WO2014190866A1 - 肌肉干细胞体外培养方法及其应用 - Google Patents

肌肉干细胞体外培养方法及其应用 Download PDF

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WO2014190866A1
WO2014190866A1 PCT/CN2014/077952 CN2014077952W WO2014190866A1 WO 2014190866 A1 WO2014190866 A1 WO 2014190866A1 CN 2014077952 W CN2014077952 W CN 2014077952W WO 2014190866 A1 WO2014190866 A1 WO 2014190866A1
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muscle stem
cytokines
cells
cell
added
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PCT/CN2014/077952
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English (en)
French (fr)
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胡苹
傅鑫
肖俊
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中国科学院上海生命科学研究院
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Priority to JP2016515626A priority Critical patent/JP6378321B2/ja
Priority to US14/894,642 priority patent/US10287549B2/en
Publication of WO2014190866A1 publication Critical patent/WO2014190866A1/zh

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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0658—Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts
    • C12N5/0659—Satellite cells
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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
    • A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells
    • C12N2502/1114—T cells

Definitions

  • the present invention relates to the field of biotechnology and medicine, and more particularly to the field of cell culture and cell therapy related to muscle stem cells. Background technique
  • Muscle stem cells are responsible for the growth and regeneration of muscle tissue after birth, and muscle stem cells have no carcinogenic effects in the body. There are no ethical problems encountered when applying embryonic stem cells, and thus various muscle degenerative diseases such as muscle atrophy and myasthenia gravis. There are broad application prospects in treatment.
  • An important obstacle to the clinical application of muscle stem cells is the inability to culture muscle stem cells in vitro for long periods of time. Like most adult stem cells, muscle stem cells can only be split 2-4 times in the in vitro culture system after being isolated from the body, and can hardly be passaged.
  • the existing methods for culturing muscle stem cells are mostly derived from the method established by Dr. Bischoff in 1986, and then modified by Beauchamp et al. to form an existing culture medium supplemented with 10 ng/ml FGF in F10 basal medium. method. Under this culture condition, muscle stem cells can proliferate 3-5 times. During passage, the muscle stem cells undergo a period of severe crisis in which most cells die and the remaining cells differentiate into non-dry precursor cells. Therefore, after in vitro culture for 1-3 passages (3-12 days), the isolated muscle stem cells are all differentiated into non-dry precursor cells, and molecular markers of muscle stem cells are not detected in these precursor cells.
  • the first aspect of the present invention provides a method for in vitro culture of muscle stem cells, which comprises culturing muscle stem cells in vitro using a cell culture medium supplemented with blood cell cytokines or a conditioned medium of blood cells.
  • the in vitro culture method of the muscle stem cells of the present invention is suitable for long-term culture of muscle stem cells in vitro.
  • a second aspect of the present invention provides a muscle stem cell culture medium, which is a fine factor of a cytokine to which a blood cell is added Cell culture medium or blood cell conditioned medium.
  • the cytokine of the blood cell added to the cell culture medium to which the cytokine of the blood cell is added, and the blood cell for preparing the blood cell conditioned medium are all derived from the same animal species as the muscle stem cell to be cultured. .
  • the cytokine of the blood cell added to the cell culture medium to which the blood cell-containing cytokine is added does not include the cytokine originally contained in the serum of the animal to be used.
  • the cell culture medium to which the cytokine of the blood cell is added can be obtained by a method of adding a cytokine of blood cells to a general cell culture medium.
  • the blood cell conditioned medium is a lymphocyte conditioned medium. Further, it is B cell conditioned medium or T cell conditioned medium.
  • the cytokine of the added blood cell is selected from the plurality of cytokines: GM-CSF (granulocyte colony stimulating factor), sICAM-lC human soluble intercellular adhesion molecule 1); IFN gamma (j interferon) ), IL1C interleukin-1), IL-1 alpha receptor (interleukin 1 alpha receptor), IL1 alpha (interleukin la), IL-3 (interleukin 3), IL2 (interleukin 2), IL-10 (interleukin 10), IL- 16 (interleukin 16), IL13 (interleukin 13), IL-17 (interleukin 17), IP-10 (interferon-inducible protein 10), SCYA2 (small induced cytokine A2), MIG (interferon gamma induced mononuclear) Factor), MIP-1 alpha (macrophage inflammatory protein la), TGF-beta (transforming growth factor beta;), IL-4 (interleukin 4
  • the total concentration of the cytokine of the added blood cell is not less than 6 ng/ml, preferably 50-4500 ng / ml. .
  • the concentration of the cytokine of any added blood cell is 0.5 ng/ml or more, preferably 1 ng/liter. More preferably, it is 10 ng/ml or more, more preferably 25 ng/ml or more, and further, 50 ng/ml or more.
  • the cytokine of the added blood cell includes at least IL1, IL4, IL13, TNF alpha, IL2 and P IFN gamma o
  • the sum of the concentrations of IL1, IL4, IL13, TNF alpha, IL2 and IFN gamma is not less than 6 ng/ml. The best is 50-1250ng/ml.
  • the concentration of any one of the IL 1 , IL 4 , IL 13 , TNF alpha , IL 2 and IFN gamma is not It is less than 0.5 ng/ml, preferably 1 ng/ml or more, more preferably 10 ng/ml or more, and usually 50-500 ng/ml.
  • the cytokine or blood cell conditioned medium of the aforementioned blood cells is provided in muscle dry Use of cells in vitro culture.
  • the cytokine or blood cell conditioned medium of the blood cells promotes proliferation of muscle stem cells in vitro and keeps the expanded muscle cells in vitro dry.
  • a preparation for treating a muscle degenerative disease which comprises a muscle stem cell obtained by culturing the muscle stem cell in vitro culture method of the present invention as a main active ingredient.
  • the preparation is a muscle active stem cell cultured by the muscle stem cells of the present invention in a muscle stem cell derived from a patient's body as a main active ingredient.
  • the preparation is an injection or a surgical implant.
  • the formulations generally comprise conventional formulation excipients.
  • a method for treating a muscle degenerative disease cell of a patient comprising the steps of:
  • the obtained muscle stem cells are administered to the muscle injury site of the patient.
  • step 3 is performed by intramuscular injection of muscle stem cells into the muscle injury site of the patient by injection.
  • the expanded muscle stem cells can be removed from the culture dish by trypsinization and resuspended in physiological saline.
  • the in vitro culture and passage method of the muscle stem cells of the invention enables the muscle stem cells to be expanded and kept dry in vitro, thereby greatly increasing the number of stem cells that can be used for regenerative medicine treatment, so that a small amount of muscle tissue is provided by the patient, and the separation is performed.
  • the existing muscle stem cell culture method cannot culture muscle stem cells with differentiation potential for a long time in vitro, and cannot be passaged. After 12 days of culture, all cells differentiate into progenitor cells and lose the ability to repair muscle damage in vivo. Under the current culture conditions, muscle stem cells can only divide 3-5 times, that is, up to 32 times the number of original cells, that is, the ability to divide, so to get enough muscle stem cells for treatment, you must take very Large muscle tissue is used to isolate and purify muscle stem cells, which is not clinically feasible. In the medium of the present invention, the muscle stem cells can be propagated more than ten times, that is, each generation can be expanded to 1000 times or more of the original cells.
  • muscle stem cells can be continuously passaged and continue to maintain dryness and differentiation potential.
  • the resulting number of muscle stem cells is 2 m x 2 n times the number of isolated and purified muscle stem cells, m is the number of divisions per generation, and n is the number of passages.
  • the m value obtained by the method of the invention is at least 12, and the value of n is at least 40, that is, the number of muscle stem cells can be at least 15 times as large as 4.5 ⁇ l0 of the original isolated and purified muscle stem cells, and the muscle stem cells after expansion are maintained. Dry and complete differentiation potential to repair muscle damage in the body.
  • these stem cells After injection into mice, these stem cells can participate in the repair of muscle damage more efficiently, and can also supplement the stem cell reserve in the body, and have the ability to repair secondary damage after transplantation. This makes it possible to extract a small amount of muscle tissue (grams) by minimally invasive surgery, to isolate and purify a small amount of muscle stem cells, and then to obtain a sufficient amount of muscle stem cells for in vitro regenerative medical treatment.
  • FIG. 1 P0 generation muscle stem cells (Pax7 staining) cultured in F10 medium supplemented with cytokines.
  • FIG. 1 Control fibroblasts (Pax7 staining) cultured in F10 medium supplemented with cytokines.
  • FIG. 1 P8 generation muscle stem cells (Pax7 staining) cultured in F10 medium supplemented with cytokines.
  • FIG. 1 P22 generation muscle stem cells (Pax7 staining) cultured in F10 medium supplemented with cytokines.
  • FIG. 7 RFP-expressing muscle stem cells cultured in cytokine-added F10 medium are involved in the repair of muscle damage in vivo.
  • RFP-labeled muscle stem cells cultured in cytokine-added F10 medium can participate in the repair of muscle damage.
  • RFP-labeled muscle stem cells were injected into muscles without fluorescently labeled lesions for 7 days, and sections were observed for red cell integration, and red muscle fibers derived from injected RFP-expressing muscle stem cells were found at the lesion site.
  • Figure A shows the repair of muscle stem cells cultured by cytokine injection
  • Figure B shows the repair of muscle stem cells cultured in T cell conditioned medium for injection.
  • the present inventors have found that co-culture with blood cells can promote the proliferation of muscle stem cells, and this promotion does not depend on cell contact between blood cells and muscle stem cells.
  • the blood cell conditioned medium promotes the proliferation of muscle stem cells.
  • B cell and T cell conditioned media can promote the proliferation of muscle stem cells.
  • the muscle stem cells can be divided more than 10 times per generation and can be passaged for at least 40 generations in an in vitro culture system.
  • the cytokines secreted by T cells are isolated, and the combination of these cytokines can promote the proliferation of muscle stem cells in vitro.
  • the cytokine-treated muscle stem cells express the molecular markers that should be expressed by muscle stem cells, and their proliferative ability is significantly improved, and can be continuously passaged for more than 40 generations. Each generation of cells after passage expresses molecular markers of muscle stem cells, has strong proliferative ability, and can be efficiently differentiated into mature myotubes. More importantly, when these muscle stem cells cultured with T cell conditioned medium or cytokines are injected into mice that induce muscle damage, the injected muscle stem cells can repair muscle damage and form new muscle fibers, indicating that the present invention It is a true muscle stem cell.
  • the in vitro culture method of the muscle stem cells of the present invention is a cell culture using a cytokine to which blood cells are added Muscle stem cells are cultured in vitro on conditioned medium of blood or blood cells.
  • the key to the present invention is the improvement of the medium for the culture of muscle stem cells in vitro, and other aspects of in vitro culture of muscle stem cells are consistent with conventional cell culture.
  • Optimal culture conditions Culture at 37 ° C in a C0 2 incubator.
  • C0 2 incubator C0 2 concentration is preferably 5% (v / v).
  • the in vitro culture of the muscle stem cells is adherent culture.
  • the muscle stem cell culture medium used in the present invention is a cell culture medium or a blood cell conditioned medium to which cytokines of blood cells are added.
  • the cytokine of the blood cell added to the cell culture medium to which the cytokine of the blood cell is added and the muscle stem cell to be cultured should be derived from the same animal species.
  • the blood cells used to prepare the blood cell conditioned medium and the muscle stem cells to be cultured should also be derived from the same animal species.
  • a cell culture medium supplemented with a mouse blood cell cytokine or a mouse blood cell conditioned medium such as a cultured human muscle stem cell, a cell culture medium or a human blood cell supplemented with a human blood cell cytokine.
  • Conditioned medium, and the rest of the analogy is derived from the same animal species.
  • the species is the basic unit of taxonomy. Further, the animal species is selected from the group consisting of mammals. Further, the species is selected from the group consisting of rodents, cloven-hoofed, hoofed, rabbit-shaped, primate, and the like in mammals, such as rats, rabbits, sheep, pigs, monkeys, humans, and the like.
  • the cytokine of the blood cell added to the cell culture medium to which the blood cell-containing cytokine is added does not include the cytokine originally contained in the serum of the animal to which the medium is added. That is, when animal serum cultured cells are added, the present invention requires additional addition of cytokines of blood cells of the same species as the cultured muscle stem cells.
  • the cell culture medium to which the cytokine of the blood cell is added in the present invention refers to a cell culture medium in which a cytokine of a blood cell is additionally added in addition to a component required for conventional cell culture.
  • the components and contents of the muscle stem cell culture medium of the present invention other than the cytokine of the added blood cells are consistent with the conventional cell culture medium.
  • Conventional cell culture medium components are generally selected from the group consisting of: balanced saline, pH adjusting solution, antibiotics, animal serum, amino acids necessary for cell growth, vitamins, glucose, pH indicators, and the like.
  • the balanced brine component such as calcium chloride, ferric nitrate, magnesium sulfate, potassium chloride, sodium fluoride, sodium chloride, sodium phosphate, etc.; the pH adjusting solution such as 3.7% sodium hydrogencarbonate, HEPES solution (dihydroxyl B) Epirubicin sulfonate, sodium pyruvate, etc.; the antibiotics such as penicillin, streptomycin, etc.; commonly used animal serum mainly has bovine serum and horse serum. Vitamins such as choline chloride, folic acid, inositol, nicotinamide, calcium pantothenate, pyridoxal hydrochloride, vitamin B6, riboflavin, thiamine, and the like.
  • the cell culture medium to which the cytokine of the blood cell is added can be obtained by a method of adding a cytokine of blood cells to a general cell culture medium.
  • the universal cell culture medium may be selected from various conventional cell culture media such as DMEM, RPMI 1640, MEM, DEME/F12, F10, CD293, medium 231, medium 106.
  • the embodiment of the present invention specifically enumerates a muscle stem cell culture medium in which cytokines of various blood cells are added to the F10 medium.
  • the blood cell conditioned medium is referred to as: a cell culture medium in which blood cells are cultured.
  • the blood cells are lymphocytes.
  • the blood cells are B cells and/or T cells.
  • the blood cell conditioned medium can be isolated after culturing blood cells in a universal cell culture medium.
  • B cells were cultured in a common cell culture medium, and B cell conditioned medium was isolated, and T cells were cultured in a universal cell culture medium to obtain T cell conditioned medium.
  • the cytokine of the added blood cells is selected from the plurality of cytokines: GM-CSF, sICAM-1; IFN gamma, ILK IL-1 alpha, IL-1 alpha receptor IL-3, IL2, IL-10 , IL-16, IL13, IL-17, IP-10, SCYA2, MIG, MIP-1 alpha TGF-beta, IL-4, TRAF6, FGF, IGF, PDGF, LIF, mTOR, LPS, TLR1, IL12, IL23 , NGF, TNF alpha IL1 beta.
  • All of the above cytokines can be detected in lymphocyte conditioned medium, especially in T cell conditioned medium.
  • the cytokine of the blood cell is selected from at least six of the above cytokines; or the cytokine of the blood cell is selected from at least seven of the above cytokines; or the cell of the blood cell
  • the factor is selected from at least eight of the above cytokines; or, the cytokine of the blood cell is selected from at least nine of the above cytokines; or the cytokine of the blood cell is at least ten selected from the above cytokines Kind.
  • the total concentration of cytokines of the blood cells may not be too low in the cell culture medium or the conditioned medium of the blood cells to which the cytokines of blood cells are added. Generally it should not be less than 6 ng/ml, preferably 50-4500 ng / ml.
  • the concentration of the cytokine of any added blood cell is 0.5 ng/ml or more, preferably 1 ng/ml or more. More preferably, it is lOng/ml or more, and is generally selected in the range of 50-500 ng/ml.
  • concentration of the cytokine cannot be higher than 500 ng/ml, but that the cytokine can exert a positive effect in this concentration range, the effect of the concentration is too low, and the concentration is too high, resulting in waste.
  • the cytokines of six blood cells are closely related to the maintenance of muscle stem cell proliferation and dryness.
  • the cytokines of the six blood cells must be added, and the cytokines of the remaining blood cells may or may not be added.
  • the sum of the concentrations of IL1, IL4, IL13, TNF alpha, IL2 and IFN gamma is not less than 6 ng/ml. The best is 50-1250ng/ml.
  • the concentration of any one of the IL1, IL4, IL13, TNF alpha, IL2 and IFN gamma is 0.5 ng. Above /ml, it is preferably 1 ng/ml or more, more preferably 10 ng/ml or more, and generally 50-500 ng/ml is optional.
  • concentration of the cytokine above 500 ng/ml does not have a serious negative effect on the proliferation of muscle stem cells, but in consideration of cost factors, it is not necessary to add excessive cytokines.
  • the concentration of each of the above cytokines refers to a final concentration of a cytokine derived from a blood cell of the same species as the cultured muscle stem cell in a cell culture medium to which a cytokine of a blood cell is added or a conditioned medium of a blood cell.
  • cytokine or blood cell conditioned medium of blood cells can be used for in vitro culture of muscle stem cells, can promote proliferation of muscle stem cells in vitro and keep dry muscle cells in vitro dry.
  • the present invention also provides a preparation for treating a muscle degenerative disease, which is a preparation containing muscle stem cells obtained by the in vitro culture method of the muscle stem cells of the present invention as a main active ingredient.
  • the preparation is a muscle active stem cell derived from a muscle stem cell derived from a patient's body using the muscle stem cell in vitro culture method of the present invention as a main active ingredient.
  • the recommended dose is about 3xl0 6 th muscle stem cells / times. It can be administered in a single dose or it can be administered multiple times depending on the patient's muscle recovery.
  • a typical mode of administration is by intramuscular injection. However, the invention does not exclude other possible modes of administration.
  • the formulations generally comprise conventional formulation excipients.
  • formulation excipients include, but are not limited to, saline, buffer, dextrose, water, glycerin, ethanol, polyols, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration.
  • the preparation of the present invention is preferably prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Other possible forms of preparation can be prepared by conventional methods.
  • the formulations of the invention are preferably manufactured under sterile conditions. Furthermore, the formulations of the invention may also be used with other therapeutic agents.
  • the invention further provides a cell treatment method for a muscle degenerative disease in a patient, comprising the following steps: 1) collecting muscle stem cells of the patient;
  • the collection of the patient's muscle stem cells can be performed by minimally invasive surgery to extract a small amount of muscle samples from the patient's body, and then the muscle stem cells are isolated and purified from the muscle samples. This technique is well known to those skilled in the art.
  • the muscle stem cells after collecting the muscle stem cells of the patient, the muscle stem cells can be further genetically engineered by using known genetic engineering methods, the defective gene can be repaired, the gene can be optimized, and the muscles can be further expanded. Stem cells to achieve the goal of overcoming some genetic or mutation-related muscle diseases or further optimizing muscle tissue.
  • the obtained muscle stem cells are administered to the muscle injury site of the patient.
  • muscle stem cells can be intramuscularly injected into the muscle injury site of the patient by injection. After the injection, the patient should be moderately exercised to promote the integration of muscle stem cells. Check the repair effect after 4-8 weeks.
  • the embodiments of the present invention are described below by way of specific examples, and those skilled in the art can readily understand other advantages and effects of the present invention from the disclosure of the present disclosure. The invention may also be added by other different embodiments The details of the present invention can be variously modified or changed without departing from the spirit and scope of the invention.
  • the experimental methods, detection methods, and preparation methods disclosed in the present invention employ molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields conventional in the art. Conventional technology. These techniques are well described in the existing literature. For details, see Sambrook et al.
  • Wild type C57/B6 mice were sacrificed, and the spleen was taken out, placed in a 70 um filter, moistened with 2-3 ml of PBS, and the spleen was honed with a honing stick, and an appropriate amount of PBS was added thereto to collect the cell suspension. After centrifugation at 100 rpm for 5 minutes, the supernatant was discarded, the cells were resuspended in 5 ml of red blood cell lysate, 5 ml of RPMI-1640 medium was added, and the cell debris was again removed by filtration through a sieve, and the cells were collected by centrifugation at 100 rpm for 5 minutes.
  • the cells were washed twice with RPMI-1640 medium, adjusted to a cell density of 1 ⁇ 10 9 cells per liter, seeded in cell culture flasks, added to ConA (final concentration 5 mg/L), and cultured in a 37 ° C0 2 incubator for 48 hours. After the addition of RPMI-1640 medium, the cells were centrifuged at 3000 rpm for 5 minutes, and the cell supernatant was transferred to a new centrifuge tube and stored at -80 °C.
  • the obtained T cell conditioned medium contains the following concentrations of cytokines:
  • ELISA test see R&D Company Mouse Cytokine Array, Panel A (Catalog # ARY006) Component A GM-CSF 200ng/ml
  • the cytokine content in the tau cell conditioned medium may fluctuate slightly, but this fluctuation is not sufficient to affect the practice of the present invention.
  • the human T cell conditioned medium was prepared by the method of referring to the mouse T cell conditioned medium except that the cultured cells were human T cells.
  • the wild type C57/B6 mice were sacrificed, the spleen was taken out, placed in a 70 um filter, and 2-3 ml of PBS was added to wet the sieve, and the spleen was honed using a honing stick, and an appropriate amount of PBS was added thereto to collect the cell suspension. Centrifuge at 100 rpm for 5 minutes, discard the supernatant, resuspend the cells with 5 ml of red blood cell lysate, add 5 ml of RPMI-1640 medium, and filter again to remove cell debris. Centrifuge at 100 rpm for 5 minutes to collect the cells, using RPMI-1640 medium.
  • the human B cell conditioned medium was prepared by the method of referring to the mouse B cell conditioned medium except that the cultured cells were human B cells.
  • Each blood cytokine is commercially available.
  • Muscle stem cells can also be cultured in both 1640 medium and DMEM medium.
  • Cytokines were added according to the following table.
  • the cytokines of each blood cell in the table below were derived from mice or derived from humans.
  • TGF-beta 200 2 20 50 5 20 200
  • IL-1 alpha 10 0.5 5 2 50 2 20 mTOR 20 0.2 2 5 1 2 20
  • TLR1 50 0.5 5 10 5 10 50
  • Rat muscle stem cells Several 3 day old mice were sacrificed. The muscles of the limbs were placed in DMEM medium supplemented with 0.2% D-type collagenase, digested at 37 degrees for 1.5 hours, and then the muscles were washed three times with PBS, each time naturally. The muscles were set a few minutes to collect muscles. The muscles were sucked several times using Pasteur pipettes and 18G needles until the muscle masses were broken, 40um filter was used to remove non-muscle impurities, centrifuged at 1000 rpm for 5 minutes, resuspended in F10 medium and inoculated in 10 cm culture.
  • the supernatant was transferred to a 0.05% type 1 collagen coated 10cm dish, while adding 5ng / mlFGF cytokines, and incubated at 37 ° C0 2 incubator overnight. On the next day, the muscle cells were trypsinized, centrifuged at 1500 rpm for 5 minutes, and the cells were washed twice with PBS.
  • the cells were resuspended in PBS containing 1.5% BSA, and CD34 antibody (BD Pharmingen, cat No: 553733, dilution ratio: 1 : 20), integrin ⁇ 7 (R&D, cat No: FAB3518A, dilution ratio: 1 : 10), incubate at 37 degrees for 45 minutes, centrifuge at 5000 rpm for 5 minutes to collect cells, wash cells twice with PBS, and sort by Influx flow cytometry.
  • CD34 and integrina7 double positive cells, namely mice Muscle stem cells.
  • Human muscle stem cells After obtaining human muscle tissue, use sterile surgical instruments to remove all non-muscle tissue such as skin, fat and bone. After weighing, cut the muscle into small pieces, add 3.5 ml of dispase ll and D-type collagenase mixture per gram of muscle, and digest for 15 minutes in a 37 ° C0 2 incubator. Blow the muscle tissue with a 5 ml pipette. Repeat the above procedure 2-3 times until all muscle masses have been digested. The digestion was terminated by adding 2 times the total volume of the complete growth medium, and the lOOum filter was filtered and centrifuged at 329 g for 10 minutes.
  • Example 1 Each medium prepared in Example 1 was separately added to the isolated and purified muscle stem cells, and cultured in a 37 ° C 5% CO 2 incubator. The cells were passaged every 48 hours, and the passaged cells were cultured in a 37 ° C 5% CO 2 incubator.
  • pancreatin pre-warmed at 37 ° C was digested for 1-2 minutes, and the reaction was terminated by adding a pre-warmed medium at 37 °C.
  • the cells were resuspended and centrifuged at 3000 rpm for 6 minutes at room temperature. The supernatant was discarded, and the pelleted cells were resuspended in a pre-warmed medium at 37 ° C, diluted 3 times, and divided into 3 culture dishes, and cultured in a 37 ° C 5% CO 2 incubator for 48 hours.
  • Each generation of muscle stem cells was subjected to immunofluorescence staining of the molecular marker of muscle stem cells, Pax7, to detect the expression level.
  • Each generation of muscle stem cells was subjected to in vitro differentiation experiments to determine their differentiation potential.
  • Each generation of cells is introduced into the muscle of a mouse that induces muscle damage by intramuscular injection to detect its ability to repair muscle damage.
  • Fluorescence staining at the nucleus was observed to be positive. Fluorescence staining at the nucleus was not observed to be negative.
  • Mouse animal model for induction of muscle damage wild type mice purchased from Charlse River.
  • the staining method was as follows: Frozen sections were washed 3 times with PBS. Fix with 4% formaldehyde at room temperature for 15 minutes. Wash 3 times with PBS and add 1% Tween 20 at room temperature for 10 minutes. The cells were washed 3 times with PBS, and Laminin antibody (purchased from Abeam Co., Ltd.) diluted in 1% BSA in PBS was added, and incubated at room temperature for 1 hour.
  • Test results (1#-14# respectively correspond to F10 cell culture medium supplemented with cytokines of rat blood cells prepared using the formula of ##-14# in Table 1 and Table 2, respectively.
  • Aa maintains good differentiation potential
  • ab partially maintains differentiation potential
  • bb no differentiation potential
  • Cc muscle damage repaired well; cd: minor muscle repair; dd: muscles not repaired.

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Abstract

提供了一种肌肉干细胞体外培养方法，即采用添加有血液细胞的细胞因子的细胞培养基或血液细胞的条件培养基体外培养肌肉干细胞。还提供了所述肌肉干细胞体外培养方法所用的培养基及其应用。

Description

肌肉干细胞体外培养方法及其应用
技术领域
本发明涉及生物技术与医学领域， 特别涉及肌肉干细胞相关的细胞培养与细胞治疗领 域。 背景技术
肌肉干细胞负责出生后肌肉组织的生长和再生， 且肌肉干细胞在体内没有致癌效 果， 不存在应用胚胎干细胞时会碰到的伦理问题， 因而在肌肉萎缩和重症肌无力等各种 肌肉退行性疾病的治疗方面有广阔的应用前景。 肌肉干细胞临床应用的一个重要障碍是 无法在体外长期培养肌肉干细胞。 和大多数成体干细胞一样， 肌肉干细胞从体内分离之 后， 在体外培养系统中只能再分裂 2-4次， 几乎无法传代。
现有培养肌肉干细胞 (卫星细胞)的方法几乎都来源于 Bischoff博士在 1986年建立的 方法，后经过 Beauchamp等人的改进，形成现有的在 F10基础培养基中添加 10ng/ml FGF 进行培养的方法。 在此培养条件下， 肌肉干细胞可以增殖 3-5次。 在传代时， 肌肉干细 胞会经过一个严重的危机时期， 在这一危机时期， 绝大多数细胞都会死去， 残留的细胞 则分化为不具有干性的前体细胞。 所以体外培养 1-3代 (3-12天)后， 分离得到的肌肉干 细胞即全部分化为不具有干性的前体细胞， 肌肉干细胞的分子标记在这些前体细胞中都 无法检测到。 注射入小鼠体内后， 这些前体细胞参与肌肉损伤修复的能力非常弱， 不能 在体内形成有功能的干细胞， 完全不能进行移植后二次损伤的修复。 （Bischoff R. A satellite cell mitogen from crushed adult muscle. Dev Biol., 1986. 115(1): 140-147. Beauchamp, J R., et al., Expression of CD34 and Myf5 defines the majority of quiescent adult skeletal muscle satellite cells. J Cell Biol, 2000. 151(6): p. 1221-34.)也就是说，采用现 有的肌肉干细胞体外培养系统， 培养后的肌肉干细胞传代后要经过一个危机期， 危机期 后得到的可以增殖的细胞绝大多数已经分化为肌肉祖细胞， 丧失了肌肉损伤修复的能 力。 由于这一问题的存在， 使得通过在肌肉干细胞中进行基因修复治疗遗传性的肌肉退 行性疾病的方法难以在临床治疗中使用。 发明内容
本发明的目的就是克服现有技术的缺陷， 提供一种肌肉干细胞培养基及其应用。 本发明第一方面提供了一种肌肉干细胞体外培养方法， 为采用添加有血液细胞的细 胞因子的细胞培养基或血液细胞的条件培养基体外培养肌肉干细胞。 本发明的肌肉干细 胞体外培养方法适合体外长期培养肌肉干细胞。
本发明第二方面提供了一种肌肉干细胞培养基， 为添加有血液细胞的细胞因子的细 胞培养基或血液细胞条件培养基。
所述添加有血液细胞的细胞因子的细胞培养基中所添加的血液细胞的细胞因子， 用 于制备所述血液细胞条件培养基的血液细胞， 均与待培养的肌肉干细胞来源于相同的动 物物种。
当添加动物血清培养细胞时， 本发明所述的添加有血液细胞的细胞因子的细胞培养 基中所添加的血液细胞的细胞因子并不包括所用动物血清中本就含有的细胞因子。
所述添加有血液细胞的细胞因子的细胞培养基可由在通用细胞培养基中添加血液 细胞的细胞因子的方法制备获得。
进一步的， 所述血液细胞条件培养基为淋巴细胞条件培养基。 更进一步的， 为 B细 胞条件培养基或 T细胞条件培养基。
所述添加的血液细胞的细胞因子选自以下细胞因子中的多种： GM-CSF (粒细胞集 落剌激因子）、 sICAM-lC人可溶性细胞间粘附分子 1); IFN gamma(j干扰素)、 IL1C白介 素 1)、 IL-1 alpha receptor (白介素 1α受体）、 IL1 alpha (白介素 la)、 IL-3(白介素 3)、 IL2(白 介素 2)、IL-10(白介素 10)、IL-16(白介素 16)、IL13(白介素 13)、IL-17(白介素 17)、IP-10(干 扰素诱导蛋白 10)、SCYA2 (小诱导型细胞因子 A2)、MIG (干扰素 γ诱生单核因子）、 MIP-1 alpha (巨噬细胞炎性蛋白 la)、 TGF-beta (转化生长因子 β；)、 IL-4(白介素 4)、 TRAF6(TNF 受体相关因子 6)、 FGF (成纤维细胞生长因子；)、 IGF胰岛素样生长因子；)、 PDGF (血小板 衍生生长因子；)、 LIF (白血病抑制因子；)、 mTOR (哺乳动物雷帕霉素靶蛋白；)、 LPS (细胞内 毒素)、 TLRl(Toll-样受体 1)、 IL12(白介素 12)、 IL23(白介素 23)、 NGF (神经生长因子）、 TNFalpha(a干扰素）、 IL1 beta (白介素 1β；)。
所述添加有血液细胞的细胞因子的细胞培养基或血液细胞的条件培养基中， 所述添 加的血液细胞的细胞因子总浓度不低于 6ng/m l， 较佳地为 50-4500 ng /ml。
进一步的， 所述添加有血液细胞的细胞因子的细胞培养基或血液细胞的条件培养基 中， 任一添加的血液细胞的细胞因子的浓度均在 0.5ng/ml以上， 较佳的为 lng/ml以上， 再佳的为 10ng/ml以上， 更佳的， 25ng/ml以上， 更进一步的， 为 50ng/ml以上。
进一步的， 所添加的述血液细胞的细胞因子至少包括 IL1、 IL4、 IL13、 TNF alpha、 IL2禾 P IFN gamma o
进一步的， 所述添加有血液细胞的细胞因子的细胞培养基或血液细胞的条件培养基 中， IL1、 IL4、 IL13、 TNF alpha、 IL2和 IFN gamma的浓度之和不低于 6ng/ml， 较佳地 为 50-1250ng/ml。
进一步的， 所述添加有血液细胞的细胞因子的细胞培养基或血液细胞的条件培养基 中，所述 IL 1、 IL4、 IL 13、 TNF alpha、 IL2和 IFN gamma中任一种的浓度均不低于 0.5ng/ml， 较佳的为 lng/ml以上， 更佳为 10ng/ml以上， 一般可选择 50-500ng/ml。
本发明第三方面， 提供了前述血液细胞的细胞因子或血液细胞条件培养基在肌肉干 细胞体外培养中的用途。
所述血液细胞的细胞因子或血液细胞条件培养基可促进肌肉干细胞在体外的增殖 并使体外增殖的肌肉干细胞保持干性。
本发明第四方面， 提供了一种用于治疗肌肉退行性疾病的制剂， 为以采用本发明的 肌肉干细胞体外培养方法培养获得的肌肉干细胞为主要活性成分的制剂。
进一步的， 所述制剂以来源于病人本体的肌肉干细胞采用本发明的肌肉干细胞体外 培养方法培养获得的肌肉干细胞为主要活性成分。
进一步的， 所述制剂为注射剂、 手术植入剂。
所述制剂一般包含常用的制剂辅料。
本发明第五方面， 提供了一种对病人的肌肉退行性疾病细胞治疗方法， 包括下列步 骤：
1 ) 收集病人的肌肉干细胞；
2 ) 采用本发明的肌肉干细胞培养基体外扩增培养步骤 1)收集的肌肉干细胞至 得到足够多的肌肉干细胞；
3 ) 将获得的肌肉干细胞给药至病人肌肉损伤部位。
其中步骤 3)所述给药方式可采用注射剂的方式将肌肉干细胞肌肉注射至病人肌肉 损伤部位。
肌肉干细胞的注射剂可将扩增后的肌肉干细胞经胰酶消化从培养皿中取下后， 重悬 在生理盐水中获得。
采用本发明的肌肉干细胞体外培养、 传代方法， 使得肌肉干细胞可以在体外大量扩 增并保持干性， 从而大大提高了可以用于再生医学治疗的干细胞的数量， 使得由患者提 供少量肌肉组织， 分离肌肉干细胞， 进行基因修复， 体外扩增后将携带正确遗传信息的 肌肉干细胞注射如体内实现对多种遗传性肌肉退行性疾病的细胞治疗的策略成为可能。
现有的肌肉干细胞培养方法无法在体外长期培养具有分化潜能的肌肉干细胞， 无法 传代， 培养时间超过 12 天后全部细胞都分化为祖细胞， 丧失在体内修复肌肉损伤的能 力。 在现有培养条件下， 肌肉干细胞只能分裂 3-5 次， 即最多扩增为原始细胞数的 32 倍， 即丧失分裂能力， 因而要得到足够多的肌肉干细胞用于治疗， 就必须取非常大的肌 肉组织用来分离纯化肌肉干细胞， 在临床上基本不具有可行性。 在本发明的培养基中， 肌肉干细胞可以增殖十次以上， 即在每一代均可以扩增为原始细胞数的 1000倍以上。 更重要的是， 在本发明的培养基中， 肌肉干细胞可以连续传代， 并且继续保持干性和分 化潜能。这样最终得到的肌肉干细胞数目是起始分离纯化的肌肉干细胞数目的 2mx2n倍， m为每代分裂次数， n为传代数目。 采用本发明的方法得到的 m值至少为 12， n值至 少为 40， 即肌肉干细胞数目至少可以扩增为原始分离纯化的肌肉干细胞数目的 4.5xl015 倍， 并且扩增后的肌肉干细胞均保持干性和完整分化潜能， 能够在体内修复肌肉损伤。 注射入小鼠体内后， 这些干细胞可以较高效地参与肌肉损伤的修复， 也可以补充体内的 干细胞储备， 具有移植后二次损伤的修复能力。 这就使得通过微创手术， 提取少量肌肉 组织 (克级)， 分离纯化少量肌肉干细胞， 然后在体外扩增获得足够量的肌肉干细胞用于 再生医学治疗的策略在临床上具有可行性。 附图说明
图 1、 添加细胞因子的 F10培养基中培养的 P0 代肌肉干细胞 (Pax7染色)。
图 2、 添加细胞因子的 F10培养基中培养的对照成纤维细胞 (Pax7染色)。
图 3、 添加细胞因子的 F10培养基中培养的 P8代肌肉干细胞 (Pax7染色）。
图 4、 添加细胞因子的 F10培养基中培养的 P22代肌肉干细胞 (Pax7染色)。
图 5、 在添加细胞因子的 F10培养基中培养的 P0代细胞的分化结果。
图 6、 在添加细胞因子的 F10培养基中培养的 P22代细胞的分化结果。
图 7、在添加细胞因子的 F10培养基中培养的表达 RFP的肌肉干细胞在体内参与肌 肉损伤修复结果。
图 8、在添加细胞因子的 F10培养基中培养的 RFP标记的肌肉干细胞能够参与肌肉 损伤的修复结果。体外培养的 RFP标记的肌肉干细胞注射入没有荧光标记的损伤的肌肉 7天后， 切片观察红色细胞的整合情况， 发现在损伤部位有来源于注射的表达 RFP的肌 肉干细胞的红色肌纤维形成。
图 A为注射加入细胞因子培养的肌肉干细胞的修复情况；
图 B为注射用 T细胞条件培养基培养的肌肉干细胞的修复情况。 具体实施方式
本发明发现， 与血细胞共培养后可以促进肌肉干细胞的增殖， 且这一促进作用不依 赖于血细胞与肌肉干细胞之间的细胞接触。 血细胞条件培养基即可以促进肌肉干细胞的 增殖。 经过进一步细分， B细胞和 T细胞条件培养基均可以促进肌肉干细胞的增殖。 肌 肉干细胞在 T细胞条件培养基中培养后， 每代可连续分裂 10次以上， 并可在体外培养 系统中至少连续传代 40代。 经过一系列分离纯化步骤后， 分离到 T细胞所分泌出的细 胞因子， 这些细胞因子的组合可以促进肌肉干细胞在体外的增殖。 经细胞因子处理过后 的肌肉干细胞表达肌肉干细胞所应表达的分子标记， 其增殖能力显著提高， 可以连续传 代 40 代以上。 传代后的每一代细胞均表达肌肉干细胞的分子标记， 具有较强的增殖能 力， 可以高效分化为成熟肌管。 更重要的是， 当这些用 T细胞条件培养基或细胞因子培 养的肌肉干细胞注射入诱导肌肉损伤后的小鼠时， 注射的肌肉干细胞可以修复肌肉损 伤， 形成新的肌纤维， 表明本发明得到的是真正的肌肉干细胞。
本发明的肌肉干细胞体外培养方法， 为采用添加有血液细胞的细胞因子的细胞培养 基或血液细胞的条件培养基体外培养肌肉干细胞。
本发明的关键在于对体外培养肌肉干细胞的培养基进行了改进， 体外培养肌肉干细 胞的其他方面与常规细胞培养一致。
最佳培养条件： C02培养箱中 37°C培养。 C02培养箱中 C02浓度较佳为 5%(v/v)。 所述肌肉干细胞的体外培养是贴壁培养。
本发明所采用的肌肉干细胞培养基， 为添加有血液细胞的细胞因子的细胞培养基或 血液细胞条件培养基。
所述添加有血液细胞的细胞因子的细胞培养基中所添加的血液细胞的细胞因子与 所述待培养的肌肉干细胞应当来源于相同的动物物种。 用于制备所述血液细胞条件培养 基的血液细胞与所述待培养的肌肉干细胞也应当来源于相同的动物物种。 如培养鼠肌肉 干细胞， 则采用添加了鼠血液细胞细胞因子的细胞培养基或鼠血液细胞条件培养基， 如 培养人肌肉干细胞， 则采用添加人血液细胞的细胞因子的细胞培养基或人血液细胞条件 培养基， 其余类推。
所述物种 (Species), 为生物分类学的基本单位。 进一步的， 所述动物物种选自哺乳 动物。 更进一步的， 所述物种选自哺乳动物中的啮齿目、 偶蹄目、 奇蹄目、 兔形目、 灵 长目等的物种， 如鼠、 兔、 羊、 猪、 猴、 人等。
当添加动物血清培养细胞时， 本发明所述添加有血液细胞的细胞因子的细胞培养基 中所添加的血液细胞的细胞因子并不包括加入培养基的动物血清中本就含有的细胞因 子。 亦即， 当添加动物血清培养细胞时， 本发明还需额外添加与所培养的肌肉干细胞同 物种的血液细胞的细胞因子。
本发明所述添加有血液细胞的细胞因子的细胞培养基是指除常规细胞培养所需组分 夕卜， 还额外添加有血液细胞的细胞因子的细胞培养基。
本发明所述肌肉干细胞培养基中除添加的血液细胞的细胞因子外的其他组分及含量 均与常规细胞培养基一致。 常规细胞培养基成分一般选自： 平衡盐水、 pH 调节液、 抗 生素、 动物血清、 细胞生长必须的氨基酸、 维生素、 葡萄糖、 pH 指示剂等。 所述平衡 盐水成分如氯化钙、 硝酸铁、 硫酸镁、 氯化钾、 氟化钠、 氯化钠、 磷酸钠等； 所述 pH 调节液如 3.7%碳酸氢钠、 HEPES溶液 (二羟乙基哌嗪乙垸磺酸)、 丙酮酸钠等； 所述抗生 素如青霉素、链霉素、 等； 常用的动物血清主要有牛血清和马血清。 维生素如氯化胆碱、 叶酸、 肌醇、 烟酰胺、 泛酸钙、 盐酸吡哆醛、 维生素 B6、 核黄素、 硫胺素等。
所述添加有血液细胞的细胞因子的细胞培养基可由在通用细胞培养基中添加血液 细胞的细胞因子的方法制备获得。 其中， 所述通用细胞培养基可选自各种常规细胞培养 基，如 DMEM、 RPMI 1640、 MEM、 DEME/F12、 F10、 CD293、 medium 231、 medium 106。 本发明实施例具体列举了在 F10培养基中添加各种血液细胞的细胞因子的肌肉干细胞培 养基。 所述血液细胞条件培养基是指为： 培养过血液细胞的细胞培养基。 进一步的， 所述 血液细胞为淋巴细胞。 最佳的， 所述血液细胞为 B细胞和 /或 T细胞。
所述血液细胞条件培养基可由在通用细胞培养基中培养血液细胞后分离获得。 在通 用细胞培养基中培养 B细胞后分离获得 B细胞条件培养基，在通用细胞培养基中培养 T 细胞后分离获得 T细胞条件培养基。
所述添加的血液细胞的细胞因子选自以下细胞因子中的多种： GM-CSF、 sICAM-1 ; IFN gamma、 ILK IL-1 alpha 、 IL-1 alpha receptor IL-3、 IL2、 IL-10、 IL-16、 IL13、 IL-17、 IP-10、 SCYA2、 MIG、 MIP-1 alpha TGF-beta、 IL-4、 TRAF6、 FGF、 IGF、 PDGF、 LIF、 mTOR、 LPS、 TLR1、 IL12、 IL23、 NGF、 TNF alpha IL1 beta。
所有上述细胞因子可以在淋巴细胞条件培养基中检测到， 尤其均存在于 T细胞条件 培养基中。
更佳的， 所述血液细胞的细胞因子选自上述细胞因子中的至少六种； 或者， 所述血 液细胞的细胞因子选自上述细胞因子中的至少七种； 或者， 所述血液细胞的细胞因子选 自上述细胞因子中的至少八种； 或者， 所述血液细胞的细胞因子选自上述细胞因子中的 至少九种； 或者， 所述血液细胞的细胞因子选自上述细胞因子中的至少十种。
为了维持肌肉干细胞的增殖并使其传代细胞保持干性， 所述添加有血液细胞的细胞 因子的细胞培养基或血液细胞的条件培养基中， 所述血液细胞的细胞因子总浓度不能过 低， 一般不应低于 6ng/ml， 较佳地为 50-4500 ng /ml。
所述添加有血液细胞的细胞因子的细胞培养基或血液细胞的条件培养基中， 任一添 加的血液细胞的细胞因子的浓度均在 0.5ng/ml 以上， 较佳的为 lng/ml 以上， 更佳为 lOng/ml以上， 一般可选在 50-500ng/ml的范围内。上述描述并非指细胞因子的浓度不能 高于 500ng/ml， 而是认为在此浓度范围内细胞因子可以较好地发挥积极效果， 浓度过低 效果不明显， 浓度过高造成浪费。
经试验， 六种血液细胞的细胞因子： IL1、 IL4、 IL13、 TNF alpha、 IL2和 IFN gamma 对肌肉干细胞的增殖与干性的保持关系密切。 在本发明优选的技术方案中， 这 6种血液 细胞的细胞因子必须添加， 而其余血液细胞的细胞因子可添加也可以不添加。
进一步的， 所述添加有血液细胞的细胞因子的细胞培养基或血液细胞的条件培养基 中， IL1、 IL4、 IL13、 TNF alpha、 IL2和 IFN gamma的浓度之和不低于 6ng/ml， 较佳地 为 50-1250ng/ml。
进一步的， 所述添加有血液细胞的细胞因子的细胞培养基或血液细胞的条件培养基 中， 所述 IL1、 IL4、 IL13、 TNF alpha、 IL2和 IFN gamma中任一种的浓度均在 0.5ng/ml 以上， 较佳的为 lng/ml以上， 更佳为 10ng/ml以上， 一般可选 50-500ng/ml。 所述细胞 因子的浓度高于 500ng/ml并不会对肌肉干细胞的增殖构成严重的负面影响，但考虑到成 本因素， 无需添加过多的细胞因子。 上述各细胞因子的浓度均指： 添加有血液细胞的细胞因子的细胞培养基或血液细胞 的条件培养基中， 来源于与所培养的肌肉干细胞同物种的血液细胞的细胞因子的终浓 度。
本发明揭示了血液细胞的细胞因子或血液细胞条件培养基可用于肌肉干细胞体外 培养， 可促进肌肉干细胞在体外的增殖并使体外增殖的肌肉干细胞保持干性。
本发明还提供了一种用于治疗肌肉退行性疾病的制剂， 为以采用本发明的肌肉干细 胞体外培养方法培养获得的肌肉干细胞为主要活性成分的制剂。
所述制剂以来源于病人本体的肌肉干细胞采用本发明的肌肉干细胞体外培养方法 培养获得的肌肉干细胞为主要活性成分。
根据本发明的试验， 用于人体， 推荐的给药剂量约为 3xl06个肌肉干细胞 /次。 可单 次给药， 也可以根据病人的肌肉恢复情况多次给药。 一般的给药方式为通过肌肉注射的 方式给药。 但本发明并不排除其他可能的给药方式。
所述制剂一般包含常用的制剂辅料。
常用的制剂辅料包括 (但并不限于):盐水、 缓冲液、 葡萄糖、 水、 甘油、 乙醇、 多元 醇及其组合。 药物制剂应与给药方式相匹配。 本发明的制剂优选被制成针剂形式， 例如 用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。 其他可能的制剂 形式可通过常规方法进行制备。 本发明的制剂宜在无菌条件下制造。 此外， 本发明的制 剂还可与其他治疗剂一起使用。
本发明进一步提供了一种对病人的肌肉退行性疾病细胞治疗方法， 包括下列步骤： 1 ) 收集病人的肌肉干细胞；
病人肌肉干细胞的收集可通过微创手术从病人机体中提取少量肌肉样品， 而后从肌 肉样品中将肌肉干细胞分离纯化获得。 该技术为本领域技术人员熟知。
2 ) 采用本发明的肌肉干细胞培养基体外扩增培养步骤 1)收集的肌肉干细胞至 得到足够多的肌肉干细胞；
基于本发明的肌肉干细胞培养技术， 收集了病人的肌肉干细胞后， 可以进一步利用 已知的基因工程手段对肌肉干细胞进行基因工程改造， 修复缺陷基因或进行基因优化， 并进一步扩增改造后的肌肉干细胞， 以达到克服一些遗传或突变相关的肌肉疾病或进一 步优化肌肉组织的目的。
3 ) 将获得的肌肉干细胞给药至病人肌肉损伤部位。
一般可采用注射剂的方式将肌肉干细胞肌肉注射至病人肌肉损伤部位。 注射后应当 让病人进行适度锻炼， 促进肌肉干细胞的整合。 4-8周后检查修复效果。 以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露 的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加 以实施或应用， 本说明书中的各项细节也可以基于不同观点与应用， 在没有背离本发明的精 神下进行各种修饰或改变。
除非另外说明， 本发明中所公开的实验方法、 检测方法、 制备方法均采用本技术 领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组 DNA 技术及相关领域的常规技术。 这些技术在现有文献中已有完善说明， 具体可参见 Sambrook等 MOLECULAR CLONING ： A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press , 1989 and Third edition, 2001 ； Ausubel 等， CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates ； the series METHODS IN ENZYMOLOGY, Academic Press, San Diego ； Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998 ； METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.)， Academic Press, San Diego, 1999 ；和 METHODS IN MOLECULAR BIOLOGY , Vol.119 , Chromatin Protocols(P.B. Becker , ed.)Humana Press, Totowa, 1999等。 实施例 1、 鼠 T细胞条件培养基的制备
将野生型 C57/B6小鼠处死， 取出脾脏， 置于 70um滤网中， 加入 2-3毫升 PBS湿 润后， 使用辗磨棒辗磨脾脏， 加适量 PBS过滤， 收集细胞悬液。 lOOOrpm离心 5分钟， 弃上清， 使用 5毫升红细胞裂解液重悬细胞， 加入 5毫升 RPMI-1640培液， 并用滤网再 次过滤去除细胞碎片， lOOOrpm离心 5分钟收集细胞。 使用 RPMI-1640培液洗涤细胞两 次，调整细胞密度为 1 X 109cells每升，接种于细胞培养瓶，加入 ConA (终浓度为 5mg/L)， 于 37度 C02培养箱培养 48小时后补加等倍 RPMI-1640培液， 24小时后 3000rpm离心 5分钟， 将细胞上清转移至新离心管中， 于 -80摄氏度保存。
经检测， 获得的 T细胞条件培养基中含有下列浓度的细胞因子：
检测方法： ELISA检测,参见 R&D公司 Mouse Cytokine Array, Panel A (Catalog # ARY006) 组分 A GM-CSF 200ng/ml
组分 B sICAM-1 120ng/ml
组分 C IFN gamma 160ng/ml
组分 D IL1 lOOng/ml
组分 E IL-1 alpha receptor lOOng/ml
组分 F IL-3 250ng/ml
组分 G IL2 200ng/ml
组分 H IL-10 80ng/ml
组分 I IL-16 130ng/ml 组分 J IL13 120ng/ml
组分 κ IL-17 300ng/ml
组分 L IP- 10 250ng/ml
组分 M SCYA2 80ng/ml
组分 N MIG 150ng/ml
组分 0 MIP-1 alpha 200ng/ml
组分 P TGF-beta 150ng/ml
组分 Q IL-4 250ng/ml
组分 R TRAF6 400ng/ml
组分 S FGF 25ng/ml
组分 τ IGF 50ng/ml
组分 U PDGF 80ng/ml
组分 V LIF lOOng/ml
组分 W IL-1 alpha 200ng/ml
组分 X mTOR 30ng/ml
组分 Y LPS 60ng/ml
组分 Z TLR1 55ng/ml
组分 Z' IL12 120ng/ml
组分 Y' IL23 200ng/ml
组分 X' NGF lOOng/ml
组分 W TNFalpha lOOng/ml
组分 V' IL1 beta lOOng/ml
在不同的细胞培养基或者不同操作人员的情况下， τ细胞条件培养基中的细胞因子含量 会略有波动， 但这一波动并不足以影响本发明的实施。
人 T细胞条件培养基除培养用细胞为人 T细胞外， 其余参考鼠 T细胞条件培养基的方 法制备。
鼠 B细胞条件培养基的制备：
将野生型 C57/B6小鼠处死， 取出脾脏， 置于 70um滤网中， 加入 2-3毫升 PBS湿润滤 网后， 使用辗磨棒辗磨脾脏， 并加适量 PBS过滤， 收集细胞悬液， lOOOrpm离心 5分钟， 弃 上清， 使用 5毫升红细胞裂解液重悬细胞， 加入 5毫升 RPMI-1640培液， 并用滤网再次过滤 去除细胞碎片， lOOOrpm离心 5分钟收集细胞， 使用 RPMI-1640培液洗涤细胞两次， 调整细 胞密度为 l*109cellS每升， 接种于细胞培养瓶， 加入 LPS, 终浓度为 lmg/L， 于 37度 C02 培养箱培养 48小时后补加等倍 RPMI-1640培液， 24小时后 3000rpm离心 5分钟， 将细胞上 清转移至新离心管中， 于 -80度保存。 人 B细胞条件培养基除培养用细胞为人 B细胞外， 其余参考鼠 B细胞条件培养基的方 法制备。
添加有血液细胞的细胞因子的细胞培养基的配制：
各血细胞因子均可市购获得。
添加有血液细胞的细胞因子的肌肉干细胞培养基的配制方法：
将 F10培养基干粉溶于超纯水， 过滤除菌， 按表 1和表 2加入无菌的各细胞因子， 10%胎牛血清， 100IU青霉素， lOO g/ml链霉素后混匀。
也可采用 1640培养基和 DMEM培养基，均能培养肌肉干细胞。
按下表加入细胞因子， 下表中各血液细胞的细胞因子均来源于鼠或来源于人。
表 1
Figure imgf000011_0001
表 2
Figure imgf000011_0002
IL-17 120 1 10 20 5 5 150
IP- 10 40 0.5 5 10 1 10 80
SCYA2 60 0.5 5 10 2 5 50
MIG 50 0.5 5 10 2 5 50
MIP-1 alpha 100 1 10 20 5 10 200
TGF-beta 200 2 20 50 5 20 200
IL-4 100 100 100 100 100 100 100
TRAF6 50 0.5 5 10 2 5 50
FGF 10 0.2 2 5 1 2 20
IGF 10 0.2 2 5 1 2 20
PDGF 10 0.2 2 5 1 2 20
LIF 20 0.2 2 5 1 2 20
IL-1 alpha 10 0.5 5 2 50 2 20 mTOR 20 0.2 2 5 1 2 20
LPS 10 0.1 1 2 1 1 10
TLR1 50 0.5 5 10 5 10 50
IL12 40 0.4 4 10 5 10 50
IL23 20 0.2 2 5 2 5 20
NGF 10 0.2 2 5 2 5 20
TNFalpha 150 150 150 150 150 150 150 实施例 2、 肌肉干细胞的培养与检测
1 ) 肌肉干细胞的获取：
鼠肌肉干细胞： 将数只 3天新生小鼠处死， 取四肢肌肉置于加有 0.2%D型胶原酶 的 DMEM培养液中， 37度消化 1.5小时， 随后用 PBS洗涤肌肉三遍， 每次自然沉降肌 肉数分钟收集肌肉。先后使用巴斯德吸管和 18G针头吹吸肌肉数次， 直至肌肉团块被打 碎， 40um滤网过滤去除非肌肉杂质， lOOOrpm离心 5分钟， 重悬于加有 F10培液并接 种于 10cm培养皿中， 3小时后将上清转移至 0.05%1型胶原包被的 10cm培养皿中， 同 时加入 5ng/mlFGF细胞因子， 于 37度 C02培养箱培养过夜。 次日， 胰酶消化肌肉细胞， 1500rpm离心 5分钟， PBS洗涤细胞两次， 将细胞重悬于含有 1.5%BSA的 PBS中， 加 入 CD34抗体 (BD Pharmingen, cat No: 553733 ,稀释比： 1 :20)、 integrin α7 (R&D, cat No： FAB3518A, 稀释比： 1 : 10),37度孵育 45分钟， 5000rpm离心 5分钟收集细胞， PBS洗 涤细胞两次， 使用 Influx流式细胞分选仪分选 CD34和 integrina7双阳性细胞， 即为鼠 肌肉干细胞。
人肌肉干细胞： 获取人肌肉组织后， 使用无菌手术器械去除所有皮肤、 脂肪及骨骼 等非肌肉组织。称重后将肌肉剪成小块，每克肌肉加入 3.5毫升 dispase ll和 D型胶原酶 等比混合液， 置于 37度 C02培养箱消化 15分钟， 使用 5毫升移液管吹吸肌肉组织， 重 复上述过程 2-3次， 直至所有肌肉团块已被消化。 加入 2倍于总体积的完全生长培养基 终止消化， lOOum滤网过滤后 329g离心 10分钟。 以初始每克肌肉 3.5毫升将沉淀重悬 于完全生长培养基， 加入 7倍体积的红细胞裂解液， 上下颠倒离心管数次， 40um滤网 过滤， 329g离心 10分钟收集细胞， 调整细胞浓度至 0.5-lxlO6 cells/lOml将细胞接种于 gelatin包被的培养皿中。
2 ) 肌肉干细胞传代培养：
分别将实施例 1制备的各培养基加入分离纯化的肌肉干细胞， 在 37°C 5% C02 培 养箱中培养。 每 48小时传代一次， 传代后的细胞在 37°C5% C02 培养箱中培养。
传代方法： 肌肉干细胞生长至密度达到 70%时， 用 37°C预热的 PBS洗一次， 加入
37°C预热的胰酶， 消化 1-2分钟， 加入 37°C预热的培养基终止反应。 重悬细胞， 室温 3000rpm离心 6分钟。 弃上清， 将沉淀的细胞重悬于 37°C预热的培养基中， 稀释 3倍， 均分入 3个培养皿， 在 37°C 5% C02培养箱中培养 48小时。
3 ) 检测：
每一代肌肉干细胞均进行肌肉干细胞的分子标记 Pax7的免疫荧光染色， 检测其表达 水平。
每一代肌肉干细胞均进行体外分化实验， 检测其分化潜能。
每一代细胞均通过肌肉注射引入诱导肌肉损伤的小鼠肌肉检测其修复肌肉损伤的能 力。
每种培养基最多检测 40代细胞。
肌肉干细胞的分子标记 Pax7的免疫荧光染色检测：
传代后的细胞取其中一盘， 用 PBS (磷酸缓冲液)洗 3 次。 用 4%甲醛固定， 室温 15 分钟。用 PBS洗 3次，加入 l% Tween20，室温 10分钟。用 PBS洗 3次，加入在 1% BSA (牛 血清白蛋白） 的 PBS中稀释的 Pax7抗体 (购自 DSHB), 室温孵育 1小时。 用 PBS洗三 次， 每次 5分钟。 加入在 1%BSA PBS溶液中 1 : 1000稀释的荧光标记的驴抗鼠二抗， 室温孵育 1小时。 用 PBS洗一次， 加入 20μΜ DAPI, 室温 5分钟， 用 PBS洗三次， 每 次 5分钟。 加入抗猝灭剂后封片。 在 Zeiss荧光显微镜下观察拍照。
能够观察到位于细胞核的荧光染色判断为阳性。 不能观察到位于细胞核的荧光染色 判断为阴性。
体外分化实验：
取一盘培养后的肌肉干细胞， 将其用预热 37°C 的 PBS洗三次， 加入含有 2%马血清 的 DMEM培养基，在 37°C 5% C02培养箱中培养 72小时后，在显微镜下观察分化情况。 能够观察到 >90%的细胞分化为成熟肌管， 判断为保持良好分化潜能。 观察到部分细 胞分化为成熟肌管， 判断为部分保持分化潜能。 没有肌管形成， 判断为无分化潜能。
肌肉损伤修复试验：
诱导肌肉损伤的小鼠动物模型： 购自 Charlse River的野生型小鼠。
取一盘培养后的表达 RFP (红色荧光蛋白）的肌肉干细胞，用预热 37°C 的 PBS洗一次， 用预热 37°C 的胰酶消化 2分钟后， 取 lxlO5个肌肉干细胞， 重悬于 200μ1 无菌的 PBS 中， 转入无菌的 lml注射器， 肌肉注射入诱导肌肉损伤的不表达 RFP的小鼠腓肠肌中。 小鼠培养 1个月后， 取腓肠肌冰冻切片， 进行 laminin染色以标记肌纤维的轮廓， DAPI 染色以标记 DNA， 在激光共聚焦显微镜下观察 RFP细胞是否存在于损伤部位。 染色方 法如下： 冰冻切片用 PBS洗 3次。 用 4%甲醛固定， 室温 15分钟。 用 PBS洗 3次， 加 入 1% Tween20,室温 10分钟。用 PBS洗 3次，加入在 1% BSA 的 PBS中稀释的 Laminin 抗体 (；购自 Abeam公司）， 室温孵育 1小时。用 PBS洗三次， 每次 5分钟。加入在 1%BSA PBS溶液中 1 : 1000稀释的荧光标记的驴抗兔二抗， 室温孵育 1小时。 用 PBS洗一次， 加入 20μΜ ϋΑΡΙ(;4',6-二脒基 -2-苯基吲哚；)， 室温 5分钟， 用 PBS洗三次， 每次 5分钟。 加入抗猝灭剂后封片。 在 Zeiss激光共聚焦显微镜下观察拍照。
在损伤部位可观察到较多的有红色荧光的肌纤维存在判断为肌肉修复优良。 在损伤 部位仅观察到少数有红色荧光的肌纤维存在判断为肌肉少量修复。 无法检测到任何有红 色荧光的肌纤维判断为肌肉未修复。
4 ) 试验结果（1#-14#分别对应采用表 1和表 2中 1#-14#的配方制备的添加有鼠血液细胞 的细胞因子的 F10细胞培养基
Figure imgf000014_0001
8# (鼠） ++/aa/cc ++/aa/cc ++/aa/cc ++/aa/cc ++/aa/cc ++/ab/cd ++/ab/dd
9# (鼠） ++/aa/cc ++/aa/cc ++/aa/cc ++/aa/cc ++/aa/cc ++/ab/cd ++/ab/dd
10# (鼠） ++/aa/cc ++/ab/cd ++/ab/cd ++/ab/cd ++/ab/cd ++/ab/cd ++/ab/dd
1 1#(鼠） ++/aa/cc ++/aa/cc ++/aa/cc ++/aa/cc ++/aa/cc ++/ab/cd ++/ab/dd
12# (鼠） ++/aa/cc ++/ab/cd ++/ab/cd ++/ab/cd ++/ab/cd ++/ab/cd ++/ab/dd
13# (鼠） ++/aa/cc ++/ab/cd ++/ab/cd ++/ab/cd ++/ab/cd ++/ab/cd ++/ab/dd
14# (鼠） ++/aa/cc ++/aa/cc ++/aa/cc ++/aa/cc ++/ab/cd ++/ab/cd ++/ab/dd 的免疫荧光染色检测阴性
aa: 保持良好分化潜能； ab: 部分保持分化潜能； bb : 无分化潜能
cc: 肌肉损伤修复良好； cd: 肌肉少量修复； dd: 肌肉未修复。
-: 该项检测未进行
部分实验结果图片参见附图。
5 ) 结果分析：
上述结果可见， 采用本发明的培养基， 对 40代肌肉干细胞进行 Pax7染色后， 发现 其均为 Pax7阳性， 每一代细胞均表达肌肉干细胞的分子标记 Pax7， 均能分化为成熟的 肌管细胞。 第 1到第 30代细胞均可修复肌肉损伤。 在小鼠体内， 目前已经检测的 18代 细胞均能够参加肌肉损伤的修复。 以上的实施例是为了说明本发明公开的实施方案， 并不能理解为对本发明的限制。 此外， 本文所列出的各种修改以及发明中方法、 组合物的变化， 在不脱离本发明的范围 和精神的前提下对本领域内的技术人员来说是显而易见的。 虽然已结合本发明的多种具 体优选实施例对本发明进行了具体的描述， 但应当理解， 本发明不应仅限于这些具体实 施例。 事实上， 各种如上所述的对本领域内的技术人员来说显而易见的修改来获取发明 都应包括在本发明的范围内。

Claims

m ^
1、 一种肌肉干细胞体外培养方法， 为采用添加有血液细胞的细胞因子的细胞培养基或血液 细胞的条件培养基体外培养肌肉干细胞。
2、 如权利要求 1所述肌肉干细胞体外培养方法， 其特征在于， 所述添加有血液细胞的细胞 因子的细胞培养基中所添加的血液细胞的细胞因子， 或用于制备所述血液细胞条件培养 基的血液细胞， 均与待培养的肌肉干细胞来源于相同的动物物种。
3、 如权利要求 2所述肌肉干细胞体外培养方法， 其特征在于， 所述血液细胞条件培养基为 淋巴细胞条件培养基。
4、 如权利要求 3所述肌肉干细胞体外培养方法， 其特征在于， 所述血液细胞条件培养基为 B细胞条件培养基或 T细胞条件培养基。
5、 如权利要求 2所述肌肉干细胞体外培养方法， 其特征在于， 所述添加的血液细胞的细胞 因子选自以下细胞因子中的多种： GM-CSF、 sICAM-1 ; IFN gamma ILK IL-1 alpha receptor、 IL1 alpha IL-3、 IL2、 IL-10、 IL-16、 IL13、 IL-17、 IP-10、 SCYA2、 MIG、 MIP-1 alpha TGF-beta、 IL-4、 TRAF6、 FGF、 IGF、 PDGF、 LIF、 mTOR、 LPS、 TLR1、 IL12、
IL23、 NGF、 TNFalpha禾口 IL1 beta。
6、 如权利要求 2所述肌肉干细胞体外培养方法， 其特征在于， 所述添加有血液细胞的细胞 因子的细胞培养基或血液细胞的条件培养基中， 所述添加的血液细胞的细胞因子总浓度 不低于 6ng/m 1， 较佳地为 50-4500 ng /ml。
7、 如权利要求 2所述肌肉干细胞体外培养方法， 其特征在于， 所述添加有血液细胞的细胞 因子的细胞培养基或血液细胞的条件培养基中， 任一添加的血液细胞的细胞因子的浓度 均在 0.5ng/ml 以上， 较佳的为 lng/ml 以上， 再佳的为 10ng/ml 以上， 更佳的， 25ng/ml 以上， 更进一步的， 为 50ng/ml以上。
8、 如权利要求 2所述肌肉干细胞体外培养方法， 其特征在于， 所添加的述血液细胞的细胞 因子至少包括 IL1、 IL4、 IL13、 TNF alpha IL2和 IFN gamma。
9、 如权利要求 8所述肌肉干细胞体外培养方法， 其特征在于， 所述添加有血液细胞的细胞 因子的细胞培养基或血液细胞的条件培养基中， 添加的 IL1、 IL4、 IL13、 TNF alpha、 IL2 禾口 IFN gamma的浓度之和不低于 6ng/ml， 较佳地为 50-1250ng/ml。
10、 如权利要求 8所述肌肉干细胞体外培养方法， 其特征在于， 所述添加有血液细胞的 细胞因子的细胞培养基或血液细胞的条件培养基中， 所述 IL1、 IL4、 IL13、 TNF alpha、
IL2禾 P IFN gamma中任一种的浓度均不低于 0.5ng/ml， 较佳的为 lng/ml 以上， 更佳为 10ng/ml以上， 一般可选择 50-500ng/ml。
11、 一种肌肉干细胞培养基， 为添加有血液细胞的细胞因子的细胞培养基或血液细胞条 件培养基。
12、 如权利要求 11所述的肌肉干细胞培养基， 其特征在于， 所述添加有血液细胞的细胞 因子的细胞培养基中所添加的血液细胞的细胞因子， 或用于制备所述血液细胞条件培养 基的血液细胞， 均与待培养的肌肉干细胞来源于相同的动物物种。
13、 如权利要求 12所述的肌肉干细胞培养基， 其特征在于， 所述血液细胞条件培养基为 淋巴细胞条件培养基。
14、 如权利要求 13所述的肌肉干细胞培养基， 其特征在于， 所述血液细胞条件培养基为 B细胞条件培养基或 T细胞条件培养基。
15、 如权利要求 12所述的肌肉干细胞培养基， 其特征在于， 所述添加的血液细胞的细胞 因子选自以下细胞因子中的多种： GM-CSF、 sICAM-1 ; IFN gamma、 ILK IL-1 alpha receptor、 IL1 alpha IL-3、 IL2、 IL-10、 IL-16、 IL13、 IL-17、 IP-10、 SCYA2、 MIG、 MIP-1 alpha TGF-beta、 IL-4、 TRAF6、 FGF、 IGF、 PDGF、 LIF、 mTOR、 LPS、 TLR1、 IL12、 IL23、 NGF、 TNFalpha禾 P IL1 beta。
16、 如权利要求 12所述的肌肉干细胞培养基， 其特征在于， 所述添加有血液细胞的细胞 因子的细胞培养基或血液细胞的条件培养基中， 所述添加的血液细胞的细胞因子总浓度 不低于 6ng/m 1， 较佳地为 50-4500 ng /ml。
17、 如权利要求 12所述的肌肉干细胞培养基， 其特征在于， 所述添加有血液细胞的细胞 因子的细胞培养基或血液细胞的条件培养基中， 任一添加的血液细胞的细胞因子的浓度 均在 0.5ng/ml 以上， 较佳的为 lng/ml 以上， 再佳的为 10ng/ml 以上， 更佳的， 25ng/ml 以上， 更进一步的， 为 50ng/ml以上。
18、 如权利要求 12所述的肌肉干细胞培养基， 其特征在于， 所添加的述血液细胞的细胞 因子至少包括 IL1、 IL4、 IL13、 TNF alpha IL2和 IFN gamma。
19、 如权利要求 18所述的肌肉干细胞培养基， 其特征在于， 所述添加有血液细胞的细胞 因子的细胞培养基或血液细胞的条件培养基中， 添加的 IL1、 IL4、 IL13、 TNF alpha、 IL2 禾口 IFN gamma的浓度之和不低于 6ng/ml， 较佳地为 50-1250ng/ml。
0、 如权利要求 18所述的肌肉干细胞培养基， 其特征在于， 所述添加有血液细胞的细胞 因子的细胞培养基或血液细胞的条件培养基中， 所述 IL1、 IL4、 IL13、 TNF alpha、 IL2 禾 P IFN gamma中任一种的浓度均不低于 0.5ng/ml，较佳的为 lng/ml以上，更佳为 10ng/ml 以上， 一般可选择 50-500ng/ml。 、 血液细胞的细胞因子或血液细胞条件培养基在肌肉干细胞体外培养中的用途。 、 如权利要求 21所述的用途， 其特征在于， 所述血液细胞的细胞因子或血液细胞条件 培养基用于促进肌肉干细胞在体外的增殖并使体外增殖的肌肉干细胞保持干性。
、 一种用于治疗肌肉退行性疾病的制剂， 是以权利要求 1-10任一权利要求所述的肌肉 干细胞体外培养方法培养获得的肌肉干细胞为主要活性成分的制剂。
、 如权利要求 23所述的制剂， 其特征在于， 所述制剂以来源于病人本体的肌肉干细胞 经所述的肌肉干细胞体外培养方法培养获得的肌肉干细胞为主要活性成分。
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