WO2021147899A1 - 赤红球菌细胞壁骨架在再生医学中的用途 - Google Patents

赤红球菌细胞壁骨架在再生医学中的用途 Download PDF

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WO2021147899A1
WO2021147899A1 PCT/CN2021/072871 CN2021072871W WO2021147899A1 WO 2021147899 A1 WO2021147899 A1 WO 2021147899A1 CN 2021072871 W CN2021072871 W CN 2021072871W WO 2021147899 A1 WO2021147899 A1 WO 2021147899A1
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stem cells
mesenchymal stem
rhodococcus
cell wall
derived
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PCT/CN2021/072871
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English (en)
French (fr)
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盖波
窦春艳
金培生
张轶
张国英
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辽宁格瑞仕特生物制药有限公司
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Priority to CN202180005363.2A priority Critical patent/CN115087729A/zh
Priority to EP21744220.1A priority patent/EP4043555A4/en
Priority to JP2022541262A priority patent/JP2023510232A/ja
Priority to KR1020227027900A priority patent/KR20220130162A/ko
Priority to US17/792,396 priority patent/US20230069441A1/en
Publication of WO2021147899A1 publication Critical patent/WO2021147899A1/zh

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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
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    • C12N2500/00—Specific components of cell culture medium
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    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • This application relates to the field of regenerative medicine. Specifically, it relates to the use of Rhodococcus rubrum (especially the cell wall skeleton) in promoting the proliferation of stem cells and promoting the differentiation of stem cells.
  • Rhodococcus rubrum especially the cell wall skeleton
  • Stem cells are primitive cells with self-renewal ability and multi-directional differentiation potential. Under certain conditions, it can proliferate and differentiate into different functional cells. Therefore, the research of stem cells plays a very important role in the renewal and damage repair of various tissues and organs of the living body, and it has become the hope of many incurable diseases, especially cell and tissue loss or injury diseases.
  • Stem cells include embryonic stem cells and adult stem cells.
  • the application of embryonic stem cells is greatly restricted due to ethical issues; adult stem cells can be differentiated into functional cells and tissues, providing a basis for the wide application of stem cells; providing a new cell source for cell replacement therapy for many diseases. Therefore, adult stem cells have become the focus of research.
  • stem cells in normal adult mammals are small, and the differentiation is affected by various internal mechanisms and microenvironmental factors; it is difficult to culture in large quantities for a long time in vitro, especially serum-free expansion culture, which cannot be applied to actual treatment.
  • Stem cell drugs can prevent and treat diseases caused by cell loss or damage by regulating the proliferation and differentiation of stem cells in organisms.
  • Stem cell drugs are used to regulate the proliferation and directed differentiation potential of autologous stem cells to rebuild damaged functional cells and restore their biological functions.
  • Ad-MSC Adipose-derived mesenchymal stem cells
  • Ad-MSC Adipose-derived mesenchymal stem cells
  • Rhodococcus ruber also known as Rhodococcus ruber
  • Rhodococcus ruber a gram-positive bacteria.
  • the colony is orange or orange, round; the size of the colony is about 1mm to 2mm; the cell morphology is spherical or short rod; it can form primary branched mycelium; it has no flagella.
  • Rhodococcus is aerobic and chemically heterotrophic.
  • Rhodococcus rubrum researchers have performed whole-gene foci of Rhodococcus rubrum.
  • Fan Xin et al. sequenced the entire genome of Rhodococcus rubrum SD3 strain and performed bioinformatics analysis.
  • the whole genome length of SD3 strain is about 5.37Mb
  • GC content is about 70.63%
  • GenBank accession number is CP029146 (Fan Xin, Rhodococcus rubrum SD3 whole genome sequencing and its heat shock protein DnaK expression analysis, genomics and applied biology, January 2019).
  • Rhodococcus is a genus of Rhodococcus, which can adapt to a variety of living environments due to its strong tolerance to organic matter and a wide degradation spectrum. Therefore, Rhodococcus is widely used in pollution remediation, organic compound degradation, sewage treatment and other fields.
  • Rhodococcus rubra lies in environmental management, see CN108862590A, CN107151635A, CN102250796A, CN1519312A, CN103627653A, CN101033454A, CN108130288A, CN104830738A, CN101619299A, CN103509833A, CN106434466A, CN101580808A, CN102491, 875, CN102591106A105, CN102604466A, CN101580808A, CN102604, 875, CN101580808A, CN102491, 875, CN102591106A, CN103160.
  • CN109576180A discloses a bacterium RDC-01 screened from the red soil in the suburbs near Panyu District, Guangzhou City. After 16S rRNA gene sequence analysis and identification of culture characteristics, the strain was identified as Rhodococcus rubrum. After the bacterium was inactivated, it was added as an immune adjuvant to an inactivated vaccine for animals, and it was found to promote the production of antibodies in animals. However, the application of Rhodococcus rubrum in the field of human medicine has not yet been reported.
  • This application provides active ingredients for regulating stem cells and their applications.
  • an isolated Rhodococcus ruber is provided.
  • a Rhodococcus rubrum is provided, which was deposited at the China General Microbiological Culture Collection Center of the China General Microbiological Culture Collection Center on March 22, 2019, Beichen, Chaoyang District, Beijing No. 3, No. 1 West Road, Institute of Microbiology, Chinese Academy of Sciences; Zip Code: 100101), and the deposit number is CGMCC No.17431.
  • the deposit meets the requirements of the Budapest Treaty on the International Recognition of Microorganisms for Patent Procedures (Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure).
  • Rhodococcus rubrum and its derivative products are provided.
  • the derivative product is derived from Rhodococcus rubrum and contains the components of Rhodococcus rubrum (such as protein, nucleic acid, lipid, cell wall and its constituent components, carbohydrates, metabolites).
  • an isolated Rhodococcus rubrum cell wall is provided.
  • an isolated Rhodococcus rhodococcus cell wall is provided, and the Rhodococcus rhodococcus refers to a strain with a deposit number of CGMCC No. 17431.
  • a product derived from the cell wall of Rhodococcus rubrum is provided.
  • an isolated Rhodococcus rubrum cell wall skeleton is provided.
  • an isolated Rhodococcus Rhodococcus cell wall skeleton is provided, and the Rhodococcus Rhodococcus refers to the strain with the deposit number of CGMCC No. 17431.
  • a pharmaceutical composition comprising the cell wall of Rhodococcus or the cell wall skeleton of Rhodococcus according to the present disclosure.
  • a product derived from the cell wall of Rhodococcus rubrum which comprises a product obtained by crushing Rhodococcus rubrum.
  • a product derived from the cell wall of Rhodococcus rubrum which comprises a product obtained by crushing and purifying (removing fat, nucleic acid, and protein) of Rhodococcus rubrum.
  • a product derived from the cell wall of Rhodococcus which comprises the cell wall of Rhodococcus.
  • a product derived from the cell wall of Rhodococcus which comprises the cell wall skeleton of Rhodococcus.
  • a pharmaceutical composition or medical device comprising a product derived from the cell wall of Rhodococcus rubrum.
  • a pharmaceutical composition or medical device which comprises a product obtained by crushing and purifying Rhodococcus rubrum (degreasing, and/or denucleating, and/or deproteinizing).
  • a pharmaceutical composition or medical device which comprises the cell wall of Rhodococcus rubrum.
  • a pharmaceutical composition or medical device which comprises the cell wall skeleton of Rhodococcus rubrum.
  • a pharmaceutical composition or medical device which comprises the above-mentioned product derived from the cell wall of Rhodococcus rubrum.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.
  • the product derived from the Rhodococcus rhodochrous cell wall in the pharmaceutical composition is 1 part by weight, and the pharmaceutically acceptable excipient is 50 to 5000 parts by weight (e.g., 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 two numerical ranges Any point value).
  • the Rhodococcus rubrum cell wall in the pharmaceutical composition is 1 part by weight
  • the pharmaceutically acceptable excipient is 50 to 5000 parts by weight (e.g., 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 point value within any two numerical ranges ).
  • the Rhodococcus rhodochrous cell wall skeleton in the pharmaceutical composition is 1 part by weight
  • the pharmaceutically acceptable excipient is 50 to 5000 parts by weight (e.g., 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 point within any two numerical ranges value).
  • the pharmaceutical composition may be prepared as a liquid (liquid formulation).
  • the pharmaceutical composition can be prepared as a solid (dry powder formulation or lyophilized powder formulation).
  • the liquid preparation and the dry powder preparation can be converted into each other, and the difference lies only in the water content. Remove most or all of the water in the liquid preparation to obtain a dry powder preparation (or freeze-dried powder preparation). The dry powder preparation (or freeze-dried powder preparation) is dissolved (or reconstituted) to obtain a liquid preparation.
  • the drug or pharmaceutical composition is prepared into a dosage form selected from the group consisting of ointments, creams, emulsions, suspensions, pastes, gels, lotions, tinctures, oils, tablets, gas Fog, spray, liniment, powder; wherein, the ointment is selected from: ointment, plaster, and cream.
  • the pharmaceutically acceptable excipient relates to, but is not limited to: fillers, stabilizers, flavoring agents, disintegrants, binders, and lubricants.
  • the pharmaceutically acceptable excipient such as but not limited to: dextran, lactose, microcrystalline cellulose, trehalose, glycine, xylitol, sodium carboxymethyl cellulose, erythrose Alcohol, gelatin, magnesium stearate, propellant, humectant, solvent, solubilizer, emulsifier, antioxidant, pH regulator, preservative.
  • non-limiting examples also include: white petrolatum, carbomer, hypromellose, methyl cellulose, sodium hydroxymethyl cellulose, chitosan, sucralfate chitosan, polyvinylpyrrolidone, Polyvinyl alcohol, sodium hyaluronate, dimethyl ether, tetrafluoroethane, hydrofluoroalkane, glycerin, propylene glycol, deionized water, water for injection, distilled water, ethanol, cetyl alcohol, stearyl alcohol, p-aminobenzoic acid, ethyl Amide, isopropanol, Tween, polyoxyethyl hydrogenated castor oil, stearic acid, glyceryl monostearate, triglycerol monostearate, sucrose fatty acid esters, sucrose esters, sucrose acetate isobutyrate Esters, sorbitan tristearate, isopropyl myristate,
  • a method for preparing a product derived from the cell wall of Rhodococcus rubrum which includes or consists of the following steps:
  • remove the water in the purified product preferably by freeze-drying to remove the water in the purified product;
  • steps 5.1), 5.2), and 5.3) can be interchanged in order or can be parallel; step 6) and step 7) can be interchanged in order.
  • step 5) may further include (for example, using a non-ionic surfactant) to remove the cell membrane.
  • Rhodococcus rubrum is not limited to specific media and culturing parameters, and the skilled person can use well-known and appropriate methods for culturing, and can use petri dishes, culture bottles, and fermentation tanks according to the scale of preparation.
  • Technicians have the ability to adjust the specific parameters and equipment of culture, crushing, separation, collection, impurity removal, and packaging according to the subsequent application (such as oral, injection, topical application, etc.) of the active ingredients (cell wall and its components), so as to avoid preparation Introduce factors that affect subsequent applications in the steps.
  • an organic solvent is used to remove lipids in the fragmented product.
  • nucleases are used to remove DNA and RNA in the fragmented product.
  • a hydrolase is used to degrade the protein in the fragmented product.
  • a surfactant is used to remove the cell membrane in the disrupted product.
  • the average particle size of pulverization is 10 nm to 1000 nm; mention may be made of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190nm ⁇ 10nm, and the range between any two of the above values.
  • particle size testing Hu Songqing et al., Modern Particle Size Measurement Technology, Modern Engineering, 2002 22:1).
  • the average particle size of the comminution is 10 nm to 800 nm.
  • the average particle size of the pulverization is 10 nm to 500 nm.
  • the dispensing refers to dispensing into a container or placing on a solid support.
  • the container is selected from: bottles, tubes, bags, bags, plates, ampoules, injection devices, aluminum film packaging, dressings, and capsules.
  • the dispensing refers to dispensing into bottles/ampoules. Just before use, add solvent to the bottle/ampule.
  • a product derived from the cell wall of Rhodococcus rubrum which is prepared by the method according to the present disclosure.
  • a pharmaceutical composition or medical device which comprises a product derived from the cell wall of Rhodococcus rubrum prepared according to the method of the present disclosure.
  • an isolated Rhodococcus rubrum cell wall for regulating adult stem cells.
  • the regulation refers to one or a combination selected from the group consisting of: 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, iPSC, mesenchymal stem cells.
  • 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 Mesenchymal stem cells, amniotic membrane 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 product derived from the cell wall of Rhodococcus rubrum is provided, which is used to regulate adult stem cells.
  • a pharmaceutical composition or medical device for regulating adult stem cells for regulating adult stem cells.
  • Rhodococcus rubrum cell wall according to the present disclosure in regulating adult stem cells.
  • Rhodococcus rhodochrous cell wall of the present disclosure in the preparation of a medicine/medical device for regulating adult stem cells.
  • the use of the Rhodococcus cell wall-derived product according to the present disclosure in regulating adult stem cells and the use of the Rhodococcus cell wall-derived product of the present disclosure in the preparation of a medicament for regulating adult stem cells is also provided. /Use in medical devices.
  • the use of the pharmaceutical composition according to the present disclosure in regulating adult stem cells is provided; and the use of the pharmaceutical composition of the present disclosure in preparing drugs/medical devices for regulating adult stem cells is also provided.
  • the drug is used to modulate adult stem cells.
  • the medical device (such as dressing, patch, bandage, film, patch, etc.) is used to regulate adult stem cells.
  • a method of modulating adult stem cells comprising contacting a subject with a therapeutically effective amount (or a preventively effective amount) of any one selected from the following:
  • a method for regulating stem cells which includes the step of contacting stem cells with a product derived from the cell wall of Rhodococcus rubrum.
  • the ratio of the number of stem cells/the product derived from the Rhodococcus cell wall is: 1-1000 stem cells/1ng product derived from the Rhodococcus cell wall; preferably 5-50 stem cells/1ng derived from the Rhodococcus cell wall ⁇ The product.
  • the product for example, not limited to 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/1ng product derived from the Rhodococcus cell wall, and any range in between.
  • a method for promoting wound healing including the step of contacting a subject's wound surface with a therapeutically effective amount of mesenchymal stem cells and a product derived from the cell wall of Rhodococcus.
  • the ratio of the number of mesenchymal stem cells/the product derived from the Rhodococcus cell wall is: 1-100 stem cells/1ng product derived from the Rhodococcus cell wall; preferably 5-50 stem cells/1ng derived from the Rhodococcus The product of the cell wall.
  • the wound surface is a wound surface related to diabetes.
  • drugs are used to apply drugs (or medical devices) to the lesions according to the difference in the area and depth of the lesion.
  • drugs or medical devices
  • drugs are used to apply drugs (or medical devices) to the lesions according to the difference in the area and depth of the lesion.
  • drugs or medical devices
  • the period of contact is from 2 days to 2 months or longer.
  • the subject is administered the active ingredient for 3-4 weeks.
  • the administration is performed at the following frequency: 1 to 3 times a day, 1 to 6 times two days, 1 to 9 times three days, 1 to 14 times a week, 1 to 60 times a month. In some embodiments, it is administered twice a day, or once a day, or once every two days.
  • the dosage for each administration is different depending on the specific conditions of the subject, usually 1 ⁇ g to 1000 ⁇ g/unit dose/each administration; specifically, such as 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 ⁇ g/unit dose/ Each time, and the range between any two of the aforementioned values.
  • contact is achieved by the following methods, such as but not limited to: oral, mucosal, transdermal, transdermal, abdominal cavity, puncture, nasal spray, eye drops, suppository, sublingual.
  • the subject is an animal other than humans, such as farm animals, pets, working animals, ornamental animals, and production animals.
  • the subject is a human.
  • the subject is suspected of having, confirmed to have, already suffering from, or susceptible to the target disease or its symptoms.
  • a cell culture medium which comprises one or a combination selected from the following:
  • a cell culture medium which further contains other components known in the art suitable for culturing stem cells (especially mesenchymal stem cells).
  • stem cells especially mesenchymal stem cells.
  • technicians can add cytokines, such as FGF, PDGF, TGF- ⁇ , HGF, EGF, CTGF, VEGF, insulin, to the cell culture medium as needed (for example, to maintain dryness or promote differentiation). , One or a combination of insulin-like growth factors.
  • cytokines such as FGF, PDGF, TGF- ⁇ , HGF, EGF, CTGF, VEGF, insulin
  • the content of FGF (in terms of final concentration) is preferably 0.1 to 100 ng/ml.
  • FGF refers to a growth factor in the fibroblast growth factor family, preferably FGF-1 and FGF-2 (bFGF).
  • the content of PDGF (based on the final concentration) is preferably 0.5 to 100 ng/ml.
  • PDGF refers to a growth factor in the platelet-derived growth factor family, preferably PDGF-BB or PDGF-AB.
  • the content of TGF- ⁇ (based on the final concentration) is preferably 0.5 to 100 ng/ml.
  • TGF- ⁇ refers to a growth factor in the transforming growth factor- ⁇ family, preferably TGF- ⁇ 3.
  • the content of HGF (in terms of final concentration) is preferably 0.1 to 50 ng/ml.
  • the content of EGF (in terms of final concentration) is preferably 0.5 to 200 ng/ml.
  • a cell culture medium which further comprises at least one phospholipid and/or at least one fatty acid.
  • phospholipids examples include phosphatidic acid, lysophosphatidic acid, phosphatidylcyclohexanol, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol.
  • the total content of phospholipids is preferably 0.1 to 30 ⁇ g/ml.
  • fatty acids examples 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.
  • a cell culture medium which further comprises cholesterol.
  • a cell culture medium which further comprises ascorbic acid.
  • a cell culture medium which further contains an antioxidant.
  • antioxidants include DL- ⁇ -tocopherol acetate (vitamin E).
  • a cell culture medium which further comprises transferrin.
  • a cell culture medium which further comprises selenate.
  • a cell culture medium which further contains amino acids, nucleotides, and trace elements required for cell maintenance.
  • the composition of the present application is added to a known commercially available mesenchymal stem cell culture medium;
  • the commercially available mesenchymal stem cell culture medium is, for example, selected from: MesenPRO RS TM , MSC SFM, MSC SFM XenoFree, Human adipose-derived stem cell medium.
  • a therapeutic composition comprising:
  • the ratio of the number of mesenchymal stem cells/product derived from Rhodococcus cell wall is: 1-100 stem cells/1ng product derived from Rhodococcus cell wall; preferably 5-50 stem cells/1ng Product derived from the cell wall of Rhodococcus rubrum.
  • 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, placenta Mesenchymal stem cells, amniotic membrane 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 method for improving stem cell apoptosis including the step of contacting the stem cells with an effective amount of the above-mentioned product derived from the cell wall of Rhodococcus rubrum.
  • a method for improving the survival rate of stem cells including the step of contacting the stem cells with an effective amount of the above-mentioned product derived from the cell wall of Rhodococcus rubrum.
  • the only therapeutic (or prophylactic) active ingredient in a drug or medical device is a product derived from the cell wall of Rhodococcus, especially containing Rhodococcus components (such as protein, nucleic acid, lipid, cell wall and Its constituents, carbohydrates, metabolites), specifically products containing Rhodococcus Rhodococcus cell wall (more preferably Rhodococcus Rhodococcus cell wall skeleton or its composition).
  • Rhodococcus components such as protein, nucleic acid, lipid, cell wall and Its constituents, carbohydrates, metabolites
  • composition 1 of the present application was applied to Ad-MSC for 24h, 48h, 72h, 96h and then Ad-MSC was detected by CCK-8 at a wavelength of 450nm, and the absorbance value (OD) was measured by an enzyme-linked immunoassay.
  • Figure 2 10 ⁇ g/ml, 50 ⁇ l of the composition 1 of the application was applied to Ad-MSCs, after 72h and 96h, EdU incorporation analysis was carried out with the EDU kit, and the cell proliferation rate of each group was statistically analyzed. The experiment was repeated 3 times, and the data used errors The bar graph represents the mean ⁇ SD (*P ⁇ 0.05, **P ⁇ 0.01, vs. control).
  • Figures 3A to 3B Flow cytometry to detect the stem cell apoptosis rate of 48h, 72h blank control group, high glucose group, and composition treatment group of the present application. The experiment was repeated 3 times, and the data was represented by an error bar graph as the mean ⁇ SD (**P ⁇ 0.01, vs. high sugar).
  • Figure 4A Figure 4B: Adding high sugar to the cells induces apoptosis, and Western blotting is performed to detect the protein expression levels of c-caspase-3 and Bax. The experiment was repeated 3 times, and the data was represented by an error bar graph as the mean ⁇ SD (*P ⁇ 0.05, **P ⁇ 0.01, vs high glucose).
  • Figure 5A After cells were inoculated on the wounds of diabetic mice, the survival rate of Ad-MSC in each group was observed according to the cells labeled with the fluorescent dye CM-Dil on the 0th, 2nd, and 7th days. Statistical analysis of the fluorescence flux results of CM-Dil-labeled cells.
  • Figure 5B Measure and calculate the wound healing rate of each group (*P ⁇ 0.05, ** ⁇ 0.01, vs blank).
  • Isolation refers to the separation of the Rhodococcus rubrum of the present disclosure from its original growth environment.
  • 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 20nm to 80nm), containing about 90% peptidoglycan and about 10% teichoic acid (a polymer formed by alcohol and phosphoric acid molecules, usually with sugar Exist in the form of ester or amino acid ester).
  • the peptidoglycan layer is dense, even as many as 20 layers.
  • 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 2nm to 3nm).
  • the peptidoglycan layer is a unique component of the bacterial cell wall and is a derivative of heteropolysaccharide.
  • the monomer of each peptidoglycan consists of 3 parts: sugar unit (for example, at least two sugar molecules are connected by glycosidic bonds to form the framework structure of peptidoglycan), peptide tail (short peptide chain connected by several amino acids) , Which is connected to the N-acetylmuramic acid molecule), and the peptide bridge (crosslinking adjacent "peptide tails" to form a high-strength network structure).
  • sugar unit for example, at least two sugar molecules are connected by glycosidic bonds to form the framework structure of peptidoglycan
  • peptide tail short peptide chain connected by several amino acids
  • the peptide bridge crosslinking adjacent "peptide tails" to form a high-strength network structure.
  • Different bacteria have different peptide bridges, peptide tails, and cross-linking methods.
  • isolated Rhodococcus rhodococcus cell wall can be understood as either a complete cell wall or an incomplete cell wall (for example, broken or partially degraded).
  • the ingredients exhibiting the desired activity are derived from the cell wall of Rhodococcus (for example, the cell wall itself or its composition). Therefore, various forms such as complete cell walls, broken cell walls, incomplete degradation products of cell walls, cell wall components, cell wall extracts, etc. are allowed to be used in clinical applications, which are all included in the scope of the present disclosure.
  • Rhodococcus used in the embodiments of the present disclosure refers to Rhodococcus ruber of the genus Rhodococcus, and is not limited to a specific cell strain.
  • Non-limiting examples include TOY7 strain (Nanjing Agricultural University Agricultural Environmental Microbial Culture Collection), CGMCC No. 4795, DSM43338, CCTCC No. 2012035, CGMCC No. 16640, CGMCC No. 17431.
  • technicians can perform taxonomic identification on a strain of bacteria.
  • the available identification techniques include morphology, physiological and biochemical characteristics, 16S rRNA, and so on.
  • Technicians understand 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 this method is not high. After the advent of sequencing technology, technicians can identify strains in a more reliable way.
  • the medicine or pharmaceutical composition or active ingredient or product of the present disclosure may be embodied in but not limited to the following forms: ointment, cream, plaster, gel, lotion, tincture, liniment, oil, paste, Freeze-dried powders, aerosols, suppositories, patches, suspensions, oral liquids, lozenges, skin care products (cleansers, lotions, essences, lotions, creams, masks).
  • the medicine or pharmaceutical composition or active ingredient or product of the present disclosure can be prepared in the form of a unit preparation (unit preparation).
  • the unit dose in the drug contains:
  • Rhodococcus rubrum cell wall skeleton -1 ⁇ g to 1000 ⁇ g of the Rhodococcus rubrum cell wall skeleton.
  • unit doses 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 range between any two of the above values.
  • administering refers to drugs or medical devices and animals, humans, cells, tissues, organs or biological samples, refers to drugs or medical devices and animals, humans, cells, tissues, organs or Biological sample contact.
  • Treatment means to give a subject an internal or external drug (therapeutic agent, active ingredient or composition) (eg, the Rhodococcus erythrococcus cell wall or pharmaceutical composition thereof according to the present disclosure) or a medical device, in the treatment of the subject Alleviate (relieve, delay, improve, cure) one or more symptoms of a disease in a person (or group) to a clinically measurable degree, wherein the subject has, is suspected of suffering, or is susceptible For one or more diseases or their symptoms.
  • an internal or external drug eg, active ingredient or composition
  • a medical device eg, the Rhodococcus erythrococcus cell wall or pharmaceutical composition thereof according to the present disclosure
  • the amount of the drug (therapeutic agent, active ingredient or composition) that is effective to alleviate the symptoms of any disease is called the therapeutically effective amount. It can vary based on a variety of factors, such as the subject's disease state, age, and weight. It should be understood that the drug (therapeutic agent, active ingredient or composition) may not be effective in relieving the target disease or its symptoms of a single subject, but according to any statistical test method known in the art (such as Student's T test, card The prescription test, according to Mann and Whitney's U test), determines that the drug (therapeutic agent, active ingredient or composition) is statistically effective for the target disease or its symptoms.
  • any statistical test method known in the art such as Student's T test, card The prescription test, according to Mann and Whitney's U test
  • “Optional” means that what is described later can happen, but does not have to happen; it depends on the situation.
  • sub-package means that the product is allowed to be sub-packaged, but it is not necessary to be sub-packaged; whether the product is sub-packaged or not does not affect the realization of the technical effect.
  • Rhodococcus used in the following examples is the CGMCC No. 17431 strain, which was deposited at No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing on March 22, 2019 (Institute of Microbiology Chinese Academy of Sciences, No. 1 West Beichen Road) , Chaoyang District, Beijing China), China General Microbiological Culture Collection Center (CGMCC).
  • CGMCC General Microbiological Culture Collection Center
  • the size of the colony is about 1mm to 2mm (it varies slightly depending on the culture conditions).
  • -Mycelium divides to form regular short and thick cells (different depending on the culture conditions);
  • Gram stain is positive.
  • Nitrate reduction reaction is positive, contact enzyme is positive, tyrosinase is positive, amylase is negative, oxidase is negative, gelatin liquefaction is negative.
  • the 15 strains isolated from the working seed tube and the 10 different strains isolated from the original seed tube were subjected to genome extraction, 16Sr RNA amplification and sequencing.
  • the 16SrRNA gene identity of 25 strains in total was 100%.
  • the neighbor-joining strain phylogenetic tree constructed based on the Kimura2-parameter algorithm showed that the strain was classified as Rhodococcus ruber.
  • Rhodococcus rubrum can be cultivated by conventional microbial production methods.
  • the culture method can be solid culture or liquid culture.
  • the medium can contain carbon sources, nitrogen sources and other nutrient sources that are commonly used for microbial culture.
  • the carbon source is any carbon source that can be used by Rhodococcus rubrum, such as fructose, glucose, etc.
  • the nitrogen source can be meat extract, peptone, ammonium salt, nitrate and other organic or inorganic nitrogen-containing compounds.
  • the medium composition includes:
  • Peptone beef extract, sodium chloride, phosphate, glycerin (and, optionally, agar, when in solid culture).
  • the working strain After the working strain is recovered, it is 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 can also be used. Batch mode. Monitor pH, bacterial density, dissolved oxygen, and carbon source consumption during cultivation.
  • a common protease such as trypsin
  • Organic reagents such as but not limited to one or a combination of acetone, ether, and ethanol are added to the precipitate, and lipids are removed according to conventional operations in the art.
  • TritonX-100 was added to the precipitate, and the precipitate was collected by centrifugation according to conventional operations in the art, and rinsed with PBS.
  • the precipitate was re-dissolved in water for injection and set aside.
  • it can be sterilized at 115°C for 20-30 minutes as the original solution of the cell wall skeleton (mainly containing the cell wall skeleton and its components).
  • the pharmaceutical composition can be formulated in various forms
  • the pharmaceutical composition of item 1 is lyophilized to prepare lyophilized powders (numbered as lyophilized powder compositions 1 to 7 respectively).
  • Adipose tissue samples were obtained from liposuction aspirates (age range of 25-35 years) of subjects and Ad-MSCs were isolated and cultured. The subjects were from plastic surgery patients of the affiliated Hospital of Xuzhou Medical University, and were approved by the ethics committee and the patients informed consent.
  • the obtained fresh adipose tissue extract was digested with 0.25% trypsin-EDTA, filtered, and centrifuged to retain the cell sedimentation layer, and added to DMEM (Invitrogen) containing 10% FBS fetal bovine serum (Gibco) and 1% penicillin/streptomycin. Basically, place the cell culture dish in a 37°C, 5% CO 2 incubator for culture. Thereafter, the medium was changed every 2-3 days after washing with PBS, and the cells were grown to 80% for passage.
  • DMEM Invitrogen
  • Ad-MSC in logarithmic growth phase and inoculate it in 96-well plate at 4 ⁇ 10 3 /well; DMEM + 10% fetal bovine serum + 1% penicillin/streptomycin co-culture; use PBS solution to combine the composition of the application ( Composition 1) Dissolve and dilute to 10 ⁇ g/ml.
  • each test substance was divided into four groups: control, 25 ⁇ l, 50 ⁇ l, and 75 ⁇ l.
  • EdU incorporation analysis was performed using Cell-Light EdU 567 In Vitro Imaging Kit (RiboBio). Take Ad-MSC in logarithmic growth phase and inoculate it in 96-well plate at 4 ⁇ 10 3 /well; 10 ⁇ g/ml, 50 ⁇ l of the composition of the application acts on Ad-MSC, and after 72h and 96h, add 100 ⁇ l of EDU medium to each well and incubate twice PBS wash 1-2 times. Add 4% paraformaldehyde fixative to each well and incubate at room temperature for 30 min. Then add 2mg/ml glycine solution and shake for 5 minutes. After washing with PBS, add penetrant, shake for 10 minutes, and wash with PBS.
  • Ad-MSC When Ad-MSC grows to 80% fusion, the density of 1 ⁇ 10 4 /well is inoculated into 6-well plates. The experiment was divided into four groups. After the cells adhered, 100 ⁇ l of 50% sucrose was added to each experimental group to induce apoptosis of Ad-MSC cells, and the two experimental groups were then added 100 ⁇ l and 250 ⁇ l of the composition of the application at 10 ⁇ g/ml; 48h, After 72 hours, the cell supernatants of each group were collected in a flow tube.
  • the adherent cells were digested with EDTA-free trypsin and collected in the same flow tube, centrifuged at 2000 rpm for 5 minutes; washed twice with PBS buffer, shaken and mixed; Add 500 ⁇ l, FITC5 ⁇ l, PI5 ⁇ l of binding buffer (Annexin V-FITC Cell Apoptosis Detection Kit, Shanghai Biyuntian Biotechnology Co., Ltd.) to the tube in sequence, shake evenly, and incubate at 4°C for 5-15min in the dark; flow cytometry detection ( BD Biosciences) analysis results.
  • experiment grouping is the same as flow cytometry; cells were washed 3 times with PBS buffer, fixed with 4% paraformaldehyde for 30 min at room temperature; cells were washed 3 times with PBS buffer with 0.1% Incubate TritionX-100 for 10 minutes at 2°C-8°C; wash cells with PBS buffer 3 times, prepare 500 ⁇ l TUNEL reaction solution (TUNEL Apoptosis Kit, Roche), mix 50 ⁇ l enzyme solution and 450 ⁇ l labeling solution as reagent A; Add 50 ⁇ l Reagent A to the negative control group and incubate at 37°C in the dark for 60min; add DNase I to the positive control group and incubate for 10min at room temperature; wash the cells 3 times with PBS buffer and add 50 ⁇ l/well of TUNEL reaction mixture, 37°C Incubate for 60 min in the dark in the incubator; wash the cells 3 times with PBS buffer, add 50 ⁇ l/well DAPI staining solution, and
  • mice 4-week-old BALB/c athymic nude mice, SPF grade. All animal studies are approved by the Animal Care and Use Committee. Experimental mice are kept in SPF animal room. Before modeling, the mice were fasted for 12 hours, weighed and recorded, and induced diabetic mice by intraperitoneal injection of 2% STZ (Sigma) at a dose of 150 mg/kg.
  • mice After the diabetic mouse model is successfully established, the mice are anesthetized and wounds with a diameter of 1.5 cm are prepared.
  • the pretreated Ad-MSCs are injected into the skin of each group of mice by multi-point intradermal injection, and six points are taken from each wound and injected at each point. 0.1ml of cells. Spread the surface on sterile gauze, continue to feed and observe, use the LB983 in vivo imaging system to observe the survival of wound cells and the healing of mouse skin.
  • Ad-MSC was labeled with CM-Dil before injection. According to the method recommended by the supplier, add CM-Dil live cell stain, incubate for 30 minutes, centrifuge and discard the supernatant, wash 3 times with PBS buffer, finally add an appropriate amount of PBS to mix, and place the labeled Ad-MSC in an ice box spare.
  • the paraffin sections of the tissues were deparaffinized and incubated with 3% H 2 O 2 for 5-10 min at room temperature to eliminate the endogenous peroxidase activity.
  • the sections were washed with distilled water, immersed in PBS twice, 5min/time, and blocked with 10% normal goat serum (diluted in PBS) for 10min at room temperature, discarded the blocking solution, and do not wash. Add CD31 primary antibody (dilution ratio 1:300) working solution dropwise, overnight.
  • the sections were rinsed 3 times with PBS, 5min/time.
  • Test Example 1 Identification of morphology, surface markers and differentiation induction ability of human Ad-MSC
  • the primary Ad-MSC extracted from the adipose tissue extract is planted in a cell culture dish, and the cells grow and fuse to 80% passage. After passage, the cell proliferation speed is significantly increased, and the cell morphology is uniform and consistent. Long fusiform. Ad-MSCs with good growth in the P3 generation were used for adipogenic differentiation and osteogenic differentiation, respectively. Two weeks later, oil red staining and observation under a microscope showed that there were obvious red lipid droplets of different sizes in the cells; staining with Alizarin S staining solution showed obvious red calcium nodules deposits. This indicates that the isolated and extracted cells have the characteristics of stem cell differentiation potential.
  • Test Example 2 The effect of the composition of the present application on the activity and proliferation of Ad-MSC
  • composition of the present application was used to act on stem cells at 10 ⁇ g/ml and 50 ⁇ l. After 72h and 96h respectively, the EdU incorporation analysis was carried out with the EDU kit, and the positive cells were observed by fluorescence microscope. The results showed that the composition of the present application can increase the growth rate of Ad-MSC (Figure 2).
  • Test Example 3 The composition of the present application inhibits Ad-MSC cell apoptosis induced by high glucose
  • High glucose was added to Ad-MSC cells to induce cell apoptosis, and the composition 1 of the application was treated with different concentrations for 48 hours and 72 hours, respectively, and the FITC-PI flow cytometry apoptosis detection kit was used to detect cell apoptosis.
  • composition of the present application can inhibit stem cell apoptosis induced by high glucose ( Figure 3A, Figure 3B).
  • Test Example 4 Detect the expression level of apoptosis marker protein
  • Test Example 5 The composition of the application improves the survival rate of Ad-MSC and accelerates wound healing in nude mice
  • mice were anesthetized, the wound with a diameter of 1.5 cm was cut out, and the pretreated cells labeled with the fluorescent dye CM-Dil were injected intracutaneously into the wound skin of the mice in the treatment group, and the LB983 in vivo imaging system was used to detect Cell survival rate.
  • Ad-MSC is a kind of multidirectional differentiation potential stem cell extracted from adipose tissue (Kato Y et al., Creation and transplantation of an adipose-derived stem cell (ASC) sheetin a diameter-healing model. Jove-J Vis Exp. 2017; 12(6): 1-10). Ad-MSC can migrate to damaged parts through differentiation potential, repair damaged skin with differentiated cells, and secrete a variety of growth factors (Rehman J et al., Secretion of angiogenic and anti-apoptotic factors by human adipose stromal cells.Circulation.
  • Ad-MSC can differentiate into fibroblasts, not only showing morphological similarity, but also 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).
  • Ad-MSC can also be directly transformed into fibroblasts and keratinocytes for wound repair (Unnikrishnan S et al., Constitution of fibrin-based niche for in vitro differentiation of pose-derived mesenchymal stem cells to keratinocytes. Biores Open Access.
  • Ad-MSCs undergo apoptosis when injected into the wounds of diabetic mice, leading to delayed wound healing.
  • Ad-MSC cells cultured in a high glucose environment undergo apoptosis and are time-dependent (Li Q et al., Stromal cell-derived factor-1 promoted human adipose tissue-derived stem cell survival and chronic wound healing. Exp Ther Med. 2016 ; 12:45-50).
  • CCK-8 and EdU were used to detect the in vitro activity and proliferation ability of the composition of the present application on Ad-MSC.
  • the results show that the composition of the present application can improve the activity and proliferation ability of Ad-MSC.
  • flow cytometry to detect cell apoptosis, it is found that the composition of the present application can inhibit the apoptosis of Ad-MSC induced by high glucose in a time and concentration dependent manner.

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Abstract

提供了赤红球菌细胞壁骨架在促进干细胞的增殖、促进干细胞的生长、促进干细胞的分化、促进干细胞的迁移、提高干细胞的存活率方面的用途；其中干细胞选自：成体干细胞、iPSC、间充质干细胞。

Description

赤红球菌细胞壁骨架在再生医学中的用途
本申请要求2020年01月21日提交的中国专利申请(申请号CN 202010068249.1)的优先权。
技术领域
本申请涉及再生医学领域。具体而言，涉及赤红球菌(尤其是细胞壁骨架)在促进干细胞的增殖、促进干细胞的分化中的用途。
背景技术
干细胞是一种具有自我更新能力和多向分化潜能的原始细胞。在特定条件下，可以增殖并定向分化成不同的功能细胞。因此，干细胞的研究对生命体各个组织器官的更新及损伤修复，起着非常重要的作用，成为许多无法治愈的疾病，特别是细胞及组织缺失或损伤性疾病的希望。
干细胞包括胚胎干细胞和成体干细胞。胚胎干细胞由于伦理问题，使其应用受到极大的限制；而成体干细胞由于可以分化为功能细胞和组织，为干细胞的广泛应用提供了基础；为许多疾病的细胞替代疗法提供了新的细胞来源。因此，成体干细胞成为研究的重点。
但正常成年哺乳动物的干细胞数量少，且分化受多种内在机制和微环境因素的影响；在体外则很难长期大量培养，特别是无血清扩增培养，无法应用于实际治疗。
许多疾病可追溯为功能性细胞的丢失或损伤所致，而细胞替代疗法是治疗这些疾病的有效方法。干细胞药物可以通过调节生物体内干细胞的增殖与分化，来防治由于细胞缺失或损伤而引起的疾病。用干细胞药物来调节自体干细胞的增殖与定向分化潜能，以重建受损的功能细胞，恢复其生物学功能。
脂肪间充质干细胞(Ad-MSC)是衍生自脂肪组织基质的成体干细胞，具有相当大的自我更新能力，可以分化为各种类型的功能性细胞。已有证据表明，Ad-MSC在基于干细胞的慢性伤口治疗中具有巨大潜力。但是，在潜在细胞疗法中成功使用Ad-MSC的一个重大障碍是植入后细胞的存活率。当将细胞移植到受损的皮肤组织中时，它们会经历包括缺氧、炎症、氧化应激或其他不利条件，这不可避免地导致植入后种子细胞的不良存活，干扰了Ad-MSC的治疗作用。
赤红球菌(Rhodococcus ruber)(也称为红色红球菌)，革兰氏阳性菌。通常而言，菌落呈桔黄色或呈桔红色，圆形；菌落大小约1mm至2mm；细胞形态为球状或短杆状；可形成初级分枝菌丝体；无鞭毛。赤红球菌好氧，化能异养。
目前，已有研究人员对赤红球菌进行了全基因侧学。例如，樊欣等人对赤红 球菌SD3株的全基因组进行测序，并进行生物信息学分析。SD3株的全基因组长度大约为5.37Mb，GC含量约为70.63％，GenBank登录号为CP029146(樊欣，赤红球菌SD3全基因组测序及其热休克蛋白DnaK的表达分析，基因组学与应用生物学，2019年1月)。
红球菌属Rhodococcus，因其自身具有非常强的有机物耐受性，以及较宽的降解谱，能够适应多种生存环境。因此，红球菌属被广泛应用于污染修复、有机化合物降解、污水处理等领域。目前，赤红球菌的主要应用领域在于环境治理，参见CN108862590A、CN107151635A、CN102250796A、CN1519312A、CN103627653A、CN101033454A、CN108130288A、CN104830738A、CN101619299A、CN103509833A、CN106434466A、CN101580808A、CN102604875A、CN103160491A、CN106591168A、CN106591172A、CN105820982A。
CN109576180A中公开了一种从广州市番禺区附近郊野红土中筛选到的菌RDC-01，经16S rRNA基因序列分析和培养特性鉴定，该菌株鉴定为赤红球菌。将该菌灭活后，作为免疫佐剂添加入动物用的灭活疫苗中，发现可以促进动物生产抗体。然而，赤红球菌在人类医学领域中的应用，尚未有报道。
因此，寻找特异性调节成体干细胞增殖与分化的活性成分是干细胞药物的研究热点。
发明内容
本申请提供了用于调节干细胞的活性成分及其应用。
根据本公开的一些实施方案，提供了一种分离的赤红球菌(Rhodococcus ruber)。
根据本公开的一些具体的实施方案，提供了一种赤红球菌，其于2019年03月22日在中国微生物菌种保藏管理委员会普通微生物中心保藏(China General Microbiological Culture Collection Center，北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所；邮政编码：100101)，保藏编号为CGMCC No.17431。该保藏满足《国际承认用于专利程序的微生物保存布达佩斯条约(Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure)》的规定。
根据本公开的一些实施方案，提供了赤红球菌及其衍生产品。所述衍生产品源自赤红球菌，包含赤红球菌的组成成分(如蛋白、核酸、脂质、细胞壁及其组成成分、碳水化合物、代谢物)。
在具体的实施方案中，提供了一种分离的赤红球菌细胞壁。
在具体的实施方案中，提供了一种分离的赤红球菌细胞壁，所述赤红球菌是指保藏号为CGMCC No.17431的株。
在具体的实施方案中，提供了一种源自赤红球菌细胞壁的产物。
在具体的实施方案中，提供了一种分离的赤红球菌细胞壁骨架。
在具体的实施方案中，提供了一种分离的赤红球菌细胞壁骨架，所述赤红球菌是指保藏号为CGMCC No.17431的株。
根据本公开的一些实施方案，提供了药物组合物，其包含根据本公开的赤红球菌的细胞壁或赤红球菌的细胞壁骨架。
根据本公开的一些实施方案，提供了一种源自赤红球菌细胞壁的产物，其包含赤红球菌经粉碎后所得的产物。
根据本公开的另一些实施方案，提供了一种源自赤红球菌细胞壁的产物，其包含赤红球菌经粉碎并经过纯化(除脂、除核酸、除蛋白)所得的产物。
根据本公开的另一些实施方案，提供了一种源自赤红球菌细胞壁的产物，其包含赤红球菌的细胞壁。
根据本公开的另一些实施方案，提供了一种源自赤红球菌细胞壁的产物，其包含赤红球菌的细胞壁骨架。
根据本公开的一些实施方案，提供了一种药物组合物或医疗装置，其包含源自赤红球菌细胞壁的产物。
根据本公开的另一些实施方案，提供了一种药物组合物或医疗装置，其包含赤红球菌经粉碎和经过纯化(除脂、和/或除核酸、和/或除蛋白质)后所得的产物。
根据本公开的另一些实施方案，提供了一种药物组合物或医疗装置，其包含赤红球菌的细胞壁。
根据本公开的另一些实施方案，提供了一种药物组合物或医疗装置，其包含赤红球菌的细胞壁骨架。
根据本公开的另一些实施方案，提供了一种药物组合物或医疗装置，其包含上述源自赤红球菌细胞壁的产物。
在具体的实施方案中，药物组合物还包含药学上可接受的赋形剂。
在一些实施方案中，药物组合物中所述源自赤红球菌细胞壁的产物为1个重量份，药学上可接受的赋形剂为50至5000个重量份(例如，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以及任意两个数值范围内的任意点值)。
在另一些实施方案中，药物组合物中赤红球菌细胞壁为1个重量份，所述药学上可接受的赋形剂为50至5000个重量份(例如，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以及任意两个数值范围内的任意点值)。
在又一些实施方案中，药物组合物中赤红球菌细胞壁骨架为1个重量份，所述药学上可接受的赋形剂为50至5000个重量份(例如，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以及任意两个数值范围内的任意点值)。
在一些实施方案中，药物组合物可以制备成液态的(液体制剂)。
在另一些实施方案中，药物组合物可以制备成固体的(干粉制剂或冻干粉制剂)。
技术人员理解，对于本公开的药物组合物而言，液体制剂和干粉制剂(或冻干粉制剂)，二者可以相互转化，差别仅在于含水量。除去液体制剂中的绝大部分或全部水，得到干粉制剂(或冻干粉制剂)。干粉制剂(或冻干粉制剂)溶解(或复溶)后得到液体制剂。
在一些实施方案中，药物或药物组合物被制备成选自以下的剂型：膏剂、霜剂、乳液、混悬剂、糊剂、凝胶剂、洗剂、酊剂、油剂、片剂、气雾剂、喷雾剂、搽剂、粉剂；其中，所述膏剂选自：软膏剂、硬膏剂、乳膏剂。
在一些实施方案中，所述药学上可接受的赋形剂涉及但不限于：填充剂、稳定剂、矫味剂、崩解剂、粘合剂、润滑剂。
在一些实施方案中，所述药学上可接受的赋形剂，例如但不限于：右旋糖酐、乳糖、微晶纤维素、海藻糖、甘氨酸、木糖醇、羧甲基纤维素钠、赤藓糖醇、明胶、硬脂酸镁、抛射剂、保湿剂、溶剂、增溶剂、乳化剂、抗氧化剂、pH调节剂、防腐剂。具体而言，非限制实例还包括：白凡士林、卡波姆、羟丙甲纤维素、甲基纤维素、羟甲基纤维素钠、壳聚糖、硫糖铝壳聚糖、聚乙烯吡咯烷酮、聚乙烯醇、玻璃酸钠、二甲醚、四氟乙烷、氢氟烷烃、甘油、丙二醇、去离子水、注射用水、蒸馏水、乙醇、十六醇、十八醇、对氨基苯甲酸、乙酰胺、异丙醇、吐温、聚氧乙基氢化蓖麻油、硬脂酸、单硬脂酸甘油酯、三聚甘油单硬脂酸酯、脂肪酸蔗糖酯、蔗糖酯、乙酸异丁酸蔗糖糖酯、山梨醇酐三硬脂酸酯、肉豆蔻酸异丙酯、胆固醇、角鲨烯、角鲨烷、正丁醇、乙二醇、乙醇、丙二醇、聚甘油酯、亚硫酸盐、半胱氨酸、二叔丁基羟基甲苯、山梨酸钾、磷酸缓冲溶液、三乙醇胺、氢氧化钠、乙二胺、月桂胺、碳酸氢钠、盐酸、尼泊金类、硫柳汞、氯甲酚、三氯叔丁醇、苯甲酸及其钠盐。
根据本公开的一些实施方案，提供一种源自赤红球菌细胞壁的产物的制备方法，其包括以下步骤或由以下步骤组成：
1)提供赤红球菌；
2)任选，培养所述赤红球菌；
3)任选，收集经培养的赤红球菌；
4)粉碎所述经培养的赤红球菌，得到粉碎产物；
5.1)任选，对所述的粉碎产物进行去除脂质的操作；
5.2)任选，对所述的粉碎产物进行去除核酸的操作；
5.3)任选，对所述的粉碎产物进行去除蛋白质的操作；
5.4)得到纯化产物；
6)任选地，除去所述纯化产物中的水，优选通过冷冻干燥除去所述纯化产物中的水；
7)任选地，进行分装；
8)收获所述源自赤红球菌细胞壁的产物；
其中，步骤5.1)、5.2)、5.3)可互换顺序或可并行；步骤6)和步骤7)可互换顺序。
任选地，步骤5)中还可以包含(例如用非离子型表面活性剂)去除细胞膜的步骤。
赤红球菌的培养不限于具体的培养基和培养参数，技术人员可以采用公知的适当方式进行培养，可以根据制备规模采用培养皿、培养瓶、发酵罐。
对于赤红球菌的粉碎，其目的在于去除细胞内的物质，因此可以采用超声破碎、溶菌酶等技术。技术人员理解，任何适用于破碎革兰氏阳性菌的已知或未来方法，均适用于本公开技术方案。
技术人员有能力根据活性成分(细胞壁及其组成成分)的后续应用(例如口服、注射、外敷等)，来调整培养、破碎、分离、收集、除杂质、分装的具体参数和设备，以免制备步骤中引入影响后续应用的因素。
在一些实施方案中，利用有机溶剂去除破碎产物中的脂质。在一些实施方案中，利用核酸酶去除破碎产物中的DNA和RNA。在一些实施方案中，利用水解酶降解破碎产物中的蛋白质。在一些实施方案中，利用表面活性剂去除破碎产物中的细胞膜。
在一些实施方案中，粉碎的平均粒度为10nm至1000nm；可以提及10、20、30、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190nm±10nm，以及上述任意两个数值之间的范围。粒度的测试方法有很多(胡松青等人，现代颗粒粒度测量技术，现代化工，2002年22：1)。
在一些具体的实施方案中，粉碎的平均粒度为10nm至800nm。
在另一些具体的实施方案中，粉碎的平均粒度为10nm至500nm。
在一些实施方案中，所述分装是指分装至容器或置于固体支持物上。所述容器选自：瓶、管、包、袋、板、安瓿、注射装置、铝膜包装、敷料、胶囊。
例如，在具体的实施方案中，所述分装是指分装至瓶/安瓿中。临用前，向瓶/安瓿中添加溶剂。
根据本公开的一些实施方案，提供了一种源自赤红球菌细胞壁的产物，其是通过根据本公开的方法制备所得。
根据本公开的一些实施方案，提供了一种药物组合物或医疗装置，其包含根据本公开的方法制备所得的源自赤红球菌细胞壁的产物。
根据本公开的一些实施方案，提供了一种分离的赤红球菌细胞壁，其用于调 节成体干细胞。所述调节是指选自以下的一项或其组合：促进干细胞的增殖、促进干细胞的生长、促进干细胞的分化、促进干细胞的迁移、提高干细胞的存活率；所述干细胞选自：成体干细胞、iPSC、间充质干细胞。
在一些实施方案中，所述间充质干细胞选自：骨髓间充质干细胞、脂肪间充质干细胞、滑膜间充质干细胞、脐带间充质干细胞、脐带血间充质干细胞、胎盘间充质干细胞、羊膜间充质干细胞、肝脏间充质干细胞、肌肉间充质干细胞、肺间充质干细胞、胰腺间充质干细胞、牙髓间充质干细胞。
根据本公开的一些实施方案，提供了一种源自赤红球菌细胞壁的产物，其用于调节成体干细胞。
根据本公开的一些实施方案，提供了一种药物组合物或医疗装置，其用于调节成体干细胞。
根据本公开的一些实施方案，提供根据本公开的赤红球菌细胞壁在调节成体干细胞中的用途。
还提供了本公开的赤红球菌细胞壁在制备用于调节成体干细胞的药物/医疗装置中的用途。
根据本公开的一些实施方案，提供根据本公开的源自赤红球菌细胞壁的产物在调节成体干细胞中的用途；还提供了本公开的源自赤红球菌细胞壁的产物在制备用于调节成体干细胞的药物/医疗装置中的用途。
根据本公开的一些实施方案，提供根据本公开的药物组合物在调节成体干细胞中的用途；还提供了本公开的药物组合物在制备用于调节成体干细胞的药物/医疗装置中的用途。
根据本公开的一些实施方案，提供选自以下的任一项在制备药物(或医疗装置)中的用途：
-根据本公开的赤红球菌、
-根据本公开的分离的赤红球菌细胞壁、
-根据本公开的源自赤红球菌细胞壁的产物、
-根据本公开的药物组合物。
在一些具体的实施方案中，所述药物用于调节成体干细胞。
在一些具体的实施方案中，所述医疗装置(如敷料、贴片、绷带、膜、贴片等)用于调节成体干细胞。
根据本公开的一些实施方案，还提供了一种调节成体干细胞的方法，包括使受试者接触治疗有效量(或预防有效量)的选自以下的任一项：
-根据本公开的分离的赤红球菌细胞壁、
-根据本公开的源自赤红球菌细胞壁的产物、
-根据本公开的药物组合物、
-根据本公开的医疗装置。
在一些具体的实施方案中，提供一种调节干细胞的方法，包括使干细胞接触源自赤红球菌细胞壁的产物的步骤。
在一些具体的实施方案中，干细胞数目/源自赤红球菌细胞壁的产物之比为：1-1000个干细胞/1ng源自赤红球菌细胞壁的产物；优选5-50个干细胞/1ng源自赤红球菌细胞壁的产物。例如，不限于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个干细胞/1ng源自赤红球菌细胞壁的产物，及其之间的任意范围。
根据一些实施方案，提供了一种促进创面愈合的方法，包括步骤：使受试者的创面接触治疗有效量的间充质干细胞和源自赤红球菌细胞壁的产物。
在一些实施方案中，间充质干细胞数目/源自赤红球菌细胞壁的产物之比为：1-100个干细胞/1ng源自赤红球菌细胞壁的产物；优选5-50个干细胞/1ng源自赤红球菌细胞壁的产物。在一些具体的实施方案中，所述创面是和糖尿病相关的创面。
在一些具体的实施方案中，针对病灶的面积和深度的不同，采用药物(或医疗装置)施用至病灶。例如，但不限于：
-用包含赤红球菌细胞壁骨架的药物涂抹；或者
-用浸有赤红球菌细胞壁骨架的贴片(膜或纱布)覆盖在病灶；或
-包含赤红球菌细胞壁骨架的冻干粉在病灶直接施用；或
-在病灶上施用包含赤红球菌细胞壁骨架的膏体/霜。
在一些具体的实施方案中，接触的周期为：持续2天至2个月或更长。具体而言，例如2、4、6、8、10、15、20、25、30、35、40、45、50、55、60天；再比如，可以提及1周、2周、3周、4周、5周、6周、7周、8周、9周、10周、11周、12周、13周、14周、15周、16周、17周、18周或更长。在具体的实施方案中，向受试者施用3-4周活性成分。
在一些实施方案中，按照以下频率进行施用：一天施用1至3次、两天施用1至6次、三天施用1至9次、一周施用1至14次、一月施用1至60次。在一些实施方案中，一天施用两次，或一天施用一次，或两天施用一次。
每次施用量视受试者具体情况的不同采用不同的剂量，通常为1μg至1000μg/单位剂量/每次施用；具体而言，例如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、200μg/单位剂量/每次、以及前述任意两个数值之间的范围。
在一些具体的实施方案中，接触是通过以下方式实现的，例如但不限于：口服、粘膜、经皮、透皮、腹腔、穿刺、喷鼻、滴眼、栓剂、舌下。
在一些具体的实施方案中，受试者是人以外的动物，例如用于农场动物、宠物、工作动物、观赏动物、生产动物。
在具体的实施方案中，受试者是人。
在一些具体的实施方案中，受试者疑似患有、确诊患有、已经患有、或易感于目标疾病或其症状。
在一些实施方案中，提供一种细胞培养介质，其包含选自以下的一项或组合：
-根据本公开的赤红球菌、
-根据本公开的分离的赤红球菌细胞壁、
-根据本公开的源自赤红球菌细胞壁的产物、
-根据本公开的药物组合物。
在一些具体的实施方案中，提供一种细胞培养介质，其还包含适用于培养干细胞(尤其是间充质干细胞)的本领域公知的其他成分。当应用于人时，为了提供更安全的细胞，在培养过程中建议不含异源动物成分；例如无血清培养介质。
在一些具体的实施方案中，技术人员可以根据需要(比如，维持干性，或促进分化)向细胞培养介质加入细胞因子，例如FGF、PDGF、TGF-β、HGF、EGF、CTGF、VEGF、胰岛素、胰岛素样生长因子中的一种或组合。
在一些具体的实施方案中，FGF的含量(以终浓度计)优选为0.1至100ng/ml。FGF是指成纤维细胞生长因子家族中的生长因子，优选为FGF-1、FGF-2(bFGF)。
在一些具体的实施方案中，PDGF的含量(以终浓度计)优选为0.5至100ng/ml。PDGF是指血小板衍生生长因子家族中的生长因子，优选为PDGF-BB或PDGF-AB。
在一些具体的实施方案中，TGF-β的含量(以终浓度计)优选为0.5至100ng/ml。TGF-β是指转化生长因子-β家族中的生长因子，优选为TGF-β3。
在一些具体的实施方案中，HGF的含量(以终浓度计)优选为0.1至50ng/ml。
在一些具体的实施方案中，EGF的含量(以终浓度计)优选为0.5至200ng/ml。
在一些具体的实施方案中，提供一种细胞培养介质，其还包含至少1种磷脂、和/或至少1种脂肪酸。
磷脂可列举，例如：磷脂酸、溶血磷脂酸、磷脂酰环己六醇、磷脂酰丝氨酸、磷脂酰乙醇胺、磷脂酰胆碱及磷脂酰甘油。磷脂的总含量(以终浓度计)优选为0.1至30μg/ml。
脂肪酸，可列举例如：亚油酸、油酸、亚麻酸、花生四烯酸、十四烷酸、棕榈油酸、棕榈酸及硬脂酸等。脂肪酸的总含量优选为介质的1/1000至1/10。
在一些具体的实施方案中，提供一种细胞培养介质，其还包含胆固醇。
在一些具体的实施方案中，提供一种细胞培养介质，其还包含抗坏血酸。
在一些具体的实施方案中，提供一种细胞培养介质，其还包含抗氧化剂。抗氧化剂可列举，例如：DL-α-生育酚醋酸酯(维生素E)。
在一些具体的实施方案中，提供一种细胞培养介质，其还包含运铁蛋白。
在一些具体的实施方案中，提供一种细胞培养介质，其还包含硒酸盐。
在一些具体的实施方案中，提供一种细胞培养介质，其还包含维持细胞所需的氨基酸、核苷酸、微量元素。
在一个具体示例中，向已知的市售间充质干细胞培养介质中加入本申请的组合物；市售间充质干细胞培养介质例如选自：MesenPRO RS TM、
Figure PCTCN2021072871-appb-000001
MSC SFM、
Figure PCTCN2021072871-appb-000002
MSC SFM XenoFree、
Figure PCTCN2021072871-appb-000003
人脂肪源性干细胞介质。
根据一些实施方案，提供了一种治疗组合物，其包含：
-间充质干细胞、和
-源自赤红球菌细胞壁的产物。
在一些治疗组合物的实施方案中，间充质干细胞数目/源自赤红球菌细胞壁的产物之比为：1-100个干细胞/1ng源自赤红球菌细胞壁的产物；优选5-50个干细胞/1ng源自赤红球菌细胞壁的产物。
在一些治疗组合物的实施方案中，间充质干细胞选自：骨髓间充质干细胞、脂肪间充质干细胞、滑膜间充质干细胞、脐带间充质干细胞、脐带血间充质干细胞、胎盘间充质干细胞、羊膜间充质干细胞、肝脏间充质干细胞、肌肉间充质干细胞、肺间充质干细胞、胰腺间充质干细胞、牙髓间充质干细胞。
根据一些实施方案，提供了一种改善干细胞凋亡的方法，包括步骤：使干细胞接触有效量的上述源自赤红球菌细胞壁的产物。
根据一些实施方案，提供了一种提高干细胞存活率的方法，包括步骤：使干细胞接触有效量的上述源自赤红球菌细胞壁的产物。
在本申请的上下文中，药物或医疗装置中的唯一治疗性(或预防性)活性成分是源自赤红球菌细胞壁的产物，尤其是包含赤红球菌组成成分(如蛋白、核酸、脂质、细胞壁及其组成成分、碳水化合物、代谢物)的产品，具体而言包含赤红球菌细胞壁(更优选赤红球菌细胞壁骨架或其组成)的产品。
附图说明
图1：以10μg/ml本申请组合物1作用于Ad-MSC 24h、48h、72h、96h后用CCK-8检测Ad-MSC在450nm波长下，酶联免疫检测仪测定吸光度值(OD)。
图2：以10μg/ml、50μl本申请组合物1作用于Ad-MSCs，72h、96h后用EDU试剂盒进行EdU掺入分析，统计分析各组细胞增值率，实验重复3次，数据用误差条形图表示平均值±SD(*P<0.05，**P<0.01，vs.对照)。
图3A至图3B：流式细胞仪检测48h、72h空白对照组、高糖组、本申请组合物处理组的干细胞凋亡率。实验重复3次，数据用误差条形图表示平均值±SD(**P<0.01，vs.高糖)。
图4A、图4B：细胞中加入高糖诱导细胞凋亡，进行Western印记实验，检测c-半胱天冬蛋白酶-3、Bax的蛋白表达水平。实验重复3次，数据用误差条形图表示平均值±SD(*P<0.05，**P<0.01，vs高糖)。
图5A：细胞接种于糖尿病小鼠创面后，根据第0、2、7天荧光染料CM-Dil标记的细胞观察Ad-MSC在各组中存活率。统计分析CM-Dil标记细胞的荧光通量结果。
图5B：测量和计算各组的创面愈合率(*P<0.05，**<0.01，vs空白)。
具体实施方式
“分离”是指本公开的赤红球菌脱离其原始生长环境。
技术人员知晓，革兰氏阳性菌和革兰氏阴性菌的细胞壁结构不同。具体而言，革兰氏阳性菌的细胞壁较厚(通常20nm至80nm)，含有约90％的肽聚糖和约10％的磷壁酸(一种由醇和磷酸分子形成的聚合物，通常以糖酯或氨基酸酯的形式存在)。肽聚糖层致密，甚至多达20层。然而，革兰氏阴性菌的细胞壁比革兰氏阳性菌的细胞壁要薄很多，结构较复杂，分为外膜(outer membrane)和肽聚糖层(通常2nm至3nm)。
肽聚糖层是细菌细胞壁中特有成分，是一种杂多糖的衍生物。每一个肽聚糖的单体包括3部分：糖单元(例如，至少两种糖分子通过糖苷键连接起来，构成肽聚糖的框架性结构)、肽尾(由若干氨基酸连接成的短肽链，其连接在N-乙酰胞壁酸分子上)、和肽桥(将相邻“肽尾”交联形成高强度的网状结构)。不同细菌的肽桥、肽尾、交联方式是不同的。
分离的赤红球菌细胞壁
在本公开中，“分离的赤红球菌细胞壁”既可以理解为完整的细胞壁，也可以理解为不完整的细胞壁(例如，破碎的、或部分降解的)。在本公开的教导下，技术人员将理解，显示出所需活性的成分来自赤红球菌的细胞壁(例如，是细胞壁本身或其组成)。因此，在临床应用中允许采用完整的细胞壁、经破碎的细胞壁、细胞壁的不完全降解产物、细胞壁的组成成分、细胞壁的提取物等各种形式，这些都包含在本公开范畴之内。
细胞壁骨架
构成细胞壁主体结构的组成成分；但不能理解为仅仅表示细胞壁当中的交联网状实体，技术人员理解不排除交联网状实体上所吸附、结合、携带的其他细胞壁成分。
赤红球菌
本公开实施方案中所用的赤红球菌是指红球菌属(Rhodococcus)的赤红球菌种(Rhodococcus ruber)，不限于特定的细胞株。
非限制性示例包括TOY7株(南京农业大学农业环境微生物菌种保藏中心)、CGMCCNo.4795、DSM43338、CCTCC No.2012035、CGMCC No.16640、CGMCC No.17431。
赤红球菌的鉴定
根据已知的或未来的微生物鉴定技术，技术人员可以对一株细菌进行分类学鉴定，例如可用的鉴定技术包括形态学、生理生化特征、16S rRNA等。技术人员理解，随着科技的发展，鉴定技术涉及不同的手段，在较早的时期主要采用形态学和生化鉴定方式，但是这种方法的可靠程度不高。测序技术出现后，技术人员可以利用更为可信的方式鉴定菌株。例如，当16S rRNA的DNA序列被鉴定为具有97％(含)以上相似性时，判定两个菌属于相同的种(华苟根等人，红球菌属的分类及应用研究进展，微生物学通报，2003：30(4))。就赤红球菌而言，将保藏在国际(或国家级)菌种保藏单位中的已知菌株作为模式菌株，并与其进行比对。
剂型
本公开的药物或药物组合物或活性成分或产品，可以体现为但不限于以下形式：软膏剂、乳膏剂、硬膏剂、凝胶剂、洗剂、酊剂、搽剂、油剂、糊剂、冻干粉、气雾剂、栓剂、贴片、悬液、口服液、口含片、护肤品(洁面乳、化妆水、精华、乳液、面霜、面膜)。
制剂单元
本公开的药物或药物组合物或活性成分或产品，可以制备成单位制剂(单元制剂)的形式。
在一些实施方案中，所述药物(或制剂、或治疗剂、或医疗装置)中的单位剂量含有：
-1μg至1000μg所述的源自赤红球菌细胞壁的产物；或
-1μg至1000μg所述的赤红球菌细胞壁；或
-1μg至1000μg所述的赤红球菌细胞壁骨架。
单位剂量的具体示例是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、500mg±10％、以及上述任意两个数值之间的范围。
“施用”、“给予”、“提供给”、“处理”当应用于动物、人、细胞、组织、器官或生物样品时，是指药物或医疗装置与动物、人、细胞、组织、器官或生物样品接触。
“治疗”意指给予受试者内用或外用药物(治疗剂、活性成分或组合物)(如，根据本公开的赤红球菌细胞壁或其药物组合物)或医疗装置，在被治疗的受试者(或群体)中缓解(减轻、延迟、改善、治愈)一种或多种疾病症状，以至于达到临床可测量的程度，其中所述的受试者已经患有、疑似患有或易感于一种或多种疾病或其症状。
有效缓解任何疾病症状的药物(治疗剂、活性成分或组合物)的量称为治疗有效量。可根据多种因素变化，例如受试者的疾病状态、年龄和体重。应当理解，在缓解单个受试者的目标疾病或其症状时，药物(治疗剂、活性成分或组合物) 可能无效，但是根据本领域已知的任何统计学检验方法(如Student T检验、卡方检验、依据Mann和Whitney的U检验)确定，药物(治疗剂、活性成分或组合物)在统计学意义上对目标疾病或其症状是有效的。
“任选”意味着其随后所描述的事项可以发生，但不必须发生；需要视情况而定。例如，“任选地，进行分装”意味着允许对产品进行分装，但是不是必须进行分装；分装与否不影响技术效果的实现。
“一个”、“一”、“单个”、“该”，如没有明确说明，也包括复数形式。
以下结合实施例、制备例和测试例，进一步描述本公开。但这些实施例、制备例和测试例并非限制着本公开的范围。当未注明具体条件时，按照常规条件、按照原料供应商所建议的条件操作。未注明具体来源的试剂，为市场购买的常规试剂。
实施例
实施例1.菌株来源
以下实施例所用赤红球菌为CGMCC No.17431株，其于2019年03月22日保藏在北京市朝阳区北辰西路1号院3号(Institute of Microbiology Chinese Academy of Sciences，No.1 West Beichen Road，Chaoyang District，Beijing China)，中国微生物菌种保藏管理委员会普通微生物中心(China General Microbiological Culture Collection Center，CGMCC)。
技术人员尤其理解，虽然以下具体示例采用了特定的细胞株，但是技术效果的实现不限于该特定的细胞株，任何属于红球菌属赤红球菌种的(Rhodococcus ruber)物种均适用。
实施例2.菌株鉴定
1.菌落形态特征的裸眼观测
在甘油琼脂培养基上，30-37℃(具体为32-35℃)培养12至72(具体为36至60，例如40-50)小时，可见：
-菌落隆起；
-呈桔红色(受光线、培养基颜色等影响，则略有差别)；
-表面干燥皱起、稍显光泽(随培养条件差别，则略有差别)；
-轻触易碎；
-菌落大小约1mm至2mm(随培养条件差别，则略有差别)。
2.显微镜观察
-菌体呈分枝状，有横隔膜，形成菌丝体(随培养条件差别，则略有差别)；
-菌丝分裂形成规则的短粗细胞(随培养条件差别，则略有差别)；
-培养4至5天时，菌体成短杆状或球形(随培养条件差别，则略有差别)。
3.染色性
革兰氏染色阳性。
4.生化反应
取甘油琼脂斜面培养基上，30-37℃(具体为32-35℃)培养12至72(具体为36至60，例如40-50)小时培养。然后，对培养物进行以下各项测试。
4.1碳水化合物产酸
表1.产酸测试
Figure PCTCN2021072871-appb-000004
4.2酶活性测定(API ZYM)
表2.酶活性测定
Figure PCTCN2021072871-appb-000005
4.3硝酸盐还原反应阳性、接触酶阳性、酪氨基酸酶阳性、淀粉酶阴性、氧化酶阴性、明胶液化阴性。
4.4唯一碳源
表3.碳源
Figure PCTCN2021072871-appb-000006
4.5. 16S rRNA鉴定
对工作种子管中分离的15株菌和原始种子管中分离的10株不同菌株，进行基因组提取、16Sr RNA扩增并测序。总计25个菌株的16SrRNA基因同一性为100％。
同时，基于Kimura2-参数算法构建的neighbor-joining菌株进化树，结果显示菌株归属为Rhodococcus ruber。
制备例
制备例1.培养方法
1.可以通过常规的微生物生产方法，培养赤红球菌。
2.培养方式既可为固体培养，也可为液体培养。
3.对培养基中的营养源并无特殊的规定，可使培养基中含有通常用于微生物培养的碳源、氮源及其它营养源。
-碳源是赤红球菌可以利用的任何碳源，例如果糖、葡萄糖等。
-氮源可为肉膏、蛋白胨、铵盐、硝酸盐以及其它有机或无机含氮化合物。
-其它营养源则可适当添加一些无机盐类，例如NaCl、磷酸盐类。
4.对培养条件(温度、时间等)并无严格的限制，技术人员可以根据初步的小规模的中试数据，自行选择使其产量最高的条件。
5.作为一个示例，采用以下培养条件发酵赤红球菌：
(1)培养基组成包含：
蛋白胨、牛肉膏、氯化钠、磷酸盐、甘油(以及，任选琼脂，当固体培养时)。
(2)培养的方法参数：
工作菌种复苏后，转移至固体培养介质上维持3-5天，再转移至液体培养(30-37℃，维持3-5天)，可以采用补料分批的半连续模式，也可以采用分批模式。培养期间监控pH、细菌密度、溶解氧、碳源消耗。
制备例2.菌体破碎
收集制备例1所得到的菌，对细胞进行粉碎(例如但不限于通过超声波破碎)。也允许采用本领域任何适当的公知方法对菌体进行破碎，例如CN101250490A或CN101323865A。
显微镜下检查粉碎的情况，每个视野有形菌不得超过5个，检查若干(10至30个)视野均达到此标准为合格。
制备例3.除去核酸、除去脂质、除去杂蛋白、除去细胞膜
1.除核酸：
将破碎上清液进行离心，获得的沉淀物中加入DNA酶和RNA酶，按照酶的供应商建议的操作去除核酸。
2.除蛋白质：
沉淀物加入常见的蛋白酶(例如胰蛋白酶)，按照酶的供应商建议的操作去除蛋白质。
3.除脂质：
沉淀物中加入有机试剂(如但不限于丙酮、乙醚、乙醇中的一种或组合)，按照本领域常规操作去除脂质。
4.除细胞膜：
沉淀物中加入TritonX-100，按照本领域常规操作，离心收集沉淀物，用PBS漂洗。
应当理解，上述除去杂质的步骤之间，技术人员可以调整先后顺序，使得步骤之间兼容。
去除非细胞壁成分后，将沉淀物复溶于注射用水，待用。任选地，可以在115℃下灭菌20-30分钟，作为细胞壁骨架的原液(主要包含细胞壁骨架及其组成成分)。
制备例4.药物组合物的制备方法
1.液体组合物
向制备例3所得产物中加入赋形剂(如右旋糖酐40、甘露醇或海藻糖)。灌装后，即为药物组合物。
表4.药物组合物可以配制成多种形式
Figure PCTCN2021072871-appb-000007
2.干粉组合物
将第1项的药物组合物冻干，制得冻干粉(分别编号为冻干粉组合物1至7)。
3.质量检验(以冻干粉组合物1为例)
表5.质量检验项目
Figure PCTCN2021072871-appb-000008
测试例
材料和方法
1.人Ad-MSC的分离培养和传代
从受试者的吸脂抽吸物(年龄范围25-35岁)获得脂肪组织样品并分离培养Ad-MSC。受试者来自徐州医科大学附属医院整形外科手术患者，并经伦理委员会批准及患者知情同意。
将获得的新鲜脂肪组织抽取液经0.25％胰酶-EDTA消化、过滤、离心保留细胞沉淀层，加入含10％FBS胎牛血清(Gibco)和1％青/链霉素的DMEM(Invitrogen)培养基，将细胞培养皿置于37℃、5％CO 2培养箱培养。此后每2-3天PBS洗涤后换培养基一次，待细胞生长至80％传代。
2.细胞活性测定
取对数生长期Ad-MSC，以4×10 3/孔接种于96孔板；DMEM+10％胎牛血清+1％青霉素/链霉素共培养；用PBS液将本申请的组合物(组合物1)溶解稀释至10μg/ml。待Ad-MSC贴壁后，各待测物，将实验分为对照、25μl、50μl、75μl四组。分别在24h、48h、72h、96h后每孔加CCK-8试剂10μL，继续孵育2h后在450nm波长下，酶联免疫检测仪测定各孔的吸光度。以培养时间为横轴，细胞数(吸光度)为纵轴，描绘细胞生长曲线。
3.EdU掺入实验
使用Cell-Light EdU 567体外成像试剂盒(RiboBio)进行EdU掺入分析。取对数生长期Ad-MSC，以4×10 3/孔接种于96孔板；10μg/ml、50μl本申请组合物作用于Ad-MSC，72h、96h后每孔加入EDU培养基100μl孵育两小时，PBS清洗1-2次。每孔加入4％多聚甲醛固定液，室温孵育30min。然后添加2mg/ml甘氨酸溶液，摇床5分钟。PBS洗涤后，加入渗透剂，摇床10min，PBS清洗。加入Apollo染色反应液，室温避光温育30分钟。弃染色反应溶液，加入渗透剂(0.5％ TritonX-100的PBS)摇床2-3次，每次10分钟，弃渗透剂。再次用PBS洗涤，加入Hoechest 33342反应液，避光，室温下孵育30分钟，弃反应液。PBS洗涤1-3次后，通过荧光显微镜观察阳性细胞。
4.细胞凋亡检测
4.1流式细胞法：
Ad-MSC生长融合至80％时，1×10 4/孔密度接种于6孔板。将实验分为四组，细胞贴壁后，各实验组中加入50％庶糖100μl诱导Ad-MSC细胞凋亡，其中两个实验组再分别加入10μg/ml本申请组合物100μl、250μl；48h、72h后收集各组细胞上清于流式管中，贴壁细胞用不含EDTA的胰酶消化收集到同组流式管中，2000rpm离心5min；PBS缓冲液洗涤两次，震荡混匀；每管依次加入结合缓冲液(Annexin V-FITC细胞凋亡检测试剂盒，上海碧云天生物技术有限公司)500μl、FITC5μl、PI5μl，震荡均匀，4℃避光孵育5-15min；流式细胞仪检测(BD Biosciences)分析结果。
4.2 TUNEL法：
使用75％乙醇浸泡消毒18mm×18mm盖玻片数片，使用时取盖玻片置于6孔板内，PBS缓冲液反复冲洗数遍至乙醇残液完全去除；将Ad-MSC培养于六孔板，每孔细胞密度为1×10 4个，实验分组同流式细胞法；PBS缓冲液洗涤细胞3次，4％多聚甲醛室温下固定30min；PBS缓冲液洗涤细胞3次，用0.1％TritionX-100在2℃-8℃条件下孵育10min；PBS缓冲液洗涤细胞3次，配制500μl TUNEL反应液(TUNEL凋亡试剂盒，罗氏)，将50μl酶溶液和450μl标记溶液混合为试剂A；阴性对照组加入50μl试剂A，置于37℃培养箱避光孵育60min；阳性对照组加入DNA酶I，室温孵育10min；PBS缓冲液洗涤细胞3次，加入TUNEL反应混合液50μl/孔，37℃培养箱中避光孵育60min；PBS缓冲液洗涤细胞3次，加入50μl/孔DAPI染液，室温下孵育3min；样本置于荧光显微镜下拍照分析，检测光波长范围570-620nm(最大波长580nm)。
5. Western印记分析
取出处理完毕的细胞，每皿加入300μl细胞裂解混合液(冰上溶解PIPA细胞裂解液，以100：1比例加入PMSF)，然后置于冰上充分裂解后，刮取贴壁细胞，使用Centrifuge-5810R冷冻高速离心机4℃条件下，12000rpm离心20min；收集上清。
在SDS-聚丙烯酰胺凝胶上分离细胞提取物，然后将蛋白质转移到硝酸纤维素膜上，并与以下抗体一起孵育；兔抗人半胱天冬蛋白酶-3单克隆抗体(1：400；美国CST)，兔抗人Bax单克隆抗体(1：400；美国CST)一抗孵育结束后，洗涤5min×3次，然后加入稀释的二抗(1：10000)室温避光孵育2h；弃二抗，洗涤5min×3次，分别吸取ECL发光液A液和B液等量混匀，配制成ECL工作液。在膜片上均匀滴加ECL工作液，放入TANON凝胶成像仪中进行曝光显影，拍照， ImageJ软件分析。
6.糖尿病创面动物模型的建立
实验动物为4周大的BALB/c无胸腺裸鼠，SPF级。所有动物研究均经动物保护和使用委员会批准。实验小鼠在SPF级动物房饲养。造模前小鼠禁食12h，称体重并记录，以150mg/kg的剂量通过腹膜内注射2％的STZ(Sigma)诱导糖尿病小鼠。
注射后第7天测血糖，通过尾静脉采血，用血糖仪测定小鼠血糖浓度并记录。从第7天开始小鼠血糖浓度大于16.7mmol/L，并且出现典型的糖尿病“多饮、多食、多尿和体重减轻”症状，视为造模成功。
将实验动物分为3组(每组5只)：
-空白对照组、
-Ad-MSC组、
-本申请组合物+Ad-MSC组。
糖尿病小鼠模型建立成功后，麻醉小鼠，制备直径1.5cm的创面，将预处理的Ad-MSC以多点皮内注射方式注入各组小鼠皮肤，每个创面取六点，每点注射细胞0.1ml。表面敷于无菌纱布，继续饲养观察，使用LB983活体成像系统观察创面细胞的存活情况、和小鼠皮肤愈合情况。
7. CM-Dil活细胞染色剂染色
注射前用CM-Dil标记Ad-MSC。按照供应商推荐的方法，加入CM-Dil活细胞染色剂，孵育30分钟，离心后弃上清，PBS缓冲液洗涤3遍，最后加入适量PBS混匀，标记好的Ad-MSC置于冰盒备用。
8.组织免疫荧光染色
将组织石蜡切片脱蜡，3％H 2O 2室温孵育5-10min，以消除内源性过氧化物酶的活性。切片用蒸馏水冲洗，PBS浸泡2次，5min/次，10％正常山羊血清(PBS稀释)室温封闭10min，弃去封闭液，勿洗。滴加CD31一抗(稀释比例1:300)工作液，过夜。切片使用PBS冲洗3次，5min/次。滴加适量生物素标记荧光二抗工作液(稀释比例1:400)(避光)，37避光孵育1h后PBS冲洗3次，5min/次，适量滴加DAPI染液，室温避光孵育3min，PBS冲洗3次，5min/次。最后每张切片滴加防荧光淬灭封片液，盖上盖玻片后，固定，避光保存。
9.统计分析
采用SPSS软件(SPSS16.0)进行统计分析，实验结果用平均值±SD进行表示，两组间比较采用独立样本t检验，多组均数比较采用单因素方差分析(One-way ANOVA)。α＝0.05为显著性水准，P<0.05被认为差异有统计学意义。
测试例1.人Ad-MSC的形态学、表面标记和诱导分化能力鉴定
胶原酶消化法，从脂肪组织抽取液中提取得到的原代Ad-MSC种植于细胞培 养皿中，细胞生长融合至80％传代，传代后的细胞增殖速度明显增快，细胞形态均匀一致，呈长梭形。P3代生长良好的Ad-MSC分别用于脂诱导分化和成骨诱导分化。2周后，油红染色，显微镜下观察，显示细胞内存在明显的大小不一的红色脂滴；茜红素S染液染色，可见明显的红色钙结节沉积。这表明分离提取到的细胞具有干细胞分化潜能的特性。
采用流式细胞仪检测6种不同的细胞表面标记物。结果显示：CD105、CD90和CD44呈阳性；CD31、CD34和CD106呈阴性。符合Ad-MSC免疫表型的特征。
测试例2.本申请组合物对Ad-MSC活性及增殖的影响
为了检测本申请组合物对Ad-MSC活性的影响，分别在本申请组合物1作用于Ad-MSC后24h、48h、72h、96h，用CCK-8试剂检测干细胞在450nm波长下，酶联免疫检测仪测定吸光度值。结果表明以浓度为10μg/ml、体积50μl的本申请组合物作用于干细胞，在72h和96h时Ad-MSC活力有较明显的增强(图1)。
为了验证本申请组合物对Ad-MSC增殖的影响，以10μg/ml、50μl本申请组合物作用于干细胞。分别在72h、96h后用EDU试剂盒进行EdU掺入分析，通过荧光显微镜观察阳性细胞，结果表明本申请组合物可提高Ad-MSC的增值率(图2)。
测试例3.本申请组合物抑制高糖诱导的Ad-MSC细胞凋亡
在Ad-MSC细胞中加入高糖诱导细胞凋亡，不同浓度本申请组合物1分别处理48h、72h后用FITC-PI流式细胞凋亡检测试剂盒检测细胞凋亡情况。
结果表明，本申请组合物可抑制高糖诱导的干细胞凋亡(图3A、图3B)。
测试例4.检测凋亡标志物蛋白表达水平
细胞中加入高糖诱导细胞凋亡，不同浓度本申请组合物分别处理48h、72h提取细胞蛋白，通过进行Western印记实验，检测c-半胱天冬蛋白酶-3、Bax(两种主要的凋亡标志物)的蛋白表达水平，β-actin作为内参。结果显示本申请组合物处理后c-半胱天冬蛋白酶-3、Bax蛋白表达水平降低(图4A、图4B)。
测试例5.本申请组合物在裸鼠体内提高Ad-MSC存活率、加快创面愈合
为了进一步探究本申请组合物对Ad-MSC修复糖尿病创面愈合的影响，在糖尿病裸鼠身上建立了模拟人体皮肤创面修复机制的创面模型。
糖尿病小鼠模型建立成功后，麻醉小鼠，切取直径1.5cm的创面，将用荧光染料CM-Dil标记的预处理细胞以皮内注射方式注入处理组小鼠创面皮肤，用LB983活体成像系统检测细胞存活率。
结果显示本申请组合物处理组的Ad-MSC比单独使用Ad-MSC组有高的存活 率(图5A)。同时评估了Ad-MSC治疗糖尿病小鼠的伤口14天的愈合过程。与空白组相比，经Ad-MSC处理的小鼠创面愈合状况良好，而经本申请组合物处理组愈合情况更好，加快了创面的愈合(图5B)，创面愈合率增加(表6)。
表6.术后第14天创面愈合情况(平均值±SD，n＝3)
Figure PCTCN2021072871-appb-000009
**P<0.01，vs.空白
糖尿病难愈创面形成的原因有很多。比如，正常创面愈合过程中所需的细胞和分子信号的缺失。此外，周围神经病变、外周循环损坏、蛋白酶平衡的紊乱都是糖尿病创面难以愈合的重要因素(Rathur HM等人，The diabetic foot.ClinDermatol.2007；25(1)：109-120)。另外一方面，高糖环境下血管微环境异常，可导致细胞生长环境异常，最终使创伤局部的血管重建受损(Guo WY等人，Acceleration of diabetic wood healing by low-dose radiation is associated with peripheral mobilization of bone marrow stem cells.Radiat Res.2010；174(4)：467-479)。此外，糖尿病高糖状态下创伤组织中成纤维细胞数量的减少、糖基化蛋白增多、异常生长因子表达、炎症过程延迟、及糖基化终末产物的堆积这些都影响骨髓源性细胞的迁徙和功能(Fiorina P等人，The mobilization and effect of endogenous bone marrow progenitor cells diabetic wound healing.Cell Transplant.2010；19(11)：1369-1381)。
Ad-MSC是一种从脂肪组织提取得到的多向分化潜能干细胞(Kato Y等人，Creation and transplantation of an adipose-derived stem cell(ASC)sheetin a diabetic wound-healing model.Jove-J Vis Exp.2017；12(6)：1-10)。Ad-MSC可通过分化潜能迁徙至受损部位，用分化细胞重新修复受损皮肤，可分泌多种生长因子(Rehman J等人，Secretion of angiogenic and anti-apoptotic factors by human adipose stromal cells.Circulation.2004；109：1292-8)，加快创面血管生成，促进创面愈合(Ebrahimian TG等人，Cell therapy based on adipose tissue-derived stromal cells promotes physiological and pathological wound healing.Arterioscler Thromb Vasc Biol.2009；29(4)：503–510)。
也有学者发现，Ad-MSC可分化为成纤维细胞，不仅表现出形态相似性，而且可表达成纤维细胞表面蛋白(包括波形蛋白和纤连蛋白)的能力(Kim WS等人，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)。同时，Ad-MSC还可以直接转化为成纤维细胞和角质形成细胞进行创面修复(Unnikrishnan S等人，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)。然而，在之前研究发现多数Ad-MSC在注射到糖尿病小鼠创面中发生凋亡，导致创面愈合延迟。高糖环境下培养的Ad-MSC细胞发生凋亡，且具有时间依赖性(Li Q等人，Stromal cell-derived factor-1promotes human adipose tissue-derived stem cell survival and chronic wound healing.Exp Ther Med.2016；12：45-50)。
在本测试例中，通过CCK-8、EdU检测了本申请组合物对Ad-MSC体外活性、增殖能力。结果显示本申请组合物可提高Ad-MSC的活性及增殖能力。通过流式细胞法检测细胞凋亡情况，发现本申请组合物可抑制高糖诱导的Ad-MSC凋亡，并有时间和浓度依赖性。
在动物体内检测了本申请组合物对Ad-MSC存活率的影响，结果显示使用经本申请组合物处理后Ad-MSC比单纯应用Ad-MSC存活率更高，创面愈合速度更快。

Claims (13)

  1. 一种调节干细胞的方法，包括使干细胞接触源自赤红球菌细胞壁的产物的步骤；
    干细胞数目/源自赤红球菌细胞壁的产物之比为：1-100个干细胞/1ng源自赤红球菌细胞壁的产物；优选5-50个干细胞/1ng源自赤红球菌细胞壁的产物；
    所述调节是指选自以下的一项或其组合：促进干细胞的增殖、促进干细胞的生长、促进干细胞的分化、促进干细胞的迁移、提高干细胞的存活率；
    所述干细胞选自：成体干细胞、iPSC、间充质干细胞；
    优选地，所述干细胞是间充质干细胞；
    所述间充质干细胞选自：骨髓间充质干细胞、脂肪间充质干细胞、滑膜间充质干细胞、脐带间充质干细胞、脐带血间充质干细胞、胎盘间充质干细胞、羊膜间充质干细胞、肝脏间充质干细胞、肌肉间充质干细胞、肺间充质干细胞、胰腺间充质干细胞、牙髓间充质干细胞；
    优选地，所述间充质干细胞是脂肪间充质干细胞；
    优选地，所述成体干细胞是上皮成体干细胞。
  2. 根据权利要求1所述的方法，所述源自赤红球菌细胞壁的产物是赤红球菌细胞壁或其组成。
  3. 根据权利要求1或2所述的方法，所述源自赤红球菌细胞壁的产物是通过以下方法获得，所述方法包括步骤：
    1)提供赤红球菌；
    2)粉碎所述赤红球菌，得到粉碎产物；
    3.1)任选地，对所述的粉碎产物进行去除脂质的操作；
    3.2)任选地，对所述的粉碎产物进行去除核酸的操作；
    3.3)任选地，对所述的粉碎产物进行去除蛋白质的操作；
    3.4)得到源自赤红球菌细胞壁的产物；
    4)任选地，除去所述源自赤红球菌细胞壁的产物中的水，优选对所述源自赤红球菌细胞壁的产物进行冷冻干燥；
    5)任选地，分装；
    其中，步骤3.1)、3.2)、3.3)能够互换顺序或并行；步骤4)和步骤5)能够互换顺序。
  4. 根据权利要求1或2所述的方法，所述赤红球菌于2019年03月22日以编号CGMCC No.17431保藏在CGMCC。
  5. 源自赤红球菌(Rhodococcus ruber)细胞壁的产物在制备试剂中的用途，其中：
    所述试剂用于选自以下的一项或其组合：促进干细胞的增殖、促进干细胞的生长、促进干细胞的分化、促进干细胞的迁移、提高干细胞的存活率；
    所述干细胞选自：成体干细胞、iPSC、间充质干细胞；
    优选地，所述干细胞是间充质干细胞；
    所述间充质干细胞选自：骨髓间充质干细胞、脂肪间充质干细胞、滑膜间充质干细胞、脐带间充质干细胞、脐带血间充质干细胞、胎盘间充质干细胞、羊膜间充质干细胞、肝脏间充质干细胞、肌肉间充质干细胞、肺间充质干细胞、胰腺间充质干细胞、牙髓间充质干细胞；
    优选地，所述间充质干细胞是脂肪间充质干细胞；
    优选地，所述成体干细胞是上皮成体干细胞。
  6. 根据权利要求5所述的用途，所述源自赤红球菌细胞壁的产物是赤红球菌细胞壁或其组成。
  7. 根据权利要求5或6所述的用途，所述源自赤红球菌细胞壁的产物是通过以下方法获得，所述方法包括步骤：
    1)提供赤红球菌；
    2)粉碎所述赤红球菌，得到粉碎产物；
    3.1)任选地，对所述的粉碎产物进行去除脂质的操作；
    3.2)任选地，对所述的粉碎产物进行去除核酸的操作；
    3.3)任选地，对所述的粉碎产物进行去除蛋白质的操作；
    3.4)得到源自赤红球菌细胞壁的产物；
    4)任选地，除去所述源自赤红球菌细胞壁的产物中的水，优选对所述源自赤红球菌细胞壁的产物进行冷冻干燥；
    5)任选地，分装；
    其中，步骤3.1)、3.2)、3.3)能够互换顺序或并行；步骤4)和步骤5)能够互换顺序。
  8. 根据权利要求5或6所述的用途，所述赤红球菌于2019年03月22日以编号CGMCC No.17431保藏在CGMCC。
  9. 一种细胞培养介质，其包含源自赤红球菌细胞壁的产物；优选地，所述源自赤红球菌细胞壁的产物是赤红球菌细胞壁或其组成。
  10. 根据权利要求9所述的细胞培养介质，所述源自赤红球菌细胞壁的产物是通过以下方法获得，所述方法包括步骤：
    1)提供赤红球菌；
    2)粉碎所述赤红球菌，得到粉碎产物；
    3.1)任选地，对所述的粉碎产物进行去除脂质的操作；
    3.2)任选地，对所述的粉碎产物进行去除核酸的操作；
    3.3)任选地，对所述的粉碎产物进行去除蛋白质的操作；
    3.4)得到源自赤红球菌细胞壁的产物；
    4)任选地，除去所述源自赤红球菌细胞壁的产物中的水，优选对所述源自赤红球菌细胞壁的产物进行冷冻干燥；
    5)任选地，分装；
    其中，步骤3.1)、3.2)、3.3)能够互换顺序或并行；步骤4)和步骤5)能够互换顺序。
  11. 根据权利要求9所述的细胞培养介质，所述赤红球菌于2019年03月22日以编号CGMCC No.17431保藏在CGMCC。
  12. 一种促进创面愈合的方法，包括步骤：使受试者的创面接触治疗有效量的干细胞和源自赤红球菌细胞壁的产物；
    其中：
    所述干细胞选自：成体干细胞、间充质干细胞、iPSC；
    干细胞数目/源自赤红球菌细胞壁的产物之比为：1-100个干细胞/1ng源自赤红球菌细胞壁的产物；优选5-50个干细胞/1ng源自赤红球菌细胞壁的产物；
    所述间充质干细胞选自：骨髓间充质干细胞、脂肪间充质干细胞、滑膜间充质干细胞、脐带间充质干细胞、脐带血间充质干细胞、胎盘间充质干细胞、羊膜间充质干细胞、肝脏间充质干细胞、肌肉间充质干细胞、肺间充质干细胞、胰腺间充质干细胞、牙髓间充质干细胞；
    优选地，所述间充质干细胞是脂肪间充质干细胞；
    优选地，所述成体干细胞是上皮成体干细胞；
    优选地，所述创面是和糖尿病相关的创面。
  13. 一种药物组合物，其包含：
    -干细胞、和
    -源自赤红球菌细胞壁的产物；
    所述干细胞选自：成体干细胞、间充质干细胞、iPSC；
    干细胞数目/源自赤红球菌细胞壁的产物之比为：1-100个干细胞/1ng源自赤 红球菌细胞壁的产物；优选5-50个干细胞/1ng源自赤红球菌细胞壁的产物；
    所述间充质干细胞选自：骨髓间充质干细胞、脂肪间充质干细胞、滑膜间充质干细胞、脐带间充质干细胞、脐带血间充质干细胞、胎盘间充质干细胞、羊膜间充质干细胞、肝脏间充质干细胞、肌肉间充质干细胞、肺间充质干细胞、胰腺间充质干细胞、牙髓间充质干细胞；
    优选地，所述间充质干细胞是脂肪间充质干细胞；
    优选地，所述成体干细胞是上皮成体干细胞。
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