Patent Application
20230165907
The invention relates to an application of muscle stem cells in the preparation of drugs for the treatment of diabetes and belongs to the field of biomedical technology.
With the development of society and economy, the change of people’s lifestyle (increase in energy intake, decrease in exercise, etc.) and the aging of the population, the incidence of type 2 diabetes mellitus is increasing year by year worldwide, especially in developing countries (it is expected to increase by 170% from now to 2025), the rate of increase will be faster, showing a strong epidemic trend. Diabetes is now the third non-communicable disease threatening the health and lives of the population, after cardiovascular diseases and neoplasms. Obesity causes the increase of adipose tissue or fatty infiltration in liver, muscle and other tissues, and the tissues of the body change from the state of inhibiting inflammation to the state of promoting inflammation, thus destroying tissue homeostasis. The occurrence of tissue inflammation will reduce the sensitivity of the tissue to insulin and produce tolerance. With increasing inflammation, insulin tolerance increases and the body loses its ability to regulate blood sugar levels, gradually developing type 2 diabetes mellitus.
Skeletal muscle is the body’s largest tissue and organ, making up about 35 percent of the body’s body weight. It provides the necessary power for individual movement, and also participates in the precise regulation of body energy metabolism and body temperature. When glucose levels rise in blood and tissue fluid, insulin stimulates the liver, fat, muscle and other tissues to take up glucose. Skeletal muscle is the largest tissues and organ of glucose consumption. The tolerance of muscle, liver and fat to insulin is the most important factor to induce metabolic disorders and type 2 diabetes mellitus. Macrophage infiltration is significantly increased in muscle and fat of obese or insulin-tolerant subjects. As obesity increases, macrophages accumulate between fat cells and muscle fibers. Type I macrophages can promote inflammation and secrete various factors such as TNF-α and IL-1β. TNF can stimulate the inflammatory signaling pathway in myocytes in vitro, which directly leads to the decreased sensitivity of insulin signaling pathway.
Skeletal muscle is highly plastic and regenerative. Understandably, its high plasticity is due to the fact that each skeletal muscle has more than 600 muscle fibers of different sizes and different contractile capacities. These muscle fibers work together to provide support for movement, postural maintenance, strength or fine movement, and even breathing. Satellite cells, i.e., muscle stem cells play a very important role in the repair function of skeletal muscle injury. Under normal conditions, satellite cells are in a resting state. Once they receive signals of injury or growth, satellite cells are rapidly activated and extensively migrate to the injured area to proliferate, differentiate and fuse into new myofiber cells. After repair of muscle injury, some satellite cells resist differentiation and return to the resting state and return to the nest. The important point is that the fate of satellite cells is greatly influenced by both endogenous and exogenous factors. The activation of satellite cells is related to inflammatory cells, stromal cells, nutrient signals and extracellular matrix composition in the environment.
With the continuous improvement of people’s living standards, chronic diseases caused by diets high in fat, sugar and salt are increasing. Diabetes is one of the diseases that seriously affect human life and health, and has seriously threatened the quality of life of the global population. At present, the number of adult diabetic patients in China has reached 92.4 million, ranking first in the world. In type 2 diabetic subjects, adipose-infiltrated muscle tissue inflammation caused by obesity is enhanced, which on the one hand producing tolerance to insulin and on the other hand having an activating effect on satellite cells in the resting state.
There is an urgent need to develop effective drugs for type 2 diabetes mellitus.
The purpose of the present invention is to provide a muscle stem cell for use in the preparation of pharmaceutical compositions that enhance muscle content and function and prevent, alleviate and/or treat metabolic disorders such as type 2 diabetes mellitus.
In the first aspect of the present invention, it provides a use of muscle stem cells for the preparation of a pharmaceutical composition for one or more applications selected from the following groups:
In another preferred example, the drug is used on humans or animals.
In another preferred example, the reduction in fat content includes reduction in subcutaneous fat content, reduction in abdominal fat content, reduction in liver tissue weight, and reduction in body weight.
In another preferred example, the improvement of insulin tolerance is to increase the subject’s sensitivity to insulin.
In another preferred example, the genes related to brown fat expression are selected from the following group: ucpl, tbxcl, pgc1α, tmem26, prdml6, cidea, pgc1β, cpt1α, cpt1β.
In another preferred example, the genes related to white fat expression are selected from the following group: leptin, fabp4, ppary, c/ebpα, c/ebpβ, c/ebpγ, glut4, fasn, adiponectin.
In another preferred example, the muscle stem cells are selected from the following groups: murine-derived muscle stem cells, human-derived muscle stem cells, monkey-derived muscle stem cells, dog-derived muscle stem cells, cat-derived muscle stem cells, horse-derived muscle stem cells, and a combination thereof.
In another preferred example, the mouse muscle stem cells are CD31-, CD34-, CD45-, Vcam1+, Intergrin-α7+ and PAX7+ cells.
In another preferred example, the human-derived muscle stem cells are CD31-, CD34-, CD45-, CD29+, EGFR+ and PAX7+ cells.
In another preferred example, other muscle stem cells of non-rodent and non-human origin are CD31-, CD34-, CD45-, CD29+, EGFR+ and PAX7+ cells.
In another preferred example, the metabolic disorders are selected from the following groups: diabetes mellitus, fatty liver disease, hypercholesterolemia, insulin resistance disease, hyperglycemia disease.
In another preferred example, the diabetes mellitus is selected from the following groups: type 1 diabetes mellitus, type 2 diabetes mellitus.
In another preferred example, the muscle stem cells are stem cells that can differentiate into striated muscle cells and have some characteristics of stellate cells.
In another preferred example, the pharmaceutical composition is used for the prevention and/or treatment of low muscle support and inadequate exercise capacity.
In another preferred example, the muscle stem cells are autologous, allogeneic, or xenogeneic.
In another preferred example, the muscle stem cells were selected from the following parts: limb skeletal muscle, trunk skeletal muscle.
In another preferred example, the dosage form of the pharmaceutical composition is selected from the following group: freshly cultured muscle stem cell injection, or thawed muscle stem cell injection from frozen storage.
In another preferred example, the muscle stem cells are extracted from the body muscle and obtained by cell sorting and expansion.
In another preferred example, the muscle stem cells are extracted from the limbs or trunk muscles of the body and obtained by cell sorting.
In another preferred example, the muscle stem cells are muscle-derived stem cells that can differentiate into striated muscle cells and have the characteristics of stellate cells.
In another preferred example, the dosage form of the pharmaceutical composition is an injection.
In another preferred example, the improvement of body metabolism includes: improvement of body glucose metabolism, improvement of body fat metabolism, improvement of body blood lipid metabolism, and a combination thereof.
In the second aspect of the present invention, it provides a pharmaceutical composition for human or animal comprising:
In another preferred example, the pharmaceutical composition also comprises a second active ingredient that regulates glucose metabolism, preferably, the second active ingredient selected from the following group:
biguanides, sulfonylureas, non-sulfonylureas insulin secretagogues, thiazolidinedione insulin sensitizers, glycosidase inhibitors, insulin, glucagon-like peptide-1 analogues or agonists.
In another preferred example, the muscle stem cell is a mouse muscle stem cell, preferably, CD31-, CD34-, CD45-, Vcam1+, Intergrin-α7+ and PAX7+ cell.
In another preferred example, the muscle stem cell, preferably, the human-derived muscle stem cell is CD31-, CD34-, CD45-, CD29+, EGFR+ and PAX7+ cell.
In another preferred example, the muscle stem cell is non-rodent derived muscle stem cell, preferably, CD31-, CD34-, CD45-, CD29+, EGFR+ and PAX7+ cell.
In another preferred example, the pharmaceutical composition is an anti-type 1 diabetes mellitus drug.
In another preferred example, the pharmaceutical composition is an anti-type 2 diabetes mellitus drug.
In another preferred example, the pharmaceutical composition is a drug that improves the body’s glucose metabolism.
In another preferred example, the pharmaceutical composition is a drug for the prevention and/or treatment of muscle loss and strength deficiency.
In another preferred example, the pharmaceutical composition is a drug that maintains the homeostasis of the body’s white adipose tissue.
In another preferred example, the pharmaceutical composition is a drug that reduces the inflammatory environment of the body.
In another preferred example, the pharmaceutical composition is a drug that reduces the extent of fatty liver disease.
In another preferred example, the pharmaceutical composition is for the prevention and/or treatment of low muscle support.
In another preferred example, the pharmaceutical composition is for the prevention and/or treatment of insufficient athletic ability.
In another preferred example, the dosage form of the pharmaceutical composition is selected from the following group: fresh or frozen muscle stem cell injection, lyophilized agent.
In the third aspect of the invention, it provides a method of prevention, alleviation and/or treatment of metabolic disorders, which comprises: administering to a subject in need thereof a muscle stem cell, collection, cryostorage and pharmaceutical compositions as the second aspect of the present invention, or a preparation comprising a muscle stem cell or a pharmaceutical composition, and a combination thereof.
It is to be understood that the various technical features of the present invention mentioned above and the various technical features specifically described hereinafter (as in the Examples) may be combined with each other within the scope of the present invention to constitute a new or preferred technical solution, which needs not be described one by one, due to space limitations.
Wherein ND was a normal diet mice group and mice were fed with 10% Kcal of normal diet, HFD was a high-fat diet mice group and mice were fed with 60% Kcal of high-fat diet, and HFD + MuSC was a group of mice injected to high-fat diet mice with mouse muscle stem cells.
Wherein ND was a normal diet mice group and mice were fed with 10% Kcal of normal diet, HFD was a high-fat diet mice group and mice were fed with 60% Kcal of high-fat diet, and HFD + MuSC was a group of mice injected to high-fat diet mice with mouse muscle stem cells.
Wherein MHC staining was myosin heavy chain staining and Hochest staining was nuclear staining. Merge is a overlap graph.
Wherein ND was the normal diet mice group and mice were fed with 10% Kcal of normal diet, HFD was the high-fat diet mice group and mice were fed with 60% Kcal of high-fat diet, and HFD + Human MuSC (HFD + HuSC) was a group of mice injected to high-fat diet mice with human-derived muscle stem cells.
Wherein ND was the normal diet mice group and mice were fed with 10% Kcal of normal diet, HFD was the high-fat diet mice group and mice were fed with 60% Kcal of high-fat diet, and HFD + HuSC was a group of mice injected to high-fat diet mice with human-derived muscle stem cells.
wherein, ND was the normal diet mice group and mice were fed with 10% Kcal of normal diet, HFD was the high-fat diet mice group and mice were fed with 60% Kcal of high-fat diet, and HFD + HuSC was a group of mice injected to high-fat diet mice with human-derived muscle stem cells.
wherein, ND was the normal diet mice group and mice were fed with 10% Kcal of normal diet, HFD was the high-fat diet mice group and mice were fed with 60% Kcal of high-fat diet, and HFD + HuSC was a group of mice injected to high-fat diet mice with human-derived muscle stem cells.
Wherein MHC staining was myosin heavy chain staining, Hochest staining was nuclear staining. Merge is a overlap graph.
In all above results, *P<0.05; **P<0.01; ***P<0.001.
Wherein ND mice were fed with 10% Kcal of normal diet, HFD mice were fed with 60% Kcal of high fat diet, and HFD + MuSC mice were a group of mice injected to high-fat diet mice with Balb/C murine derived muscle stem cells. At week 14, mice were injected with muscle stem cells.
In all above results, *P<0.05; **P<0.01; ***P<0.001.
Through extensive and intensive research, the inventors have unexpectedly discovered that activated satellite cells have an inhibitory effect on the inflammatory response caused by obesity, thereby reducing the level of inflammation, enhancing the sensitivity of tissues to insulin, regulating the level of tissue metabolism, reducing blood glucose, reducing fat enlargement and infiltration, and playing a therapeutic role in type 2 diabetes mellitus.
Specifically, a mouse model of insulin tolerance induced by a high-fat diet is selected in the present invention to investigate the use of human derived muscle stem cells in the preparation of pharmaceutical compositions for the prevention, remission and/or treatment of metabolic disorders (such as type 2 diabetes mellitus). Human cells injected into mice can be eliminated by the body in a short period of time, which excludes the possibility of cell colonization, proliferation and differentiation in the subject.
Muscle stem cells were injected into the tail vein of insulin tolerant mice induced by high-fat diet in the present invention. The experimental results show that compared with model group, the treatment group can significantly improve the glucose metabolism ability of mice, reduce the white adipose tissue, alleviate the enlargement of adipocytes, improve the white adipose tissue homeostasis, alleviate the inflammatory environment of mice, and reduce the extent of fatty liver disease. The present invention has been completed on the basis of these studies.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as would normally be understood by ordinary technicians in the field to which the invention belongs. As used in this application, when referring to a specific enumerated value, the term “approximately” means that the value can vary by no more than 1% from the enumerated value. For example, as used in this application, the statement “about 100” includes all values between 99 and 101 and (for example, 99.1, 99.2, 99.3, 99.4, and so on).
Although any method and material similar or equivalent to those described in the present invention may be used in the implementation or testing of the present invention, the preferred method and material is presented here.
Mauro et al. first proposed the discovery of muscle satellite cells, muscle stem cell precursors, in frog skeletal muscle in 1961. Subsequently, more and more studies have proved that there are a few muscle stem cells in adult mammalian skeletal muscle, and the number of muscle stem cells gradually decreases with age.
Muscle satellite cells are myogenic stem cells located between the basement membrane of skeletal muscle and the membrane of muscle fibers. In physiological condition, muscle satellite cells exist in stationary undifferentiated state, after the damage or inflammation stimulation activated muscle stem cells to divide for the spindle muscle stem cells, and along with the increasing division multicore muscular tube mutual fusion, finally developed to form raw muscle fibers or incorporate into the damaged muscle tissue. It plays an important role in skeletal muscle growth, injury repair, function maintenance and tissue regeneration.
In a preferred example of the invention, the muscle stem cells are selected from the following groups: murine muscle stem cells, human-derived muscle stem cells, monkey muscle stem cells, dog muscle stem cells, cat muscle stem cells, horse muscle stem cells, etc.
In a preferred example of the invention, the mouse muscle stem cells are cells with CD31-, CD34-, CD45-, Vcam1+, Intergrin-α7+ and PAX7+.
In a preferred example of the invention, the human-derived muscle stem cells are cells with CD31-, CD34-, CD45-, CD29+, EGFR+ and PAX7+.
Other muscle stem cells of non-rodent and non-human origin are cells with CD31-, CD34-, CD45-, CD29+, EGFR+ and PAX7+.
In a preferred embodiment of the present invention, said muscle stem cells are muscle-derived, differentiable into striated muscle cells with some of the properties of stellate cells.
In a preferred example of the invention, the muscle stem cells are autologous, allogeneic, or xenogeneic.
In a preferred example of the invention, the muscle stem cells were selected from the following parts: limb skeletal muscle, trunk skeletal muscle.
In a preferred example of the invention, the muscle stem cells are extracted from the body muscle and obtained by cell sorting and expansion.
In a preferred example of the invention, the muscle stem cells are extracted from the limbs or trunk muscles of the body and obtained by cell sorting.
Mesenchymal stem cells are found in both skeletal and smooth muscle. The main role of muscle injury repair and regeneration is muscle stem cells, but some other groups of cells, such as inflammatory cells, vascular endothelial cells and muscle-derived mesenchymal stem cells, play a fine regulatory role in the whole process of muscle injury repair and regeneration. For example, fibroblast adipose progenitor cells are a kind of mesenchymal stem cells in the muscle stroma, whose role is to support the differentiation of muscle stem cells and promote the repair and regeneration of muscle tissue. The main role of smooth muscle-derived muscle stem cells in the blood vessels is to replace smooth muscle cells, so as to fill the area of vascular lesions or injuries.
Metabolic disorder is a state of the body, is the body’s absorption, digestion, excretion of substances appear pathological disorders, resulting in imbalance between supply and demand. It can be a disorder of one substance or many substances. The various metabolic state disorders vary differently. Glucose metabolism disorder causes diabetes, lipid metabolism disorder causes hyperlipidemia, uric acid metabolism disorder causes gout and so on. Electrolyte also can appear metabolic disorder, cause corresponding disorder state, such as high potassium, hypokalemia and so on. Metabolic diseases characterized by chronic hyperglycemia are often associated with lipid metabolism disorder in clinic, which has become one of the main complications of diabetes mellitus. The direct symptom of lipid metabolism disorder is the increase of total cholesterol index.
In a preferred example of the invention, the metabolic disorders are selected from the following groups: diabetes mellitus, fatty liver disease, hypercholesterolemia, insulin resistance disease, hyperglycemia disease.
In another preferred example of the invention, the diabetes mellitus is selected from the following group: type 1 diabetes mellitus, type 2 diabetes mellitus.
The invention also provides a composition. In the preferred example, the composition is a pharmaceutical composition comprising the aforementioned muscle stem cells and a pharmaceutically acceptable carrier. In general, these substances may be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably, pH is about 6-8, although the pH value may be varied depending on the nature of the substances to be formulated and the condition to be treated. The formulated pharmaceutical composition may be administered by conventional routes, including (but not limited to): oral, respiratory, intratumoral, intraperitoneal, intravenous, or topical administration.
The pharmaceutical composition of the invention may be used directly for the treatment (for example, for the treatment of metabolic disorders). In addition, other therapeutic agents can also be used simultaneously.
The pharmaceutical composition according to the present invention comprises a safe and effective amount (e.g. 0.001-99 wt%, preferably 0.01-90 wt%, preferably 0.1-80 wt%) of the muscle stem cells according to the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffer solution, glucose, water, glycerin, ethanol, dimethyl sulfoxide (DMSO) or the combination thereof. The pharmaceutical preparation should be matched to the method of administration. The pharmaceutical composition of the present invention can be prepared in the form of injection, for example, prepared by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably prepared under sterile conditions. The dosage of active ingredient is therapeutically effective amount, for example from about 1 microgram per kilogram body weight to about 10 milligrams per kilogram body weight per day. Further, the muscle stem cells of the present invention can also be used in combination with the other therapeutic agents.
When a pharmaceutical composition is used, a safe and effective amount of the muscle stem cells is administered to a mammal, wherein the safe and effective amount is usually at least about 10 µg/Kg body weight, and in most cases does not exceed about 8 mg/kg body weight, preferably the dose is about 10 µg/Kg body weight to about 1 mg/kg body weight. Of course, the particular dose should also depend on various factors, such as the route of administration, patient healthy status, which are well within the skills of an experienced physician.
In a preferred example of the invention, the pharmaceutical composition comprising:
In a preferred example of the invention, the mouse muscle stem cells are cells with CD31-, CD34-, CD45-, Vcam1+, Intergrin-α7+ and PAX7+.
In another preferred example, the human-derived muscle stem cells are cells with CD31-, CD34-, CD45-, CD29+, EGFR+ and PAX7+.
Other muscle stem cells of non-rodent and non-human origin are cells with CD31-, CD34-, CD45-, CD29+, EGFR+ and PAX7+.
In a preferred example of the invention, the pharmaceutical composition is an anti-type 1 diabetes mellitus drug.
In a preferred example of the invention, the pharmaceutical composition is an anti-type 2 diabetes mellitus drug.
In a preferred example of the invention, the pharmaceutical composition is a drug that improves the body’s glucose metabolism.
In another preferred example, the pharmaceutical composition is for the prevention and/or treatment of low muscle support.
In another preferred example, the pharmaceutical composition is for the prevention and/or treatment of insufficient athletic ability.
In a preferred example of the invention, the pharmaceutical composition is a drug that maintains the homeostasis of the body’s white adipose tissue.
In a preferred example of the invention, the pharmaceutical composition is a drug that reduces the inflammatory environment of the body.
In a preferred example of the invention, the pharmaceutical composition is a drug that reduces the extent of fatty liver disease.
In a preferred example of the invention, the dosage form of the drug composition is selected from the following groups: injection and lyophilized agent.
In a preferred example of the invention, the dosage form of the anti-type 2 diabetes mellitus drug is selected from the following group: freshly cultured muscle stem cell injection, or thawed muscle stem cell injection from frozen storage.
In a preferred example of the invention, the anti-type 2 diabetes drug can significantly improve the glucose metabolism ability of mice and reduce blood glucose.
In a preferred example of the invention, the anti-type 2 diabetes drug is able to reduce white adipose tissue weight, relieve adipocyte hypertrophy, and improve white adipose tissue homeostasis.
In a preferred example of the invention, the anti-type 2 diabetes drug is able to reduce the level of inflammation in mice.
In a preferred example of the invention, the anti-type 2 diabetes drug is able to reduce the extent of fatty liver disease.
The main advantages of the present invention include:
The present invention will be further illustrated below with reference to the specific examples. It is to be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. For the experimental methods in the following examples, in which the specific conditions are not specifically indicated, they are performed under routine conditions, e.g., those described by Sambrook. et al., in Molecule Clone: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 1989, or as instructed by the manufacturers, unless otherwise specified. Unless indicated otherwise, parts and percentage are weight parts and weight percentage. The experimental materials involved in the invention can be obtained from commercial channels without special instructions.
1. Hind leg muscle tissue of C57/BL mice (C57/BL mice are used unless otherwise noted in the following) was cut into pieces to form tissue serosa, 10 mL collagenase type II (750 U/mL) was added, and was incubated at 37° C. for 60 min at a constant temperature shaker with a rotational speed of 70 rpm.
2. After incubation and digestion, complete medium (containing 10% FBS) was used for neutralization, and the supernatant was removed after centrifugation.
3. The mixed digestion solution of 10 mL collagenase type II (100 U/mL) and dispersive enzyme (1.1 U/mL) was added to the precipitated cell pellet again, and the cell precipitation was resuspended.
4. The cell suspension was incubated again in a constant temperature shaker for digestion. The incubation condition was 37° C. for 60 min, and the shaker speed was 70 rpm.
5. At the end of incubation, neutralizing solution was added to terminate digestion, and the obtained liquid was centrifuged for 5 min, and the supernatant was removed to obtain cell precipitation.
6. The cell precipitate was resuspended, and incubated with the mixed solution of monoclonal antibody labeled with magnetic beads in the mouse muscle satellite cell separation kit for 30 min. Non-target cells would directly bind to the antibody. After cleaning with neutralizing solution, negative sorting of magnetic beads was carried out.
7. After magnetic bead sorting, cell complete medium (80% F12 medium, 20% fetal bovine serum, 1% P/S, 100 ng/mL TNF-α, 100 ng/mL IFN-y, 50 ng/mL IL-1α, 50 ng/mL IL-13) was added. Then the mixture was transferred to petri dishes coated with collagen in advance and cultured in an incubator.
1. When the cell proliferation reached 70% confusion, the cells were digested and passaged, and the cells were implanted in a new extracellular matrix (ECM) coated petri dish.
2. Flow cytometric antibodies CD31--PE, CD34--PE, CD45--PE, Vcam1+-BV421 and Intergrin-α7+-BV421 were used to stain and identify mouse muscle stem cells. The expression of specific nuclear factor PAX 7 in mouse muscle stem cells was determined by intracellular staining and flow cytometry.
1. The muscle stem cells were cultured on collagen-coated plates until the cells were completely fused.
2. Cells were washed three times with serum-free DMEM medium, and then the medium was replaced with muscle stem cell differentiation medium (DMEM medium + 2% horse serum) for further culture. On the first day of differentiation culture, the cells began to elongate and intercellular fusion occurred. On the 2nd to 3rd day, cell fusion reached the peak, and most of the cells had completed the differentiation process into myotubes. At this time, immunofluorescence staining was performed for myogen heavy chain (MyHC), which is a mature protein specific differentiated by human-derived muscle stem cells, thus, identification experiments were performed to determine that the isolated cells had the differentiation characteristics of stem cells. The result is shown in
The result is shown in
Male C57/BL mice aged 4 weeks were randomly selected and divided intogroups, which were fed a normal diet containing 10% Kal fat and a high-fat diet containing 60% Kal fat, respectively. The body weight of mice was recorded daily to form a curve of body weight change. After 2-3 months of modeling, mice weighing about 40 g were selected for the experiment. Insulin tolerance was developed in high-fat diet fed mice due to obesity and can be used as a model of type 2 diabetes.
1. After the establishment of the model, the mice were divided into three groups: control group (normal diet group), model group (high-fat diet group) and treatment group (high-fat diet mice group injected with mouse muscle stem cells). Then the experiment was ready to begin.
2. The control group and the model group were injected with 200 µL of the injection system sterile PBS every week by tail vein injection. The treatment group was injected with mouse muscle stem cells, the injection volume was 1-5×105 cells per mouse, and the injection system was 200 µL. The injection cycle was once a week, and a total of 6 injections were given.
The result is shown in
1. Model mice in Example 2 were given a treatment of fasting without water abstinence 15 hours before the test.
2. Fasting blood glucose of mice was detected by Byino blood glucose meter and blood glucose test strip, which was recorded as G0.
3. Mice were intraperitoneally injected with glucose at a dose of 1 g/g. Immediately after injection, the time at the moment was recorded as T0.
After 15 min(T15), 30 min(T30), 45 min(T45), 60 min(T60) and 120 min(T120), the blood glucose of the mice was measured again and recorded as G15, G30, G45, G60 and G120, respectively.
1. Fasting blood glucose of mice was detected by Byino blood glucose meter and blood glucose test strip, which was recorded as G0.
2. Mice were intraperitoneally injected with short-acting insulin at a dose of 0.1 U/g mouse. Immediately after injection, the time at the moment was recorded as T0.
3. After 15 min(T15), 30 min(T30), 45 min(T45), 60 min(T60) and 120 min(T120), the blood glucose of the mice was measured again and recorded as G15, G30, G45, G60 and G120, respectively.
The hypoglycemic ability of mice was detected by glucose tolerance and insulin tolerance test, so as to evaluate the glucose metabolism level of mice.
The result is shown in
The subcutaneous adipose tissue of the model mice in Example 2 of each group was separated, weighed and detected, and the weight of adipose tissue of each group was compared.
In type 2 diabetes caused by obesity, the liver tissue was complicated with fatty infiltration, forming fatty liver. The liver tissue of mice was removed and weighed for detection, and the weight of liver tissue of each group was compared.
1. Peripheral blood of mice was collected and left for 30 min before centrifugation. The centrifugation condition was set at 300 g for 30 min.
2. Total cholesterol detection kit was used to detect the total cholesterol content in mice.
The result is shown in
1. Human muscle tissue was obtained from the hospital, which was cut into pieces to form tissue serosa, and put into a 50 mL centrifuge tube.
2. 10 mL collagenase type II (750 U/mL) was added to the tissue serosa and was placed in a constant temperature shaker and incubated at 37° C. for 60 min with a shaker rotation speed of 70 rpm.
3. After incubation and digestion, complete medium (containing 10% FBS) was used for neutralization, and the supernatant was removed after centrifugation.
4. The mixed digestion solution of 10 mL collagenase type II (100 U/mL) and dispersive enzyme (1.1 U/mL) was added to the precipitated cell pellet again, and the cell precipitation was resuspended.
5. The cell suspension was incubated again in a constant temperature shaker for digestion. The incubation condition was 37° C. for 60 min, and the shaker speed was 70 rpm.
6. At the end of incubation, 10 mL of complete medium was added for neutralization and filtered through a 40 µm cell screen.
7. After filtration, the obtained liquid was centrifuged at 500 g for 5 min, and the supernatant was removed to obtain cell precipitation.
8. Cell precipitation was resuspended with 600 µL complete medium, and flow sorting was carried out.
9. 10 µL of cell suspension was taken into a flow tube containing 190 µL complete medium for control.
10. The remaining cell suspension was stained with flow sorting antibody, and the labeled antibodies were CD31-PE, CD34-PE, CD45-PE, CD29-APC and EGFR-BV421 for 30 min. After staining, the complete medium was washed and centrifuged, and finally cells were resuspended using 200 µL of complete medium.
11. The sample was loaded and subjected to flow sorting.
1. After all cell suspensions were sorted, cells were centrifuged at 250 g for 5 min and the supernatant of washing medium was removed, and muscle stem cell growth medium (20% FBS, 40% DMEM/LOW, 40% MCDB131, 1%P/S, 1% insulin transferrin selenium, 10 µM P38 inhibitor) was added. After resuspended, cultures were transferred to extracellular Matrix coated petri dishes (Extra Cellular Matrix, ECM coated for 24 hours) for culture and amplification.
2. The fresh medium was changed every two days until the degree of cell confluence reaches 60 ~ 70%.
3. Muscle stem cells were passaged with trypsin, and then the progeny cells were grown in new ECM-coated petri dishes for further culture and expansion.
1. The cells were grown to a confluent degree of 70%, and the required cells could be obtained by trypsin digestion.
2. Flow cytometry was used to identify the marker proteins expressed on the surface of human-derived muscle stem cells, and the expression of human-derived muscle stem cell specific nuclear factor PAX7 was identified by flow cytometry.
Cryo-storage of human-derived muscle stem cells. The expanded muscle stem cells in (2) can be long-term stored in liquid nitrogen in cryogenic solution containing dimethyl sulfoxide (DMSO).
1. The muscle stem cells were cultured on collagen-coated plates until the cells were completely fused.
2. Cells were washed three times with serum-free DMEM medium, and then replaced with muscle stem cell differentiation medium (DMEM medium +2% horse serum) for further culture. On the first day of differentiation culture, the cells began to elongate and intercellular fusion occurred. On the 2nd to 3rd day, cell fusion reached the peak, and most of the cells had completed the differentiation process into myotubes. At this time, immunofluorescence staining was performed for myogen heavy chain (MyHC), which is the specific differentiation mature protein of human-derived muscle stem cells, so as to identify experiments to determine that the isolated cells have the differentiation characteristics of stem cells.
The result is shown in
Male C57/BL mice aged 4 weeks were randomly selected and divided into groups. The mice were fed a normal diet containing 10% Kal fat and a high-fat diet containing 60% Kal fat, respectively. The body weight of mice was recorded to form a body weight change curve. After 2-3 months of modeling, mice with a body weight of about 40 g were selected for grouping and experiment, and the body weight of each mouse was continuously recorded. Insulin tolerance was developed in high-fat diet mice due to obesity, and thus can be used as a human type 2 diabetes model.
1. After the establishment of the model, the mice were divided into three groups: control group (normal diet group), model group (high-fat diet group) and treatment group (high-fat diet mice group injected with human-derived muscle stem cells).
2. The control group and the model group were injected with 200 µL of the injection system sterile PBS every week by tail vein injection. The treatment group was injected with human-derived muscle stem cells (Human MuSC, HuSC), the injection volume was 1-5 ×105 cells per mouse, and the injection system was 200 µL. The injection cycle was once a week for a total of 6 injections.
The result is shown in
1. Model mice constructed in Example 6 were given a treatment of fasting without water abstinence 15 hours before the test.
2. Fasting blood glucose of mice was detected by Byino blood glucose meter and blood glucose test strip, which was recorded as G0.
3. Mice were intraperitoneally injected with glucose at a dose of 1 g/g mice. Immediately after injection, the time at the moment was recorded as T0.
4. After 15 min(T15), 30 min(T30), 45 min(T45), 60 min(T60) and 120 min(T120), the blood glucose of the mice was measured again, and recorded as G15, G30, G45, G60 and G120, respectively.
1. Fasting blood glucose of mice was detected by Byino blood glucose meter and blood glucose test strip, which was recorded as G0.
2. Mice were intraperitoneally injected with short-acting insulin at a dose of 0.1 U/g mice. Immediately after injection, the time at the moment was recorded as T0.
3. After 15 min(T15), 30 min(T30), 45 min(T45), 60 min(T60) and 120 min(T120), the blood glucose of the mice was measured again and recorded as G15, G30, G45, G60 and G120, respectively.
1. Mouse blood was taken and left for 30 min before centrifugation. The centrifugation condition was set at 300 g for 30 min. The upper serum was aspirated for later use.
2. ELISA kit was used to detect the serum insulin content and total cholesterol content in peripheral blood of mice.
According to the above values, analysis and comparison are carried out, and the results are shown in
In conclusion, human-derived muscle stem cells have a good improvement effect on glucose metabolism in mice.
In peripheral blood, the content of total cholesterol in the treatment group was significantly lower than that in the model group, indicating that human-derived muscle stem cells also have a certain regulatory function on lipid metabolism.
The body weight, subcutaneous white adipose tissue weight and abdominal white adipose tissue weight of the model mice constructed in Example 6 were weighed respectively. And the analysis was calculated according to the weighing value.
1. The mice were sacrificed under anesthesia, and the abdominal adipose tissue was obtained, and the tissue was put into 4% PFA (paraformaldehyde) for fixation for no less than 24 hours.
2. The adipose tissue was dehydrated, embedded in paraffin, and sectioned in paraffin with a thickness of 3 µm.
3. The paraffin sections were stained with HE, sealed with gum, observed under a microscope and photographed.
4. Image Pro software was used to analyze images.
1. The abdominal tissue of mice was obtained, and the bean-sized of abdominal tissue was placed in the EP tube, and Trizol was added.
2. Tissue homogenizer was used to grind tissue mass. After grinding thoroughly, EP tube was put directly into -80° C. refrigerator for frozen storage.
3. The frozen EP tube was taken out, and the upper layer of white fat mass on was scrapped off. After trizol was thawed, RNA in the sample was extracted.
4. The collected RNA was reverse transcribed by reverse transcription kit, and the target gene was subjected to QPCR and comparative analysis.
The result is shown in
The liver tissue of the model mice constructed in Example 6 was weighed, and the liver tissue of each group was photographed.
1. Mouse liver tissue was obtained, fixed for dehydration, then embedded in paraffin, and sectioned in paraffin.
2. The paraffin sections were stained with HE, sealed with gum, and observed under a microscope and photographed.
The experimental results are shown in
Peripheral blood of the model mice constructed in Example 6 was obtained and left for 30 min before centrifugation. The centrifugation condition was set at 300 g for 30 min. After centrifugation, the serum was aspirated. Levels of TNF-α and IL-10 in serum were detected according to ELISA instructions.
The result is shown in
1. Spontaneous hyperglycemic monkeys were obtained and anesthetized. The muscle tissue of the hind limb was taken, the size of the tissue was 1 cm∗1 cm∗1 cm, and flow cytometry was used to extract and obtain monkey-derived muscle stem cells.
2. The extracted muscle stem cells were subjected to proliferation and cultured and expanded in vitro.
3. The proliferating cells were stained with stem cell surface characteristic antibodies CD31, CD34, CD45, CD29, EGFR and intracellular stem cell characteristic protein PAX7, and analyzed by flow cytometry analyzer.
1. Intravenous infusion of stem cell preparation, in which the cell infusion dose was 1-5×106 cells /Kg of experimental monkeys.
2. After cell infusion, the fasting blood glucose of each monkey was measured every three days, and the fluctuation of blood glucose of each monkey was recorded.
3. The frequency of muscle stem cell infusion was once every two weeks for a total of 3-6 times.
Fasting blood glucose in the experimental monkeys was measured before and after muscle stem cell infusion, and the results were compared longitudinally.
The result is shown in
1. Male C57/BL mice aged 4 weeks were randomly selected and divided into groups. The mice were fed a normal diet containing 10% Kal fat and a high-fat diet containing 60% Kal fat, respectively, the weight of the mice was recorded and a weight change curve was formed. After 2-3 months of modeling, mice with a body weight of about 40 g were selected for grouping and experiment, and the body weight of each mouse was continuously recorded. High-fat fed mice developed insulin tolerance due to obesity, thus mimicking the human type 2 diabetes model.
2. After the establishment of the model, the mice were divided into three groups: control group (normal diet group), model group (high-fat diet group) and treatment group (high-fat diet group injected with Balb/C mouse muscle stem cells). Then the experiment was ready to begin.
3. The control group and the model group were injected with 200 µL of the injection system sterile PBS every week by tail vein injection. The treatment group was injected with Balb/C murine-derived muscle stem cells, the injection volume was 1-5×105 cells per mouse, and the injection system was 200 µL. The injection cycle was once a week, and a total of 6 injections were given.
4. Mice were given a treatment of fasting without water abstinence 15 hours before the test.
5. Fasting blood glucose of mice was detected by Bynoe blood glucose meter and blood glucose test strip, which was recorded as G0.
6. Mice were intraperitoneally injected with glucose at a dose of 1 g/g mice. Immediately after injection, the time at the moment was recorded as T0.
After 15 min(T15), 30 min(T30), 45 min(T45), 60 min(T60) and 120 min(T120), the blood glucose of the mice was measured again and recorded as G15, G30, G45, G60 and G120, respectively.
1. Fasting blood glucose of mice was detected by Byino blood glucose meter and blood glucose test strip, which was recorded as G0.
2. Mice were intraperitoneally injected with short-acting insulin at a dose of 0.1 U/g mice. Immediately after injection, the time at the moment was recorded as T0.
3. After 15 min(T15), 30 min(T30), 45 min(T45), 60 min(T60) and 120 min(T120), the blood glucose of the mice was measured again and recorded as G15, G30, G45, G60 and G120, respectively.
The hypoglycemic ability of mice was detected by glucose tolerance and insulin tolerance test, so as to evaluate the glucose metabolism level of mice.
The subcutaneous adipose tissue of mice in each group was separated, weighed and detected, and the weight of adipose tissue in each group was compared.
In the body with type 2 diabetes caused by obesity, the liver tissue is complicated with fatty infiltration, forming fatty liver. The liver tissue of mice was removed and weighed for detection, and the weight of liver tissue of each group was compared.
1. Peripheral blood of mice was collected and left for 30 min before centrifugation. The centrifugation condition was set at 300 g for 30 min.
2. Inflammatory cytokines IL-6 and IL-1β kit were used to detect the level of inflammation in mice.
The result is shown in
Balb/C murine-derived muscle stem cells via tail vein injection significantly reduced body weight and fasting blood glucose in mice fed a high-fat diet. After intraperitoneal injection of glucose solution, the blood glucose level of mice increased. Along with the body self-metabolism, the rate of hypoglycemia in the mice in the treatment group was significantly higher than that in the model group, indicating that the mice in treatment group had a better ability of hypoglycemia. The intervention of muscle stem cells derived from Balb/C mice reduced the fasting blood glucose of high-fat diet induced hyperglycemia mice, and enhanced the glucose metabolism ability of the model mice. Balb/C murine-derived muscle stem cells reduced the weight of subcutaneous fat, epididymal and inguinal fat, and liver tissue in obese mice. Moreover, in the peripheral blood, the level of peripheral inflammation in the model mice decreased significantly after infusion of Balb/C murine derived muscle stem cells.
All literatures mentioned in the present application are incorporated by reference herein, as though individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, various changes or modifications may be made by those skilled in the art, and these equivalents also fall within the scope as defined by the appended claims of the present application.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202010340419.7 | Apr 2020 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/088209 | 4/19/2021 | WO |
