Pharmaceutical composition for treating neurogenic muscular atrophy and preparation method and application thereof
阅读说明：本技术 治疗神经源性肌肉萎缩的药物组合物及其制法和应用 (Pharmaceutical composition for treating neurogenic muscular atrophy and preparation method and application thereof ) 是由 唐文洁 魏佑震 刘中民 胡苹 饶灵均 于 2018-08-08 设计创作，主要内容包括：本发明提供了治疗外周神经损伤性肌肉萎缩的药物组合物及其制法和应用。具体地,本发明提供了一种生肌细胞的用途,用于制备一制剂或组合物,所述的制剂或组合物用于治疗神经源性肌肉萎缩,其中,所述的治疗包括改善肌肉萎缩和神经损伤。本发明还提供了治疗神经源性肌肉萎缩的组合物和方法。(The invention provides a pharmaceutical composition for treating peripheral nerve injury type muscular atrophy and a preparation method and application thereof. Specifically, the invention provides a use of myogenic cells for preparing a preparation or composition for treating neurogenic muscle atrophy, wherein the treatment comprises ameliorating muscle atrophy and nerve damage. The invention also provides compositions and methods for treating neurogenic muscle atrophy.)
1. Use of myogenic cells for the preparation of a formulation or composition for the treatment of neurogenic muscle atrophy, wherein said treatment comprises amelioration of muscle atrophy and nerve damage.
2. The use of claim 1, wherein said myogenic cells express a marker gene selected from the group consisting of:
PAX3+, NCAM +, Myosin Heavy Chain +, Myogenin +, MRF4+ or CD56+/CD146+, and CD45-/CD34-/CD 144-.
3. The use of claim 1, wherein said ameliorating muscle atrophy comprises promoting muscle fiber repair, increasing muscle mass, improving muscle function, or a combination thereof.
4. The use of claim 1, wherein said amelioration of nerve damage is repair of nerve morphology and function.
5. The use of claim 1, wherein said formulation or composition comprises: (a) myogenic cells; and (b) a hydrogel, preferably, said hydrogel is a human hydrogel, more preferably, said hydrogel is selected from the group consisting of: collagen, vimentin, laminin, or a combination thereof.
6. A composition for use in the treatment of neurogenic muscle atrophy, said composition comprising (a) myogenic cells; and (b) a hydrogel, wherein said myogenic cells express a marker gene selected from the group consisting of:
PAX3+, NCAM +, Myosin Heavy Chain +, Myogenin +, MRF4+ or CD56+/CD146+, and CD45-/CD34-/CD 144-.
7. A method for non-therapeutically ameliorating muscle atrophy and nerve damage caused by neurogenic muscle atrophy, comprising the steps of:
(a) providing an embryonic stem cell, an induced pluripotent stem cell or a muscle stem cell;
(b) inducing differentiation of the cells of step (a) under conditions suitable for differentiation, thereby obtaining a cell population comprising differentiated myogenic cells; and
(c) injecting the myogenic cells obtained in step (b) into a desired site, thereby ameliorating muscular atrophy and nerve damage.
8. The method of claim 7, wherein in step (b) further comprising the step of isolating myogenic cells in said cell population, preferably myogenic cells sorted by flow cytometry for CD56+/CD146+/CD45-/CD34-/CD 144-.
9. The method of claim 7, wherein step (c) comprises the step of encapsulating said myogenic cells with hydrogel.
10. The method of claim 7, wherein the method is a method of constructing an animal model.
Technical Field
The invention relates to the field of biological treatment, in particular to a pharmaceutical composition for treating peripheral nerve injury type muscular atrophy, and a preparation method and application thereof.
Background
Muscle atrophy refers to the reduction of muscle volume caused by striated muscle dystrophy, thinning and even loss of muscle fibers, and the like. Muscle strength depends on the size of the muscle fibers, and muscle weakness occurs when muscle atrophy occurs. The classification of primary lesions that cause muscle atrophy can be mainly divided into three categories: myogenic muscular atrophy, neurogenic muscular atrophy, disuse muscular atrophy. The diseases of muscular neurogenic damage are the general names of upper motor nerve damage, lower motor nerve damage, amyotrophic lateral sclerosis, spinal muscular atrophy, progressive bulbar palsy, etc. Muscular atrophy caused by Peripheral Nerve Injury (PNI) is also common in clinic, for example, atrophy of lower limbs caused by compression of sciatic nerve, facial muscular atrophy caused by facial neuritis and the like, although the muscular atrophy is not fatal, the life quality of a patient is seriously affected. And even if the peripheral nerve is treated and repaired, the quality of the muscle and the symptoms of muscular atrophy are not improved. Therefore, there is an urgent need in the art to develop formulations and methods to ameliorate neurogenic muscle atrophy.
Disclosure of Invention
The invention aims to provide a pharmaceutical composition for treating peripheral nerve injury type muscular atrophy and a preparation method and application thereof.
In a first aspect of the invention, there is provided the use of myogenic cells for the preparation of a formulation or composition for the treatment of neurogenic muscle atrophy, wherein said treatment comprises amelioration of muscle atrophy and nerve damage.
In another preferred embodiment, the neurogenic muscle atrophy comprises peripheral nerve injury muscle atrophy.
In another preferred embodiment, the myogenic cells are differentiated from embryonic stem cells, induced stem cells and muscle stem cells.
In another preferred embodiment, the myogenic cells have the potential to differentiate into myogenic cells.
In another preferred embodiment, said myogenic cells express a marker gene selected from the group consisting of:
PAX3+, NCAM +, Myosin Heavy Chain +, Myogenin +, MRF4+ or CD56+/CD146+, and CD45-/CD34-/CD 144-.
In another preferred embodiment, said myogenic cells are human or non-human mammalian cells.
In another preferred embodiment, the improvement in muscle atrophy comprises promotion of muscle fiber repair, increased muscle mass, improved muscle function, or a combination thereof.
In another preferred embodiment, the improvement of nerve damage refers to repair of the shape and function of nerves.
In another preferred embodiment, the formulation or composition comprises: (a) myogenic cells; and (b) a hydrogel.
In another preferred embodiment, the hydrogel is a human hydrogel, and preferably, the hydrogel is selected from the group consisting of: collagen, vimentin, laminin, or a combination thereof.
In another preferred embodiment, the ratio of myogenic cells to hydrogel is (1 × 10)4-1×105) Individual cells/μ L hydrogel.
In another preferred embodiment, the composition is a pharmaceutical composition.
In another preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
In another preferred embodiment, the pharmaceutical composition is in the form of injection.
In a second aspect of the invention, there is provided a composition for use in the treatment of neurogenic muscle atrophy, said composition comprising (a) myogenic cells; and (b) a hydrogel, wherein said myogenic cells express a marker gene selected from the group consisting of:
PAX3+, NCAM +, Myosin Heavy Chain +, Myogenin +, MRF4+ or CD56+/CD146+, and CD45-/CD34-/CD 144-.
In a third aspect of the present invention, there is provided a method for non-therapeutically ameliorating neurogenic muscle atrophy and nerve damage, comprising the steps of:
(a) providing an embryonic stem cell, an induced pluripotent stem cell or a muscle stem cell;
(b) inducing differentiation of the cells of step (a) under conditions suitable for differentiation, thereby obtaining a cell population comprising differentiated myogenic cells; and
(c) injecting the myogenic cells obtained in step (b) into a desired site, thereby ameliorating muscular atrophy and nerve damage.
In another preferred embodiment, in step (b), the method further comprises the step of isolating myogenic cells in said cell population.
In another preferred embodiment, the myogenic cells are flow cytometrically sorted for CD56+/CD146+/CD45-/CD34-/CD 144-.
In another preferred embodiment, flow cytometry is performed by using FACS Aria (BD).
In another preferred embodiment, in step (b), the expression level of the marker gene is detected, and when the expression of PAX3, NCAM, myosury Chain, Myogenin, MRF4 is increased, differentiation is stopped and myogenic cells in the cell population are isolated.
In another preferred embodiment, the expression increase is the ratio of the expression level of the gene V1 to the expression level of the corresponding gene V2 in undifferentiated cells, V1/V2 is 1.2 or more, preferably 1.5 or more, more preferably 2 or more.
In another preferred embodiment, step (c) comprises the step of encapsulating said myogenic cells with hydrogel.
In another preferred embodiment, in step (c), 50 to 100 points per square centimeter of the site of muscle atrophy are injected.
In another preferred embodiment, in step (c), the number of myogenic cells transplanted per spot is 1 × 104～1×105。
In another preferred embodiment, in step (c), the hydrogel cell volume injected per spot is 0.8-1.2. mu.L.
In another preferred embodiment, the method is a method of constructing an animal model.
In another preferred embodiment, the method is directed to a non-human mammal.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows a fluorescent staining pattern of dystrophin antibody on the transverse section of gastrocnemius muscle 8 weeks after myogenic cell transplantation. Wherein, Sham: sham group, Model + mat: matrigel (Matrigel Matrix Growth Factor Reduced) treatment group, Model + cell: and a tissue regeneration cell treatment group is wrapped by the matrigel. Red: dysphilin, blue: DAPI, scale: 100 μm.
FIG. 2 shows graphs of the SABC and HE staining of the gastrocnemius cross section 8 weeks after myogenic cell transplantation. Wherein, Sham: sham group, Model + mat: matrigel (Matrigel Matrix Growth Factor Reduced) treatment group, Model + cell: and a tissue regeneration cell treatment group is wrapped by the matrigel. Brown: anti-GFP, Scale: 100 μm.
FIG. 3 shows a graph of SABC and HE staining of a longitudinal section of gastrocnemius muscle in mice 8 weeks after myogenic cell transplantation. Wherein, Sham: sham group, Model + mat: matrigel (Matrigel Matrix Growth Factor Reduced) treatment group, Model + cell: and a tissue regeneration cell treatment group is wrapped by the matrigel. Brown: anti-GFP, Scale: 100 μm.
Figure 4 shows the muscle strength measurements of mice 8 weeks after myogenic cell transplantation. Wherein, Sham: sham group, Model + mat: matrigel (Matrigel Matrix Growth Factor Reduced) treatment group, Model + cell: and a tissue regeneration cell treatment group is wrapped by the matrigel.
FIG. 4A shows the results of the maximal twitch force measurements.
Fig. 4B shows the result of the detection of maximal rigidity.
Figure 5 shows cross-section and longitudinal section HE staining of sciatic nerve 2 and 10 weeks after sciatic nerve clamping. Wherein, Sham: a sham operation group; model: constructing a muscle atrophy model group caused by peripheral nerve injury; model + mat: matrigel (Matrigel Matrix Growth Factor Reduced) treatment for the 8-week group; model + cell: the tissue regeneration cells are wrapped by the matrigel and treated for 8 weeks. A scale: 100 μm.
Detailed Description
The inventor of the present invention has extensively and deeply studied, for the first time unexpectedly found that myogenic cells can treat neurogenic muscle atrophy. Neurogenic muscular atrophy is muscular atrophy caused by nerve injury, and even if peripheral nerves are treated and repaired, the quality of muscles and symptoms of muscular atrophy cannot be improved, so that the treatment difficulty is high. The inventor applies the myogenic cells to treat the muscular atrophy, and the results show that the muscle quality and function are obviously improved, the nerve injury is also repaired, and the quite unexpected beneficial effects are achieved.
Term(s) for
In order that the disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning given below, unless explicitly specified otherwise herein. Other definitions are set forth throughout the application.
As used herein, the term "myogenic cell" is a cell that has the potential to differentiate into a muscle cell. Under physiological conditions, adult muscle stem cells can differentiate into myogenic cells and further into functional muscle cells. The myogenic cells can also be obtained by inducing human embryonic stem cells, human induced stem cells, and the like by a specific method.
As used herein, the terms "neurogenic muscle atrophy", "neurogenic damage of a muscle" have the same meaning and refer to muscle atrophy due to nerve damage, particularly peripheral nerve damage.
The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.
Muscular atrophy
Muscle atrophy refers to the reduction of muscle volume caused by striated muscle dystrophy, thinning and even loss of muscle fibers, and the like. The classification of primary lesions that cause muscle atrophy can be mainly divided into three categories: neurogenic muscular atrophy, myogenic muscular atrophy, disuse muscular atrophy. The pathogenesis and pathological mechanism of the cancer are different.
1. Differentiation is made according to the cause of the disease:
1) in neurogenic atrophy, for example, the peripheral nervous system, the muscles innervated by the peripheral nerve trunk or branches thereof are injured by external direct or indirect force.
2) In myogenic muscle atrophy, muscular dystrophy is a common symptom. Progressive Muscular Dystrophy (PMD) is a group of inherited muscle diseases, mostly due to defects in specific proteins on the muscle fibers.
3) Disuse muscle atrophy is caused by prolonged activity arrest impairing the contractility and muscle strength of skeletal muscles.
2. Differentiation is based on the difference of pathology and molecular mechanism:
1) for neurogenic muscular atrophy, after peripheral nerve injury, the broken ends of nerve fiber injury are degenerated, myelin sheaths are damaged, and the nerve fiber injury is broken into fragments;
the distal axon is subjected to Waller degeneration, cytoskeleton in the axon is collapsed, partial expansion and stenosis occur, granular decomposition occurs, granular substances in the axon plasma are accumulated, and the axon is subjected to swelling, breaking and dissolving; if the lesion is close to the neuron, apoptosis of the neuron may be caused.
Sciatic nerve injury, a common peripheral neuropathy, usually causes distal axonal degeneration and muscle neurodegeneration, and the fibrosis and scar tissue generated after injury can undergo secondary myofibroatrophy, so that the recovery of muscle function is limited, the axon of nerve innervating muscles grows slowly (1-2 mm/day), and the characteristics and prognosis after injury have some commonality regardless of the cause of the injury.
Peripheral nerves have a innervation effect on skeletal muscle and a nutritional effect on skeletal muscle, so skeletal muscle denervation is both dystrophy and disuse atrophy.
2) As for PMD, the specific pathogenesis is not clear, and the current research shows that the inflammation reaction is possibly related to inflammation reaction, oxidative stress, autophagy, apoptosis and the like, more and more evidences indicate that the inflammation reaction is involved in the pathophysiological process of PMD, and dysferlinopahies, DMD, FSHD, other LGMD subtypes and other patients have been reported to find the inflammation reaction in the muscle pathology, show that dystrophin is related to the inflammation reaction, such as dysferlin playing a crucial role in the biomembrane repair, the deleted sarcolemma repair function of the dysferlin is also damaged, some endogenous 'dangerous' molecules are continuously released, proinflammatory cytokines such as IL-1 β are stimulated, and some can activate the complement system, produce proinflammatory mediators and regulator C3b and up-regulate inflammasome, and finally cause cell injury and necrosis.
In addition, many studies have shown that genes associated with inflammatory responses and some inflammatory molecules, including cytokines and cell adhesion molecules, are highly expressed in dystrophin-deleted affected muscle tissues (mdx mice and DMD patients).
In view of the above, it is presently believed that the inflammatory response can lead to the development and progression of muscle damage.
3) In the case of disuse muscle atrophy, recent studies have shown that the signal pathway of redox reactions, which is the most important regulator of regulation of proteolysis and protein synthesis in skeletal muscle, is disturbed by the increase of reactive oxygen species ROS in muscle and the decrease of muscle's antioxidant capacity. This suggests that there may be a large relationship between disuse muscle atrophy and the molecular mechanisms described above.
Induction method
Methods for inducing differentiation of human embryonic stem cells, human induced stem cells, and human muscle stem cells into myogenic cells are performed according to the prior art in the field. The application in clinic is optimized by using proper culture reagents and methods according to clinical cell therapy standards.
Preferred methods for inducing human embryonic stem cells are as follows:
culture of hESCs
2 days before hESCs passage, irradiated CF1 feeder layer cells were seeded into Matrigel-coated culture flasks at a density of 30,000 cells/cm2. The medium for feeder cells was DMEM containing 10% fetal bovine serum. Only before hESCs passage the medium was changed to hESCs medium, containing (80% DMEM/F12, 20% Knockout serumplacement, 4ng/ml bFGF, 2 mmol/L-glutamine, 1% non-essential amino acids, 0.1mM β -mercaptoethanol), hESCs passage time of approximately 1 week/time.
Collagenase IV was used to isolate feeder layer cell clumps, and hESCs was washed twice with hESCs broth to allow cells to be cloned to the appropriate size and passaged to plate onto new feeder layer cells. Finally, the hESCs stocks contain 20% high quality FBS for embryonic stem cells, 70% hESCs culture and 10% DMSO.
2. Construction of Lentiviral vectors
rtTA, TRE and Hyg were cloned by a commercial RevTet systemrAn element; cloning of Puro by RNAi vector (pSIREN)rThe fragment is obtained by cloning IRE fragment through a bicistronic expression vector (pIRES2-GFP), obtaining an Embryoid Body (EB) through culturing an H1 cell line for 10 days, extracting cDNA to obtain a MyoD fragment, and finally using a second generation lentiviral vector with a promoter EF1 α as a framework.
3. Production of lentiviruses
1) Transfecting 293T cells with lentiviral vectors and helper vectors (Δ 8.91 and pvvg);
2) the transfection agent is FuGENE HD;
3) the fluid was changed 24 hours after transfection;
4) lentiviruses were collected 48, 72, 96 hours after transfection.
Establishment of Tet-on induced Gene expression System in hESCs
1) Establishing a Tet-on system on a HuES 17 cell line;
2) infecting the HuES-17 cell line with Lv-EF1a-rtTA-IRES-hygr lentivirus in feeder-free cell culture;
3) fluid changes were done 24 hours after infection, using Hygromycin B (50 μ g/mL) 48 hours after infection;
4) after 8 consecutive days of selection using the Hygromycin B culture, the cultured clones were isolated as single cells at 500/cm using TrypLE2The density of (a) was inoculated in a culture flask coated with CF 1;
5) after 2 weeks of passage, the single cell cultured clones were inoculated into fresh CF1 coated flasks, this batch of clones being designated H17-rtTA;
6) infecting H17-rtTA with Lv-EF1a-puro-TRE-GFP and Lv-EF1a-GFP-TRE-MyoD lentivirus, respectively, in the absence of feeder layer cell culture;
7) H17-Tet-on-GFP (H17-iGFP, GFP inducible) and H17-Tet-on-MyoD (H17-iMyoD, MyoD inducible) cell lines were obtained by the culture method of 3) -5).
Both cell lines have typical hESCs morphology (larger nuclei), while the hESCs pluripotency marker maintains high expression; and the induction kinetics of the Tet-on system was tested using the H17-iGFP line.
5. Overexpression of MyoD induces hESCs as early mesodermal cells
1) Culturing H17-iMyoD and H17-iGFP cells without trophoblast cells, and adding 1 μ g/ml of Doxycyline;
2) after 2-4 days of Doxycyline treatment, H17-iMyoD cells showed the characteristics of fibroblasts and become spindle-shaped cells with two sharp heads, while iGFP cells were not changed;
3) the expression levels of pluripotent stem markers Oct4 and Nanog of H17-iMyoD after 4 days of Doxycyline treatment are obviously reduced by immunostaining, and the mRNA expression level is the same as that of immunostaining;
4) detecting endoderm markers Sox17 and FoxA2 of H17-iMyoD cells after 4 days of Doxycyline treatment; mesodermal markers T and ACTC 1; ectodermal markers Pax6 and NFH; markers for trophectoderm, Hand1 and CDX2, were found to be significantly upregulated. Indicating that overexpression of MyoD in hESCs allows cells to lose sternness and differentiate towards the mesoderm.
6. Overexpression of MyoD can induce hESCs into myogenic cells
1) After 2 days of Doxycyline treatment of hESCs, the clones were again separated into single cells using trypsin and replated in matrigel-coated flasks;
2) continuing with hESCs conditioned medium containing 1. mu.g/ml Doxycyline, the cells became elongated and appeared similar to skeletal muscle myogenic cells upon induction of Doxycyline;
3) after 3 days of drug treatment, RT-PCR analysis shows that a satellite cell marker Pax7 is slightly up-regulated, Pax3 is obviously up-regulated, the expression of NCAM and endogenous MyoD is also obviously up-regulated, and the expression of another early myogenic regulatory factor Myf5 is not detected to be up-regulated in the differentiation process;
4) the results show that overexpression of MyoD induces differentiation of hESCs into myogenic cells.
Preferred methods for inducing differentiation of human induced stem cells and human embryonic stem cells into myogenic cells are as follows:
culturing human induced stem cells and human embryonic stem cells using irradiated mouse fibroblasts as feeder layer, the medium composition comprising F12/DMEM, 20% Knock Out Serum Replacer (KOSR), 1 × penicilin/streptomycin, 1% 100mM sodium sulfate, 1 × NEAA, 0.1% 0.1M β -mercaptoethanol and 2ng/mL FGF-2. the stem cell colony is separated from the mouse embryonic fibroblast layer using dispase and inoculated into a petrigel-coated culture dish containing mTESR1 medium day 2, adherent cells are digested into suspension using TrypLE and cultured in the form of 5000cells/cm2Inoculating to matrigel-coated Petri dishes containing mTESR1 Medium, and standing at 37 deg.C and 5% O2And 5% CO2Cultured in an incubator. On days 3-6, cells were maintained in serum-free DMEM/F12 basal medium containing 1% ITS and 0.2% penicillin/streptomycin; adding 3 μ M CHIRON99021 and 0.5 μ M LDN193189 to the culture medium at days 3-4; on days 5-6, 3. mu.M CHIRON99021, 0.5. mu.M LDN193189 and 20ng/mL FGF-2 were added simultaneously to the medium. From day 7 through day 30, the medium was changed to DMEM/F12+ 15% KOSR + 0.2% penicillin/streptomycin, and 10ng/mL HGF (Thermofisiher), 2ng/mL IGF-1 (R)&Dsystems), 20ng/mL FGF-2; in addition, 0.5 μ M LDN193189 is additionally added at 7-8 days; 2ng/mL IGF-1 was added additionally at 9-12 days; during the next 13-30 days of differentiation, cells were cultured in normal O2Concentration and 5% CO2The culture medium was supplemented with 10ng/mL HGF and 2ng/mL IGF-1. On day 31, cells were digested with TrypLE and re-seeded with DMEM/F12+ 1% penillin/streptomycin + 1% ITS + 1% N2supplement every 10 days.
A preferred method of inducing human muscle stem cell differentiation is as follows:
human skeletal muscle was minced and digested with enzyme (0.05% trypsin-EDTA, Gibco) and stirred for 60 minutes at 37 ℃. The process was terminated by the addition of 10% FCS (Life technologies). The cell suspension was then filtered using 70 μm and 40 μm nylon filters and then expanded for 5-7 days in Growth Medium (GM) containing Ham's F10(Life Technologies), 15% FCS, bovine serum albumin (Sigma-Aldrich; 0.5mg/ml), fetuin (Sigma-Aldrich; 0.5mg/ml), epidermal growth factor (Life Technologies; 10ng/ml), dexamethasone (Sigma-Aldrich; 0.39 μ g/ml), insulin (Sigma-Aldrich; 0.04mg/ml), creatine (Sigma-Aldrich; 1mM), pyruvic acid (Sigma-Aldrich; 100 μ g/ml), uridine (Sigma-Aldrich; 50 μ g/ml), gentamicin (Life Technologies; 5 μ g/ml).
To isolate pure human myoblasts, cultured cells were trypsinized and suspended in GM and incubated for 30min at 4 ℃ with anti-human monoclonal antibodies purchased from BD Biosciences (Franklin lake, N.J.), including anti CD56-AlexaFluor 488, anti CD45 PE-CF594, anti CD144-PE, anti CD34-APC, anti CD146-PECy 7. Human myoblasts were then washed and resuspended in GM. The purity was checked by flow cytometric sorting of human myoblasts using FACSAria (BD) as CD56+ CD146+ CD45-CD34-CD 144-and repeated analysis. Isotype control antibodies were PE-CF594-, APC-, PE-Cy7-, Alexa488 and PE-conjugated mouse IgG1 (both from BD Biosciences).
Flow cytometric analysis was performed by incubating myoblasts or human myogenic stock cells (MRC) with appropriately diluted mouse anti-human monoclonal antibodies (all from BD Biosciences) including CD56-Alexa488, CD146-PE-Cy7, CD73-FITC, CD90-FITC, CD105-FITC, Alkaline phosphotase-PerCP-Cy5.5, HLA ABC-PE, HLA DR-PE, CD82-PE and CD114-PE for 30 minutes on ice. The negative control samples were run at the same dose of FITC-, Alexa488-, PerCPCy5.5, PE-Cy 7-and PE-labeled isotype-matched antibodies (all from BD Biosciences).
Cell cycle analysis Fix/Perm buffer (BD Biosciences) and cells were incubated together on ice for 20 minutes to allowThe cells were permeabilized, washed twice with Perm/Wash buffer and then combined with the mouse anti-human antibody Ki67
The method for forming human muscle source reserve cells comprises the following steps:
human myoblasts were cultured in GM to aggregate and then transferred to Differentiation Medium (DM). DM is the addition of bovine serum albumin (Sigma-Aldrich; 0.5mg/ml), epidermal growth factor (Life Technologies; 0.01mg/ml), insulin (Sigma-Aldrich; 0.01mg/ml), creatine (Sigma-Aldrich; 1mM), pyruvic acid (Sigma-Aldrich; 100. mu.g/ml), uridine (Sigma-Aldrich; 50. mu.g/ml), gentamicin (Life Technologies; 10. mu.g/ml) in DMEM (Life Technologies) basal medium. After 2 days of culture in DM, human myotubes and non-confluent cells, defined as MRCs, were observed.
Human MRC was specifically obtained using a brief digestion (30 seconds) with pancreatin prior to cell transplantation experiments and flow cytometry analysis. I.e., all myotubes are specifically removed, leaving only quiescent undifferentiated adherent cells. To further eliminate the small myotubes, the pancreatinated MRCs can be filtered using a 20 μm pre-separation filter (Miltenyi Biotec, Bergisch Gladbach, germany) prior to the experiment. All cells used in vitro and in vivo experiments had a number of divisions of less than 20.
Identification of myogenic cells
1. Real-time quantitative PCR detection of myogenic cell marker gene (PAX3, NCAM, Myosin Heavy Chain, Myogenin, MRF4, etc.) expression
2. Immunocytochemical staining to identify the expression of myogenic cell marker genes (PAX3, NCAM, Myosin Heavy Chain, Myogenin, MRF4, etc.)
3. The flow cytometry signs the myogenic cells of CD56+/CD146+ and CD45-/CD34-/CD 144-.
Cell transplantation method
Injecting 50 to 100 points per square centimeter; the number of myogenic cells transplanted per spot is 1 × 104～1×105(ii) a The volume of hydrogel cells injected at each point was 1. mu.l; the cell concentration range is 1X 104～1×105/μl。
A micro single-needle syringe or a multi-needle syringe is used, and the needle is a 27G bevel needle.
Composition comprising a metal oxide and a metal oxide
The invention provides a composition for repairing neurogenic muscle atrophy, comprising the myogenic cells of the invention and a hydrogel. Wherein the myogenic cells are cells differentiated from embryonic stem cells, induced stem cells and muscle stem cells to have the potential of being differentiated into muscle cells.
The human hydrogel for encapsulating myogenic cells comprises: collagen, fibrin, fibronectin, laminin, vimentin, gelatin, proteoglycan, chitosan, hyaluronic acid, etc.
The main advantages of the invention include:
(a) the method of the invention can treat neurogenic muscle atrophy, improve muscle mass and symptoms of muscle atrophy.
(b) The methods of the invention can repair nerve damage in neurogenic muscle atrophy.
(c) The method is safe and effective, has no toxic or side effect, and is beneficial to popularization.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.