

[0184] The results showed that the Ad-MSCs in the treatment group treated with the composition of the present application had a higher viability than the Ad-MSC group using only Ad-MSCs (Figure 5A). In addition, the 14-day healing process of treating the wounds of diabetic mice with Ad-MSCs was evaluated. Compared with the blank group, the healing status of the wounds of the mice treated with Ad-MSCs was good, the healing status of the treatment group treated with the composition of the present application was even better, the wound healing was accelerated (Figure 5B), and the wound healing rate was improved (Table 6).

[0185] Table 6. Wound healing status on the 14th day after surgery (mean ± SD, n = 3)

Table 6

[0186] There are many reasons why diabetic wounds are difficult to heal. For example, the cells and molecular signals necessary for the normal wound healing process are lacking. In addition, peripheral neuropathy, peripheral circulatory damage, and protease imbalance are all factors contributing to the difficulty of diabetic wound healing (Rathur HM et al., The diabetic foot. Clin Dermatol. 2007, 25(1):109-120). Furthermore, under hyperglycemic conditions, the vascular microenvironment is abnormal, which can cause abnormal cell growth environments and ultimately interfere with local vascular remodeling of the wound (Guo WY et al., Acceleration of diabetic wound healing by low-dose radiation is associated with peripheral mobilization of bone marrow stem cells. Radiat Res. 2010, 174(4):467-479). Also, in the hyperglycemic state of diabetes, the decrease in the number of fibroblasts, the increase in glycated proteins, the expression of abnormal growth factors, the delay in the inflammatory process, and the accumulation of advanced glycation end products in the wound tissue all affect the migration and function of bone marrow-derived cells (Fiorina P et al., The mobilization and effect of endogenous bone marrow progenitor cells in diabetic wound healing. Cell Transplant. 2010, 19(11):1369-1381).

[0187] Ad-MSC is a stem cell with multi-lineage differentiation potential extracted from adipose tissue (Kato Y et al., Creation and transplantation of an adipose-derived stem cell (ASC) sheet in a diabetic wound-healing model. Jove-J Vis Exp. 2017, 12(6):1-10). Ad-MSC migrates to the damaged site due to its differentiation potential, repairs the damaged skin with differentiated cells, secretes various growth factors (Rehman J et al., Secretion of angiogenic and anti-apoptotic factors by human adipose stromal cells. Circulation. 2004, 109:1292-8), accelerates the generation of wound blood vessels, and can promote the healing of wounds (Ebrahimian TG et al., Cell therapy based on adipose tissue-derived stromal cells promotes physiological and pathological wound healing. Arterioscler Thromb Vasc Biol. 2009, 29(4):503-510).

[0188] In addition, there are researchers who have discovered that Ad-MSCs can differentiate into fibroblasts, not only showing morphological similarity, but also having the ability to express fibroblast surface proteins (including vimentin and fibronectin) (Kim WS et al., Wound healing effect of adipose-derived stem cells: a critical role of secretory factors on human dermal fibroblasts. J Dermatol Sci. 2007, 48:15-24). Also, Ad-MSCs can directly convert into fibroblasts and keratinocytes to repair wounds (Unnikrishnan S et al., Constitution of fibrin-based niche for in vitro differentiation of adipose-derived mesenchymal stem cells to keratinocytes. Biores Open Access. 2014, 3(6):339-347). However, according to previous studies, it has been discovered that apoptosis occurs after many Ad-MSCs are injected into the wounds of diabetic mice, resulting in delayed wound healing. Ad-MSC cells cultured in a high glucose environment have apoptosis that occurs and is time-dependent (Li Q et al., Stromal cell-derived factor-1 promotes human adipose tissue-derived stem cell survival and chronic wound healing. Exp Ther Med. 2016, 12:45-50).

[0189] In this test example, CCK-8 and EdU were used to detect the effects of the composition of the present application on the in vitro activity and proliferation ability of Ad-MSCs. The results showed that the composition of the present application can improve the activity and proliferation ability of Ad-MSCs. Flow cytometry was used to detect the apoptosis status of cells, and it was discovered that the composition of the present application suppresses the apoptosis of Ad-MSCs induced by high glucose and has time and concentration dependence.

[0190] The influence on the survival rate of Ad-MSCs of the composition of the present application in an animal body was detected, and the results showed that when using Ad-MSCs after treatment with the composition of the present application, the survival rate was higher and the wound healing rate was faster than when simply applying Ad-MSCs.

Claims

1. A method for regulating stem cells, comprising: contacting the stem cells with a product derived from the cell wall of Rhodococcus ruber, wherein the ratio of the number of stem cells to the product derived from the cell wall of Rhodococcus ruber is 1 to 100 stem cells / 1 ng of the product derived from the cell wall of Rhodococcus ruber, wherein the regulation refers to one or a combination selected from promoting the proliferation of stem cells, promoting the growth of stem cells, promoting the differentiation of stem cells, promoting the migration of stem cells, and improving the survival rate of stem cells, wherein the stem cells are selected from adult stem cells, iPSCs, and mesenchymal stem cells.

2. The method according to claim 1, wherein the stem cells are mesenchymal stem cells selected from bone marrow mesenchymal stem cells, adipose 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.

3. The method according to claim 1, wherein the product derived from the cell wall of Rhodococcus ruber is the cell wall of Rhodococcus ruber or its composition.

4. The product derived from the cell wall of Rhodococcus ruber is 1) a step of preparing Rhodococcus ruber, 2) a step of grinding the Rhodococcus ruber to obtain a ground product, 3.1) a step of performing a lipid removal operation on the ground product, 3.2) a step of performing a nucleic acid removal operation on the ground product, 3.3) a step of performing a protein removal operation on the ground product, 3.4) a step of obtaining a product derived from the cell wall of Rhodococcus ruber, 4) a step of removing water in the product derived from the cell wall of Rhodococcus ruber, 5) a step of individually packaging, and is obtained by a method comprising, Steps 3.1), 3.2), and 3.3) may be performed in any order or simultaneously, and steps 4) and 5) may be performed in any order. The method according to any one of claims 1 to 3.

5. The method according to any one of claims 1 to 3, wherein the Rhodococcus ruber was deposited with the China General Microbiological Culture Collection Center (CGMCC) on March 22, 2019 under the accession number CGMCC No. 17431.

6. Use of a product derived from the cell wall of Rhodococcus ruber in the manufacture of a reagent, The reagent is used for one or a combination selected from promoting the proliferation of stem cells, promoting the growth of stem cells, promoting the differentiation of stem cells, promoting the migration of stem cells, and improving the survival rate of stem cells. The stem cells are selected from adult stem cells, iPSCs, and mesenchymal stem cells for use.

7. The use according to claim 6, wherein the stem cells are mesenchymal stem cells selected from bone marrow mesenchymal stem cells, adipose 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.

8. The use according to claim 7, wherein the product derived from the cell wall of Rhodococcus rhodochrous is the cell wall of Rhodococcus rhodochrous or its composition.

9. The product derived from the cell wall of Rhodococcus rhodochrous is 1) a step of preparing Rhodococcus rhodochrous; 2) a step of pulverizing the Rhodococcus rhodochrous to obtain a pulverized product; 3.1) a step of performing a lipid removal operation on the pulverized product; 3.2) a step of performing a nucleic acid removal operation on the pulverized product; 3.3) a step of performing a protein removal operation on the pulverized product; 3.4) a step of obtaining a product derived from the cell wall of Rhodococcus rhodochrous; 4) a step of removing water in the product derived from the cell wall of Rhodococcus rhodochrous; 5) a step of individually packaging, and is obtained by a method including Steps 3.1), 3.2), and 3.3) may be performed in any order or simultaneously, and steps 4) and 5) may be performed in any order. The use according to any one of claims 6 to 8.

10. The use according to any one of claims 6 to 8, wherein the Rhodococcus rhodochrous was deposited with the China General Microbiological Culture Collection Center (CGMCC) on March 22, 2019 under the accession number CGMCC No. 17431.

11. A cell culture medium comprising a product derived from the cell wall of Rhodococcus rhodochrous, wherein the product derived from the cell wall of Rhodococcus rhodochrous is the cell wall of Rhodococcus rhodochrous or its composition, and the Rhodococcus rhodochrous was deposited with the China General Microbiological Culture Collection Center (CGMCC) on March 22, 2019 under the accession number CGMCC No. 17431.

12. The following steps: 1) a step of preparing Rhodococcus rhodochrous; 2) a step of pulverizing the Rhodococcus rhodochrous to obtain a pulverized product; 3.1) performing a lipid removal operation on the ground product; 3.2) performing a nucleic acid removal operation on the ground product; 3.3) performing a protein removal operation on the ground product; 3.4) obtaining a product derived from the cell wall of Rhodococcus rhodochrous; 4) removing water in the product derived from the cell wall of Rhodococcus rhodochrous; 5) individually packaging, wherein steps 3.1), 3.2), and 3.3) may be performed in any order or simultaneously, and steps 4) and 5) may be performed in any order. A method for obtaining a cell culture medium according to claim 11.

13. - stem cells, - a product derived from the cell wall of Rhodococcus rhodochrous, wherein the stem cells are selected from adult stem cells, mesenchymal stem cells, and iPSCs, the ratio of the number of stem cells / the product derived from the cell wall of Rhodococcus rhodochrous is 1 to 100 stem cells / 1 ng of the product derived from the cell wall of Rhodococcus rhodochrous, the mesenchymal stem cells are selected from bone marrow mesenchymal stem cells, adipose 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, dental pulp mesenchymal stem cells. A pharmaceutical composition.

14. The Rhodococcus rhodochrous was deposited with the China General Microbiological Culture Collection Center (CGMCC) on March 22, 2019, under the accession number CGMCC No. 17431. A pharmaceutical composition according to claim 13.

15. A pharmaceutical composition according to claim 13 or 14 for use in promoting wound healing.

16. The wound is a wound associated with diabetes. A pharmaceutical composition according to claim 15.

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