WO2022199632A1 - 一种治疗动脉粥样硬化的干细胞药物 - Google Patents

一种治疗动脉粥样硬化的干细胞药物 Download PDF

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WO2022199632A1
WO2022199632A1 PCT/CN2022/082558 CN2022082558W WO2022199632A1 WO 2022199632 A1 WO2022199632 A1 WO 2022199632A1 CN 2022082558 W CN2022082558 W CN 2022082558W WO 2022199632 A1 WO2022199632 A1 WO 2022199632A1
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stem cells
atherosclerosis
muscle stem
reducing
pharmaceutical composition
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French (fr)
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时玉舫
侯蓬勃
陈永井
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苏州大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the invention relates to the technical field of biomedicine, in particular, to the application of a muscle stem cell in preparing a drug for treating atherosclerosis.
  • Cardiovascular disease is the "number one killer” that seriously endangers human health, with high morbidity, disability and mortality, and atherosclerosis is its main pathological basis and potential cause.
  • Atherosclerosis is a chronic inflammatory response, more specifically, the accumulation and oxidative modification of low-density lipoprotein in the subendothelial layer of arteries, leading to the activation of endothelial cells and the activation of macrophages, secreting various cytokines, chemokines, etc. Chemokines and adhesion factors, thereby recruiting more immune cells to chemotaxis to the injury site and accelerating the process of atherosclerosis.
  • Skeletal muscle is the largest tissue organ in the human body. In addition to providing the necessary energy for individual exercise, it also participates in the regulation of the body's homeostasis and physiological functions by producing a variety of immune molecules.
  • the inflammatory cytokine interleukin-6 (IL-6) released by exercising muscles can blunt endotoxin-induced inflammatory responses.
  • Meteoroid-like protein (MTRNL) produced by skeletal muscle induces "browning" of white adipose tissue and induces IL-4-induced polarization of anti-inflammatory macrophages.
  • Muscle-derived interleukin-15 (IL-15) can promote the proliferation of naive T cells, enhance the development and cytotoxicity of natural killer cells to reduce lipid deposition and prevent visceral fat lesions.
  • skeletal muscle muscle stem cells as the core cellular components of skeletal muscle, not only participate in the repair of skeletal muscle damage, but also participate in the function regulation of immune cells, there is no report at home and abroad.
  • the purpose of the present invention is to provide a use of muscle stem cells in preparing a pharmaceutical composition for preventing, alleviating and/or treating cardiovascular diseases (eg, atherosclerosis).
  • cardiovascular diseases eg, atherosclerosis
  • a use of muscle stem cells is provided, and the muscle stem cells are used to prepare a pharmaceutical composition for one or more applications selected from the group consisting of:
  • the pro-inflammatory monocytes are pro-inflammatory Ly6C hi monocytes.
  • the medicament is used for humans or animals.
  • the anti-inflammatory macrophage-related gene is selected from the following group: Arg1.
  • genes related to pro-inflammatory macrophages are selected from the group consisting of Il1 ⁇ , Ccl2.
  • the white adipose tissue includes epididymal adipose tissue.
  • the muscle stem cells are selected from the group consisting of 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, and horse-derived muscle stem cells stem cells, or a combination thereof.
  • the human-derived muscle stem cells are CD31 - , CD34 - , CD45 - , CD29 + , CD56 + , EGFR + and PAX7 + cells.
  • the muscle stem cells are stem cells that can be differentiated into muscle fibers.
  • the muscle stem cells can be autologous, allogeneic, or xenogeneic.
  • the muscle stem cells are obtained from a site selected from the group consisting of skeletal muscle of limbs and skeletal muscle of trunk.
  • the dosage form of the pharmaceutical composition is selected from the following group: injection of freshly cultured muscle stem cells, or injection of thawed muscle stem cells from frozen storage.
  • the muscle stem cells are extracted from body muscles and obtained by cell sorting and expansion.
  • the muscle stem cells are extracted from the limbs or trunk muscles of the body and obtained by cell sorting.
  • the muscle stem cells are muscle-derived stem cells that can be differentiated into muscle fibers.
  • the dosage form of the pharmaceutical composition is an injection.
  • the pharmaceutical composition is used for preventing, relieving and/or treating cardiovascular disease, preferably, the cardiovascular disease is selected from the group consisting of atherosclerosis, myocardial infarction, cerebral infarction, Aortic aneurysm.
  • the muscle stem cells are selected from the group consisting of 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, and horse-derived muscle stem cells stem cells, or a combination thereof.
  • the cardiovascular disease is selected from the group consisting of: aortic atherosclerosis, coronary atherosclerosis, carotid artery and cerebral atherosclerosis, coronary atherosclerotic heart disease, renal artery atherosclerosis atherosclerosis and atherosclerosis of the extremities.
  • the muscle stem cells can be autologous, allogeneic, or xenogeneic.
  • the muscle stem cells are extracted from the limbs or trunk muscles of the body and obtained by cell sorting.
  • composition for humans or animals, the pharmaceutical composition comprising:
  • the pharmaceutical composition is for one or more applications selected from the group consisting of:
  • the pro-inflammatory monocytes are pro-inflammatory Ly6C hi monocytes.
  • the pharmaceutical composition further contains a second active ingredient for regulating lipid metabolism, preferably, the second active ingredient is selected from the group consisting of:
  • Statins PCSK9 inhibitors, fibrates, anticoagulants, or thrombolytics.
  • the muscle stem cells are CD31 ⁇ , CD34 ⁇ , CD45 ⁇ , CD29 + , CD56 + , EGFR + and PAX7 + cells.
  • the pharmaceutical composition is an anti-atherosclerotic drug.
  • the pharmaceutical composition is a drug for reducing the plaque area at the aortic arch and its branches.
  • the pharmaceutical composition is a medicine for reducing the inflammatory environment of the body.
  • the dosage form of the pharmaceutical composition is selected from the following group: injection, lyophilized preparation.
  • the pharmaceutical composition is a drug for preventing, relieving and/or treating cardiovascular diseases
  • cardiovascular diseases include: aortic atherosclerosis, coronary atherosclerosis, carotid arteries and cerebral arteries Atherosclerosis, coronary atherosclerotic heart disease, renal atherosclerosis, and extremity atherosclerosis.
  • a method for preventing, alleviating and/or treating cardiovascular disease comprising: administering muscle stem cells, the pharmaceutical combination according to the second aspect of the present invention to a subject in need or a preparation comprising the muscle stem cells or the pharmaceutical composition, or a combination thereof.
  • the cardiovascular diseases include: aortic atherosclerosis, coronary atherosclerosis, carotid artery and cerebral atherosclerosis, coronary atherosclerotic heart disease, renal atherosclerosis and Atherosclerosis of the extremities.
  • Figure 1 shows A. flow cytometric identification of surface markers expressed by human-derived muscle stem cells. The isolated muscle stem cells were stained for surface markers using anti-human flow cytometry antibodies CD31-PE, CD34-PE, CD45-PE, CD29-APC, CD56-BV421, EGFR-BV421, and analyzed by flow cytometry; B. Flow cytometric identification of the specific nuclear factor PAX7 expressed by human muscle stem cells. After the cells were permeabilized and fixed, muscle stem cells were stained with PAX7 protein antibody, and fluorescent secondary antibody with excitation light wavelength of 647 nm was used for cell analysis by flow cytometry. C.
  • PBS was the model group, which was fed with 40% Kcal high-fat diet for 6 weeks, and 200 ⁇ L of PBS was injected through the tail vein in the third week, once a week, a total of four times;
  • hMuSC was the treatment group, which was fed with 40% Kcal high-fat diet.
  • the mice were fed with a fat diet for 6 weeks, and each rat was injected with 5 ⁇ 10 5 human-derived muscle cells via the tail vein once a week for a total of four times in the third week.
  • PBS was the model group, which was fed with 40% Kcal high-fat diet for 8 weeks, and 200 ⁇ L of PBS was injected via tail vein in the fifth week, once a week, for a total of four times;
  • hMuSC was the treatment group, which was fed with 40% Kcal high-fat diet.
  • the mice were fed with fat diet for 8 weeks, and each rat was injected with 5 ⁇ 10 5 human-derived muscle cells via tail vein once a week for a total of four times at the 5th week.
  • FIG 4 shows A. Using Mac-3 immunofluorescence staining to compare the aggregation of macrophages at the aortic valve, Mac-3 can be used as a marker of macrophages. Among them, Hoechst is the nucleus staining; B. Using flow cytometry to compare the number of macrophages (CD11b + F4/80 + ) in peripheral blood and their proportion to immune cells (CD45 + ); C. Using flow cytometry Technical comparison of the number of pro-inflammatory monocytes (Ly6C hi CD11b + ) and the proportion of immune cells (CD45 + ) in peripheral blood; D. Determination of the content of chemokine CCL2 in mouse serum; E. For Expression detection of pro-anti-inflammatory macrophage-related genes Arg1, Ccl2 and Il1b in mouse aorta.
  • Figure 5 shows that A.ApoE -/- (6-8 weeks) mice were divided into control group (Normal chow), model group (PBS), treatment group (hMuSC), and were given normal diet and 40% Kcal high-fat diet, respectively , 40% Kcal high-fat diet for a total of 10 weeks of continuous feeding.
  • control group Normal chow
  • model group PBS
  • treatment group hMuSC
  • mice were removed from the control group
  • PBS model group
  • the aorta was stripped to determine the degree of atherosclerosis.
  • 200 ⁇ L of PBS, 200 ⁇ L of PBS and 5 ⁇ 10 5 muscle stem cells were injected into the tail vein of the control group, the model group and the treatment group, respectively.
  • the inventors found for the first time that activated skeletal muscle muscle stem cells have an inhibitory effect on chronic inflammation caused by high-fat diet, reduce the level of inflammation, significantly reduce the area of aortic plaque, and reduce the weight of white adipose tissue , has a relieving and therapeutic effect on atherosclerosis.
  • the present invention selects a high-fat diet-induced atherosclerosis mouse model, and studies the preparation of drug combinations based on human-derived muscle stem cells for preventing, alleviating and/or treating cardiovascular diseases (such as atherosclerosis). use in things.
  • human-derived cells injected into mice can be eliminated by the body in a short period of time, excluding the possibility of cell colonization, proliferation and differentiation in the recipient. The present invention has been completed on this basis.
  • muscle stem cells were injected into the tail vein of atherosclerotic mice induced by high-fat diet feeding.
  • the experimental results showed that, compared with the model group, the treatment group could significantly delay the progression of atherosclerotic plaques. It can reduce the plaque area of the aortic arch and its branches in mice, reduce lipid deposition at the aortic valve, reduce epididymal adipose tissue, and relieve the inflammatory environment in mice.
  • the hMuSC of the present invention reduced the levels of CCL2 and IL-6 in mouse serum, and significantly reduced the number and percentage of macrophages and pro-inflammatory monocytes, thereby further illustrating that the hMuSC of the present invention reduced the The level of inflammation in the body exerts an anti-atherosclerotic effect.
  • plaque in “plaque area” refers to inflammatory lesions occurring in the intima of the aorta, rich in a large amount of lipid substances (which can be stained red by Oil Red O dye), so “plaque area” To a certain extent, it represents the size of the inflammatory lesions of the aortic intima.
  • At the aortic valve is at the aortic root valve, and the plaque area at the aortic root valve is indicative to some extent of the degree of lipid deposition.
  • human-derived muscle stem cells of the present invention can be used interchangeably, and all refer to the highly expressed stem cell markers CD29 and CD56, epidermal growth factor receptor of the present invention
  • the isolated cell populations of EGFR, and high expression of muscle stem cell marker PAX7 namely CD31 ⁇ , CD34 ⁇ , CD45 ⁇ , CD29 + , CD56 + , EGFR + and PAX7 + cells.
  • Muscle satellite cells are myogenic stem cells located between the basement membrane of skeletal muscle and myofibrocyte membrane. Under physiological conditions, muscle satellite cells exist in a quiescent and undifferentiated state, and are activated to divide into spindle-shaped muscles after injury or inflammatory stimulation. Stem cells fuse with each other into multinucleated myotubes with continuous differentiation, and finally develop to form new muscle fibers or integrate into damaged muscle tissue, which play an important role in the growth, repair, function maintenance and tissue regeneration of skeletal muscle.
  • the muscle stem cells are selected from the group consisting of: mouse muscle stem cells, human muscle stem cells, monkey muscle stem cells, dog muscle stem cells, cat muscle stem cells, and horse muscle stem cells. muscle stem cells, etc.
  • the human muscle stem cells are CD31 - , CD34 - , CD45 - , CD29 + , CD56 + , EGFR + and PAX7 + cells.
  • the muscle stem cells are muscle-derived stem cells that can be differentiated into muscle fibers.
  • the muscle stem cells may be autologous, allogeneic, or xenogeneic.
  • the muscle stem cells are obtained from a site selected from the group consisting of skeletal muscle of limbs and skeletal muscle of trunk.
  • the muscle stem cells are extracted from body muscles, sorted and expanded by flow cytometry.
  • the muscle stem cells are extracted from limbs or trunk muscles of the body and sorted by flow cytometry.
  • Mesenchymal stem cells are present in both skeletal and smooth muscle.
  • the protagonist of muscle injury repair and regeneration is muscle stem cells, but some other types of cells, such as inflammatory cells, vascular endothelial cells and muscle-derived mesenchymal stem cells, play a role in fine regulation and coordination in the whole process of muscle injury repair and regeneration.
  • fibroblast progenitor cells are a type of mesenchymal stem cells in the muscle interstitium, and their role is to support the differentiation of muscle stem cells and promote the repair and regeneration of muscle tissue.
  • the smooth muscle progenitor cells derived from smooth muscle in blood vessels mainly function to replace smooth muscle cells, thereby filling the vascular lesions or damaged areas.
  • the present invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above-mentioned muscle stem cells and a pharmaceutically acceptable carrier.
  • these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 5-8, preferably at a pH of about 6-8, although the pH may vary depending on the This will vary depending on the nature of the formulation material and the condition to be treated.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, oral, respiratory, intratumoral, intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition of the present invention can be used directly for treatment (eg, treatment of atherosclerosis), and in addition, other therapeutic agents can be used concurrently.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (eg, 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned muscle stem cells of the present invention and a pharmaceutically acceptable carrier or excipient agent.
  • a pharmaceutically acceptable carrier or excipient agent include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the drug formulation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, eg, from about 1 [mu]g/Kg body weight to about 10 mg/Kg body weight per day.
  • a safe and effective amount of muscle stem cells is administered to the mammal, wherein the safe and effective amount is generally at least about 10 ⁇ g/Kg body weight, and in most cases no more than about 8 mg/Kg body weight, preferably the Doses are about 10 ⁇ g/Kg body weight to about 1 mg/Kg body weight.
  • the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.
  • the pharmaceutical composition contains:
  • the pharmaceutical composition is for one or more applications selected from the group consisting of:
  • the human-derived muscle stem cells are CD31 - , CD34 - , CD45 - , CD29 + , CD56 + , EGFR + and PAX 7 + cells.
  • the pharmaceutical composition is an anti-atherosclerotic drug.
  • the pharmaceutical composition is a drug for reducing the plaque area at the aortic arch and its branches.
  • the pharmaceutical composition is a medicine for reducing the inflammatory environment of the body.
  • the dosage form of the pharmaceutical composition is selected from the group consisting of injection and lyophilized preparation.
  • the dosage form of atherosclerosis is a freshly cultured muscle stem cell injection, or a thawed muscle stem cell injection from frozen storage.
  • the anti-atherosclerotic drug can significantly delay atherosclerosis in mice and reduce the plaque area at the aortic arch and its branches.
  • the anti-atherosclerotic drug can reduce the weight of white adipose tissue.
  • the anti-atherosclerotic drug can reduce the level of inflammation in mice.
  • the present invention fully studies the effect of muscle stem cells in the treatment of cardiovascular diseases by injecting muscle stem cells into the tail vein of high-fat diet-induced atherosclerotic mice;
  • the present invention focuses on finding that muscle stem cells have the following functions:
  • the present invention can achieve a relatively long-term therapeutic effect through a limited number of muscle stem cell injections, thereby producing the following advantages:
  • 2Few injections of muscle stem cells can have a long-term effect, which has the advantage of saving time and effort.
  • Oil Red O dye liquor mother liquor Dissolve 0.5g of Oil Red O powder in 100mL of isopropanol (concentration is 100%), fully dissolve in a water bath at 37°C, and store at room temperature away from light for later use.
  • the aorta will be stripped out, put into an EP tube containing 2 mL of 60% isopropanol, and mordant for 10 minutes, which is beneficial to the coloring of Oil Red O dye.
  • the mouse heart was taken, the apex of the heart was excised, and after OCT embedding, it was placed in a -80°C refrigerator overnight.
  • the frozen sections were fixed in a PBS solution containing 4% paraformaldehyde for 10 minutes, washed with ddH 2 O, and then placed in a dye vat containing 60% isopropanol for mordant dyeing for 5 minutes.
  • washing solution DMEM LOW medium containing 20% fetal bovine serum + 1% double antibody
  • crude digestion solution type II collagenase is added to the washing solution, so that the final concentration of type II collagenase is 750U) /mL
  • sperm digestion solution type II collagenase and dispase were added to the washing solution in turn, so that the final concentrations were 1000U/mL, 11U/mL
  • muscle stem cell growth complete medium (20% fetal bovine serum + 40 %DMEM LOW+40%MCDB 131+Insulin-transferrin-X+1% double antibody+10 ⁇ mol p38 inhibitor).
  • human-derived muscle tissue blocks were obtained from the hospital, placed in a 10cm petri dish, 2 mL of crude digestion solution was added dropwise, and shredded to form a tissue original slurry, and then the petri dish was fully rinsed with 13 mL of crude digestion solution. Fragments were transferred to 50mL centrifuge tubes.
  • the obtained liquid was centrifuged at 500 g for 5 min, and the supernatant was aspirated to obtain a cell pellet.
  • the remaining cell suspension was stained by flow sorting antibody, and the labeled antibodies were CD31-PE, CD34-PE, CD45-PE, CD29-APC, CD56-BV421, EGFR-BV421 staining for 40min, shaking every 20min, Four degrees of storage. After staining was completed, complete medium was washed and centrifuged, and cells were finally resuspended in 200 ⁇ L of complete medium.
  • the cells are grown to 70% confluence, and the desired cells can be obtained by trypsin digestion.
  • the marker proteins expressed on the surface of human muscle stem cells were identified by flow cytometry, and the specific nuclear factor PAX7 of human muscle stem cells was identified by flow cytometry.
  • the cells were washed three times with serum-free DMEM medium, and then replaced with muscle stem cell differentiation medium (DMEM medium + 2% horse serum) to continue culturing.
  • DMEM medium + 2% horse serum muscle stem cell differentiation medium
  • immunofluorescence staining of human muscle stem cell-specific differentiation maturation protein myosin heavy chain (MyHC) was performed to identify experiments to determine that the isolated cells had the differentiation properties of stem cells.
  • Figure 1 shows that the isolated cells of the present invention highly express the muscle stem cell marker PAX7.
  • the isolated cells were negative for the endothelial cell marker CD31, negative for the hematopoietic stem cell marker CD34, and negative for the leukocyte marker CD45.
  • the isolated cells highly expressed stem cell markers CD29 and CD56, and the expression ratio was 100%. And the cells highly expressed epidermal growth factor receptor EGFR up to 90%. So the isolated cells are CD31 - , CD34 - , CD45 - , CD29 + , CD56 + , EGFR + and PAX7 + muscle stem cells.
  • Muscle stem cells cultured and expanded in vitro can well maintain the expression of stem cell surface markers and nuclear specific factor PAX7, and can differentiate into mature and functional muscle fiber structures, indicating that the muscle stem cells cultured in the above system have excellent stem cells. Sex and muscle fiber production capacity.
  • ApoE -/- mice In the third week of feeding high-fat diet, the model group was injected with sterile PBS every week by means of tail vein injection, and the injection system was 200 ⁇ L. The treatment group was injected with human-derived muscle stem cells (Human MuSC, hMuSC), and the injection volume was 5 ⁇ 10 5 cells per mouse, and the injection system was 200 ⁇ L. The injection cycle was once a week, with a total of 4 injections. On the fourth day after the last injection, the mice were euthanized, and the samples were dissected, and the follow-up blood lipid levels, inflammatory factors in serum, plaque size or area, and histopathological indicators were completed (see panel A in Figure 2). Right).
  • Human MuSC human-derived muscle stem cells
  • hMuSC human-derived muscle stem cells
  • Fig. 2B shows a visual comparison of plaques in the mouse aortic arch and its branches in situ.
  • the aorta was stained with Oil Red O, and the blood vessels were opened to take pictures to visually compare the area of the plaque. ImageJ was used to quantify the stained area, and the plaque area and the proportion of the plaque area to the entire aortic area were calculated ( See Figure 2, Panel C).
  • Figure D of Figure 2 The experimental results are shown in Figure D of Figure 2. After the aortic root was frozen sectioned, oil red O was stained and photographed, and the degree of lipid deposition was compared.
  • Figure 2E shows a comparison of plaque area at different distances from the aortic root valve.
  • mice peripheral blood of mice was obtained, and centrifuged after standing for 30 min. The centrifugation conditions were set to 3000 rpm for 30 min. After centrifugation, aspirate serum. The content of IL-6 in serum was detected according to the instructions of the ELISA reagent (see Figure G in Figure 2).
  • LDLR -/- mice were used as a model for inducing atherosclerosis (there was no significant difference in blood lipid levels between the PBS group and the hMuSC group, data not shown).
  • Example 3 Delaying effect of human muscle stem cells on the progression of atherosclerosis in LDLR -/- mice
  • LDLR -/- mice In the fifth week of feeding with high-fat diet, the model group was injected with sterile PBS every week by means of tail vein injection, and the injection system was 200 ⁇ L. The treatment group was injected with human-derived muscle stem cells (Human MuSC, hMuSC), and the injection volume was 5 ⁇ 10 5 cells per mouse, and the injection system was 200 ⁇ L. The injection cycle was once a week, with a total of 4 injections. Mice were euthanized on the fourth day after the last injection, dissections were performed, and follow-up plaque size or area, inflammatory factors in serum, histopathology, and myeloid cells (mainly macrophages) in peripheral blood were completed. and pro-inflammatory Ly6C hi monocytes) and other detection indicators (see the lower side of Figure A in Figure 3).
  • the aortic root was frozen sectioned and then stained with Oil Red O and photographed to compare the degree of lipid deposition at the aortic root valve.
  • the aorta was stained with Oil Red O, and the blood vessels were opened to take pictures to visually compare the area of the plaque. ImageJ was used to quantify the stained area, and the plaque area and plaque area were calculated. percentage of the total aortic area.
  • mice peripheral blood 300uL
  • place it in an anticoagulant tube add five times the volume of schistin for 8 minutes, add 5mL of PBS containing 2% FBS to neutralize, and at 4°C, Centrifuge at 500g for 5min.
  • hMuSC treatment significantly reduced the number and percentage of macrophages and pro-inflammatory monocytes, thereby further reducing the level of inflammation in the diseased mice. Since a large number of literatures have reported that all atherosclerotic mice induced by high-fat diet are often accompanied by an increase in myeloid cells (mainly macrophages and pro-inflammatory monocytes), the hMuSC treatment of the present invention The progression of the disease can be slowed down by inhibiting the proliferation of myeloid cells.
  • CCL2 not only measures the level of inflammation in the body, but also induces the recruitment of immune cells (especially monocyte-macrophage recruitment). Therefore, the experimental results that hMuSC treatment significantly reduced the level of CCL2 in the serum of mice further supported the anti-atherosclerosis effect of hMuSC by reducing the level of inflammation in the body.
  • tissue homogenizer to grind the tissue block, and after the grinding is complete, put the EP tube directly into a -80°C refrigerator and freeze it.
  • RNA was reverse transcribed using a reverse transcription kit, and the target gene was subjected to QPCR and comparative analysis.
  • hMuSC human-derived muscle stem cells
  • mice 6-8 week old male ApoE -/- mice were randomly selected and divided into control group (normal diet), model group (high-fat diet group) and treatment group (high-fat diet mice injected with human muscle stem cells). group) were fed a high-fat diet with 40% Kal fat content for 10 weeks (see panel A of Figure 5).
  • ApoE -/- mice In the 7th week of feeding high-fat diet, the control group and model group were injected with sterile PBS every week by means of tail vein injection, and the injection system was 200 ⁇ L.
  • the treatment group was injected with human-derived muscle stem cells (Human MuSC, hMuSC), and the injection volume was 5 ⁇ 10 5 cells per mouse, and the injection system was 200 ⁇ L.
  • the injection cycle is once a week, with a total of 4 injections. This experiment can be used as the therapeutic effect of muscle stem cells on atherosclerosis.
  • mice Observe the in situ pictures of the aortic arch in mice treated with 6-week normal diet, 6-week high-fat diet, 10-week normal diet + PBS, 10-week high-fat diet + PBS, and 10-week high-fat diet + hMuSC treatment (see panel B of Figure 5). .
  • mice After the mice were weighed, the mice were sacrificed and the epididymal fat of the mice was collected.
  • the mouse epididymal fat index the weight of the epididymal fat/the body weight of the mouse (see Figure 5, panel D).
  • muscle stem cells also have a good effect on the treatment of atherosclerosis.

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Abstract

本发明提供了一种肌肉干细胞在预防、缓解和治疗心血管疾病中的用途。实验表明，在动脉粥样硬化小鼠尾静脉注射肌肉干细胞能显著延缓小鼠的动脉粥样硬化进程，有效减少主动脉弓及颈动脉分支处斑块面积，减少体内白色脂肪组织，降低小鼠机体炎症反应等。此外，本发明还提供一种肌肉干细胞对于动脉粥样硬化的预防和治疗方法。

Description

一种治疗动脉粥样硬化的干细胞药物 技术领域
本发明涉及生物医学技术领域，具体地，涉及一种肌肉干细胞在制备治疗动脉粥样硬化药物中的应用。
背景技术
心血管疾病是严重危害人类健康的“头号杀手”，具有较高的发病率、致残率和致死率，而动脉粥样硬化是其主要的病理基础及潜在病因。近年来，我国心血管病患病率呈上升趋势，全国每年约有350万人死于心血管病，心血管病已成为中国居民死亡的首要原因。动脉粥样硬化是一种慢性炎症反应，更具体地说是由低密度脂蛋白在动脉内皮下层发生聚集及氧化修饰，导致内皮细胞的激活和巨噬细胞的活化，分泌各种细胞因子、趋化因子和黏附因子，从而招募更多的免疫细胞趋化到损伤部位，加速动脉粥样硬化的进程。
骨骼肌是人体最大的组织器官，除了为个体运动提供所必需的能量，同时还通过产生多种免疫分子，参与机体的稳态和生理功能的调控。运动性肌肉释放的炎症细胞因子白介素-6(IL-6)能够钝化内毒素诱导的炎症反应。骨骼肌产生的类流星蛋白(MTRNL)可诱导白色脂肪组织的“褐变”，并诱导IL-4诱导的抗炎型巨噬细胞的极化。肌肉来源的白介素-15(IL-15)能够促进幼稚T细胞的增殖，增强自然杀伤细胞的发育和细胞毒性，以减少脂质沉积，预防内脏器官脂肪病变。然而，骨骼肌肌肉干细胞作为骨骼肌的核心细胞成分，除了参与着骨骼肌的损伤修复，是否也参与了免疫细胞的功能调控，国内外尚无报道。
随着经济社会的发展、人们生活方式的改变(不健康饮食和缺乏运动)及人口老龄化，动脉粥样硬化性心血管疾病的发病率日益升高，不仅对居民健康有着重大影响，而且经济负担也相当沉重，因此，本领域亟待开发针对动脉粥样硬化的有效药物。
发明内容
本发明的目的在于提供一种肌肉干细胞在制备预防、缓解和/或治疗心血管疾病(例如动脉粥样硬化)的药物组合物中的用途。
在本发明的第一方面，提供了一种肌肉干细胞的用途，所述肌肉干细胞用于 制备药物组合物，所述药物组合物用于选自下组的一种或多种应用：
(a)延缓动脉粥样硬化进程
(b)减少主动脉弓及其分支处斑块面积；
(c)减少主动脉瓣膜处脂质的沉积；
(d)减少主动脉瓣膜处巨噬细胞的聚集；
(e)减少外周血中促炎型单核细胞的数量和比例；
(f)减少外周血中巨噬细胞的数量和比例；
(g)减少白色脂肪组织重量；
(h)缓解机体炎症反应；
(i)降低血清中IL-6和CCL2的水平；
(j)差异性调节斑块中巨噬细胞内促抗炎基因的表达水平；
(k)提高抗炎型巨噬细胞相关基因的表达水平；
(l)降低促炎型巨噬细胞相关基因的表达水平；
(m)预防、缓解和/或治疗动脉粥样硬化；
(n)预防、缓解和/或治疗心血管疾病。
在另一优选例中，所述促炎型单核细胞为促炎型Ly6C hi单核细胞。
在另一优选例中，所述药物用于人或动物。
在另一优选例中，所述抗炎型巨噬细胞的相关基因选自下组：Arg1。
在另一优选例中，所述促炎型巨噬细胞的相关基因选自下组：Il1β、Ccl2。
在另一优选例中，所述白色脂肪组织包括附睾脂肪组织。
在另一优选例中，所述肌肉干细胞选自下组：鼠源性肌肉干细胞、人源性肌肉干细胞、猴源性肌肉干细胞、狗源性肌肉干细胞、猫源性肌肉干细胞、马源性肌肉干细胞、或其组合。
在另一优选例中，所述人源性肌肉干细胞为CD31 -，CD34 -，CD45 -，CD29 +，CD56 +，EGFR +和PAX7 +的细胞。
在另一优选例中，所述肌肉干细胞为可分化为肌纤维的干细胞。
在另一优选例中，所述的肌肉干细胞可以是自体的，也可以是同种异体的，或异种的。
在另一优选例中，所述的肌肉干细胞获自选自下组的部位：四肢骨骼肌、躯干骨骼肌。
在另一优选例中，所述药物组合物的剂型选自下组：新鲜培养的肌肉干细 胞注射剂，或来自于冷冻储存的解冻肌肉干细胞注射剂。
在另一优选例中，所述的肌肉干细胞是从机体肌肉中提取并通过细胞分选、扩增所获得。
在另一优选例中，所述的肌肉干细胞是从机体四肢或躯干肌肉中提取并通过细胞分选所获得。
在另一优选例中，所述肌肉干细胞为肌肉来源的可分化为肌纤维的干细胞。
在另一优选例中，所述药物组合物的剂型为注射剂。
在另一优选例中，所述药物组合物用于预防、缓解和/或治疗心血管疾病，较佳地，所述心血管疾病选自下组：动脉粥样硬化、心肌梗死、脑梗死、主动脉瘤。
在另一优选例中，所述肌肉干细胞选自下组：鼠源性肌肉干细胞、人源性肌肉干细胞、猴源性肌肉干细胞、狗源性肌肉干细胞、猫源性肌肉干细胞、马源性肌肉干细胞、或其组合。
在另一优选例中，所述心血管疾病选自下组：主动脉粥样硬化、冠状动脉粥样硬化、颈动脉和脑动脉粥样硬化、冠状动脉粥样硬化性心脏病、肾动脉粥样硬化和四肢动脉粥样硬化。
在另一优选例中，所述的肌肉干细胞可以是自体的，也可以是同种异体的，或异种的。
在另一优选例中，所述的肌肉干细胞是从机体四肢或躯干肌肉中提取并通过细胞分选所获得。
在本发明的第二方面，提供了一种用于人或动物的药物组合物，所述药物组合物含有：
(i)肌肉干细胞作为第一活性成分；和
(ii)药学上可接受的载体；
所述药物组合物用于选自下组的一种或多种应用：
(a)延缓动脉粥样硬化进程
(b)减少主动脉弓及其分支处斑块面积；
(c)减少主动脉瓣膜处脂质的沉积；
(d)减少主动脉瓣膜处巨噬细胞的聚集；
(e)减少外周血中促炎型单核细胞的数量和比例；
(f)减少外周血中巨噬细胞的数量和比例；
(g)减少白色脂肪组织重量；
(h)缓解机体炎症反应；
(i)降低血清中IL-6和CCL2的水平；
(j)差异性调节斑块中巨噬细胞内促抗炎基因的表达水平；
(k)提高抗炎型巨噬细胞相关基因的表达水平；
(l)降低促炎型巨噬细胞相关基因的表达水平；
(m)预防、缓解和/或治疗动脉粥样硬化；
(n)预防、缓解和/或治疗心血管疾病。
在另一优选例中，所述促炎型单核细胞为促炎型Ly6C hi单核细胞。
在另一优选例中，所述的药物组合物还含有调节脂代谢的第二活性成分，较佳地，所述的第二活性成分选自下组：
他汀类药物、PCSK9抑制剂、贝特类药物、抗凝血类药物或溶栓类药物。
在另一优选例中，所述肌肉干细胞，较佳地为人源性肌肉干细胞，为CD31 -，CD34 -，CD45 -，CD29 +，CD56 +，EGFR +和PAX7 +的细胞。
在另一优选例中，所述药物组合物为抗动脉粥样硬化的药物。
在另一优选例中，所述药物组合物为降低主动脉弓及其分支处斑块面积的药物。
在另一优选例中，所述药物组合物为减轻机体炎症环境的药物。
在另一优选例中，所述药物组合物的剂型选自下组：注射剂、冻干剂。
在另一优选例中，所述药物组合物为预防、缓解和/或治疗心血管疾病的药物，所述心血管疾病包括：主动脉粥样硬化、冠状动脉粥样硬化、颈动脉和脑动脉粥样硬化、冠状动脉粥样硬化性心脏病、肾动脉粥样硬化和四肢动脉粥样硬化。
在本发明的第三方面，提供了一种预防、缓解和/或治疗心血管疾病的方法，所述方法包括：给需要的对象施用肌肉干细胞、如本发明的第二方面所述的药物组合物、或包含所述肌肉干细胞或药物组合物的制剂、或其组合。
在另一优选例中，所述心血管疾病包括：主动脉粥样硬化、冠状动脉粥样硬化、颈动脉和脑动脉粥样硬化、冠状动脉粥样硬化性心脏病、肾动脉粥样硬化和四肢动脉粥样硬化。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了A.人源性肌肉干细胞表达的表面标志流式鉴定。使用抗人的流式抗体CD31-PE，CD34-PE，CD45-PE，CD29-APC，CD56-BV421，EGFR-BV421对分离的肌肉干细胞进行表面标志物染色，并使用流式细胞分析仪分析；B.人源性肌肉干细胞表达的特异性核因子PAX7的流式鉴定。细胞经破膜固定后，使用PAX7蛋白抗体对肌肉干细胞进行染色，使用激发光波长为647nm的荧光二抗染色，流式细胞分析仪进行细胞分析。C.人源性肌肉干细胞特异性分化表达成熟肌纤维标志肌球蛋白重链(MyHC)的免疫荧光染色鉴定，红色为肌球蛋白重链，蓝色为细胞核。比例尺(Scale bar)＝25μm；其中，MyHC为肌球蛋白重链染色，Hoechst为细胞核染色；Merge为叠加图，Isotype为阴性对照，Specific Ab为特异性的流式抗体，PAX7-APC为用荧光二抗(在APC通道被检测到荧光)偶联PAX7蛋白抗体。
图2显示了A.ApoE -/-小鼠(12-14周)动脉粥样硬化模型建立、人源干细胞移植方案和实施的检测指标；B.对小鼠主动脉弓及其分支处斑块原位直观比较(PBS组与hMuSC组)；C.对主动脉进行油红O染色，剖开血管并拍照，直观的比较斑块的面积大小，并对着色区域进行量化，从而比较主动脉斑块面积和面积占比；D.对主动脉根进行冰冻切片后进行油红O染色并拍照，比较主动脉根瓣膜处脂质沉积程度。比例尺(Scale bar)＝500μm；E.比较不同距离主动脉根瓣膜处斑块面积；F.对小鼠的附睾脂肪指数进行统计和比较，附睾脂肪指数＝附睾脂肪的质量/小鼠体重；G.对小鼠血清中炎症因子IL-6含量的检测。
其中，PBS为模型组，使用40％Kcal的高脂饲料喂养6周，并在第三周经尾静脉注射200μL PBS，每周一次，共四次；hMuSC为治疗组，使用40％Kcal的高脂饲料喂养6周，并在第三周每只鼠经尾静脉注射5×10 5个人源性肌肉细胞，每周一次，共四次。
图3显示了A.LDLR -/-小鼠(12-14周)动脉粥样硬化模型建立、人源干细胞移植方案和实施的检测指标；B.对小鼠主动脉弓及其分支处斑块原位直观比较；C.对主动脉根进行冰冻切片后进行油红O染色并拍照，比较主动脉根瓣膜处脂质沉积程度。比例尺(Scale bar)＝500μm；D.比较不同距离主动脉根瓣膜处斑 块面积；E.对主动脉进行油红O染色，剖开血管并拍照，直观的比较斑块的面积大小,并对着色区域进行量化，从而比较主动脉斑块面积和面积占比；F.对小鼠的附睾脂肪指数进行统计和比较，附睾脂肪指数＝附睾脂肪的质量/小鼠体重；G.对小鼠血清中炎症因子IL-6含量的检测。
其中，PBS为模型组，使用40％Kcal的高脂饲料喂养8周，并在第5周经尾静脉注射200μL PBS，每周一次，共四次；hMuSC为治疗组，使用40％Kcal的高脂饲料喂养8周，并在第5周每只鼠经尾静脉注射5×10 5个人源性肌肉细胞，每周一次，共四次。
图4显示了A.利用Mac-3免疫荧光染色，比较主动脉瓣膜处巨噬细胞的聚集情况，Mac-3可做为巨噬细胞的marker。其中，Hoechst为细胞核染色；B.利用流式细胞技术比较外周血中巨噬细胞(CD11b +F4/80 +)的数量及其所占免疫细胞(CD45 +)的比例；C.利用流式细胞技术比较外周血中促炎型单核细胞(Ly6C hiCD11b +)的数量及其所占免疫细胞(CD45 +)的比例；D.对小鼠血清中趋化因子CCL2含量的测定；E.对小鼠主动脉中促抗炎型巨噬细胞相关基因Arg1、Ccl2及Il1b的表达检测。
图5显示了A.ApoE -/-(6-8周)小鼠分为对照组(Normal chow)、模型组(PBS)、治疗组(hMuSC)，分别给予正常饮食、40％Kcal高脂饮食，40％Kcal高脂饮食，共连续喂养10周。在第六周结束后，分别从对照组(Normal chow)取出两只小鼠、模型组(PBS)取出三只小鼠，安乐死后剥离主动脉判断小鼠动脉粥样硬化的程度。在第七周，分别给与对照组、模型组和治疗组尾静脉注射200μL PBS、200μL PBS、5×10 5个肌肉干细胞。每周注射一次，共四次。B.从左到右，依次是6周正常饮食小鼠、6周高脂饮食、10周正常饮食+PBS、10周高脂饮食+PBS、10周高脂饮食+hMuSC治疗的主动脉弓原位图片(箭头指示分支斑块的位置)；C.从左到右，依次是6周正常饮食小鼠、6周高脂饮食、10周正常饮食+PBS、10周高脂饮食+PBS、10周高脂饮食+hMuSC治疗的主动脉油红O染色图片；D.对小鼠的附睾脂肪指数进行统计和比较，附睾脂肪指数＝附睾脂肪的质量/小鼠体重；E.对主动脉斑块面积所占主动脉面积的比例进行统计。
在以上所有结果中，*P<0.05；**P<0.01；***P<0.001。
具体实施方式
本发明人经过广泛而深入的研究，首次发现激活的骨骼肌肌肉干细胞对于高脂饮食引起的慢性炎症反应有抑制作用，降低了炎症水平，可显著减少主动 脉斑块面积，减少白色脂肪组织重量，对动脉粥样硬化有缓解和治疗作用。具体地，本发明选取高脂饲料诱导的动脉粥样硬化小鼠模型，研究出了基于人源性肌肉干细胞在制备预防、缓解和/或治疗心血管疾病(例如动脉粥样硬化)的药物组合物中的用途。特别地，人源性细胞注射进入小鼠体内，可在短期内被机体清除，排除了细胞在受体体内定植、增殖和分化的可能性。在此基础上完成本发明。
具体地，本发明中对高脂饲料喂养诱导形成的动脉粥样硬化小鼠进行尾静脉注射肌肉干细胞，实验结果表明，与模型组相比，治疗组能显著延缓动脉粥样硬化斑块进程，减少小鼠的主动脉弓及其分支处斑块面积，减少主动脉瓣膜处脂质沉积，减少附睾脂肪组织，缓解小鼠机体炎症环境等。特别地，本发明的hMuSC降低了小鼠血清中CCL2和IL-6的水平，并且显著地降低了巨噬细胞和促炎型单核细胞的数量和百分比，从而进一步说明本发明的hMuSC通过降低机体炎症水平发挥抗动脉粥样硬化的作用。
术语
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
虽然在本发明的实施或测试中可以使用与本发明中所述相似或等价的任何方法和材料，本文在此处列举优选的方法和材料。
如本文所用，“斑块面积”中“斑块”指发生于主动脉内膜的炎症病变，富含大量脂质性物质(可被油红O染料染成红色)，故“斑块面积”在一定程度上代表主动脉内膜炎症病变的大小。
如本文所用，“主动脉瓣膜处”为主动脉根瓣膜处，主动脉根瓣膜处的斑块面积在一定程度上表明了脂质沉积程度大小。
如本文所用，“本发明的人源性肌肉干细胞”、“hMuSC”、“本发明的肌肉干细胞”可以互换使用，均指本发明的高表达干细胞标志物CD29和CD56、表皮生 长因子受体EGFR、和高表达肌肉干细胞标志物PAX7的分离的细胞群，即为CD31 -，CD34 -，CD45 -，CD29 +，CD56 +，EGFR +和PAX7 +的细胞。
肌肉干细胞
Mauro等人于1961年首次提出在蛙骨骼肌中发现了肌卫星细胞，即肌肉干细胞前体细胞。随后，越来越多的研究证明在成年哺乳类动物骨骼肌中也有少量肌肉干细胞，其数目随年龄的增长而逐渐下降。肌卫星细胞是位于骨骼肌基膜与肌纤维细胞膜之间的肌源性干细胞，在生理状态下，肌卫星细胞以静止未分化状态存在，在受到损伤或炎症刺激后被激活分裂为纺锤形的肌肉干细胞，并随着不断分化相互融合为多核肌管，最后发育形成新生肌纤维或者融入受损肌组织，在骨骼肌的生长、损伤修复、功能维持以及组织再生过程中有着重要作用。
在本发明的一个优选例中，所述肌肉干细胞选自下组：小鼠肌肉干细胞、人源性肌肉干细胞、猴源性肌肉干细胞、狗源性肌肉干细胞、猫源性肌肉干细胞、马源性肌肉干细胞等。
在本发明的一个优选例中，所述人源性肌肉干细胞为CD31 -，CD34 -，CD45 -，CD29 +，CD56 +，EGFR +和PAX7 +的细胞。
在本发明的一个优选例中，所述肌肉干细胞为肌肉来源的、可分化为肌纤维的干细胞。
在本发明的一个优选例中，所述的肌肉干细胞可以是自体的，也可以是同种异体的，或异种的。
在本发明的一个优选例中，所述的肌肉干细胞获自选自下组的部位：四肢骨骼肌、躯干骨骼肌。
在本发明的一个优选例中，所述的肌肉干细胞是从机体肌肉中提取并通过流式细胞术分选、扩增所获得。
在本发明的另一个优选例中，所述的肌肉干细胞是从机体四肢或躯干肌肉中提取并通过流式细胞术分选所得到。
肌肉来源的间充质干细胞
骨骼肌与平滑肌中都存在间充质干细胞。肌肉损伤修复再生的主角是肌肉干细胞，但是一些其他类群的细胞，例如炎症细胞，血管内皮细胞和肌肉来源的间充质干细胞在整个肌肉损伤修复再生过程中，发挥着精细调节配合的作用。例如成纤维脂肪祖细胞是肌肉间质内的一类间充质干细胞，其作用是支持肌肉干细胞的分化，促进肌肉组织的修复再生。而在血管内的平滑肌来源的平滑肌祖细胞，主要作用是替代平滑肌细胞，从而填充血管病变或损伤区域。
药物组合物
本发明还提供了一种组合物。在优选例中，所述的组合物是药物组合物，它含有上述的肌肉干细胞，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：口服、呼吸道、瘤内、腹膜内、静脉内、或局部给药。
本发明的药物组合物可直接用于治疗(例如动脉粥样硬化的治疗)，此外，还可同时使用其他治疗剂。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的肌肉干细胞以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，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。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约1μg/Kg体重-约10mg/Kg体重。此外，本发明的肌肉干细胞还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的肌肉干细胞施用于哺乳动物，其中该安全有效量通常至少约10μg/Kg体重，而且在大多数情况下不超过约8mg/Kg体重，较佳地该剂量是约10μg/Kg体重-约1mg/Kg体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
在本发明的一个优选例中，所述药物组合物含有：
(i)肌肉干细胞作为活性成分；和
(ii)药学上可接受的载体；
所述药物组合物用于选自下组的一种或多种应用：
(a)延缓动脉粥样硬化进程
(b)减少主动脉弓及其分支处斑块面积；
(c)减少主动脉瓣膜处脂质的沉积；
(d)减少主动脉瓣膜处巨噬细胞的聚集；
(e)减少外周血中促炎型Ly6C hi单核细胞的数量和比例；
(f)减少外周血中巨噬细胞的数量和比例；
(g)减少白色脂肪组织重量；
(h)缓解机体炎症反应；
(i)降低血清中IL-6和CCL2的水平；
(j)差异性调节斑块中巨噬细胞内促抗炎基因的表达水平；
(k)提高抗炎型巨噬细胞相关基因的表达水平；
(l)降低促炎型巨噬细胞相关基因的表达水平；
(m)预防、缓解和/或治疗动脉粥样硬化；
(n)预防、缓解和/或治疗心血管疾病。
在另一优选例中，所述人源性肌肉干细胞为CD31 -，CD34 -，CD45 -，CD29 +，CD56 +，EGFR +和PAX 7 +的细胞。
在本发明的一个优选例中，所述药物组合物为抗动脉粥样硬化药物。
在本发明的一个优选例中，所述药物组合物为降低主动脉弓及其分支处斑块面积的药物。
在本发明的一个优选例中，所述药物组合物为减轻机体炎症环境的药物。
在本发明的一个优选例中，所述药物组合物的剂型选自下组：注射剂、冻干剂。
在本发明的一个优选例中，所述的动脉粥样硬化的剂型为新鲜培养的肌肉干细胞注射剂，或来自于冷冻储存的解冻肌肉干细胞注射剂。
在本发明的一个优选例中，所述的抗动脉粥样硬化药物能够明显延缓小鼠 的动脉粥样硬化，减少主动脉弓及其分支处的斑块面积。
在本发明的一个优选例中，所述的抗动脉粥样硬化药物能够减轻白色脂肪组织重量。
在本发明的一个优选例中，所述的抗动脉粥样硬化药物能够降低小鼠机体炎症水平。
本发明的主要优点包括
(1)本发明通过给高脂饲料诱导的动脉粥样硬化小鼠尾静脉注射肌肉干细胞，充分研究了肌肉干细胞在心血管疾病治疗方面的效果；
(2)本发明重点发现了肌肉干细胞具备下列功能：
①延缓小鼠动脉粥样硬化进程；
②减少主动脉弓及其分支处斑块面积；
③减少白色脂肪组织重量；
④降低机体炎症水平；
⑤改善机体各项生理机能；
⑥降低了巨噬细胞和促炎型单核细胞的数量和百分比。
(3)本发明能够通过有限次数的肌肉干细胞注射达到相对长久的治疗效果，从而产生以下优点：
①细胞注射次数少，减少机体遭受疼痛的次数，提高机体生存质量；
②少次肌肉干细胞注射就能起到长效作用，具有省时、省力的优势。
下面结合具体实施例，进一步陈述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
通用方法
(一)原位主动脉弓及分支处斑块面积观察
1、在处死小鼠后，通过心脏灌流预冷的PBS冲出血管内残留的血液。
2、在体式显微镜下，利用显微器械仔细剥离出主动脉及其分支，除去粘连的脂肪。
3、在主动脉下放置黑色纸片作为背景，相机聚焦于主动脉弓处并拍照。
(二)主动脉的油红O染色
1、油红O染液母液的配制：取0.5g油红O粉末溶于100mL的异丙醇(浓度为100％)中，37℃水浴使其充分溶解，室温避光保存，备用。
2、油红O工作液的配制：按油红O染液母液：ddH 2O＝3:2比例混匀,0.22μm有机系滤头过滤后使用，现用现配。
3、将剥离出主动脉，放入盛有2mL浓度为60％异丙醇的EP管中，媒染10min，有利于油红O染料的着色。
4、再将其转移至盛有2mL油红O工作液的EP管中，染色30min后，再用浓度为60％的异丙醇洗去残余的油红O浮色。
5、在体式显微镜下，剔除血管外粘连的脂肪(已被染成红色)，利用维纳斯剪剪开血管，并将其置于洁净的双面胶上，迅速展开血管，防止主动脉干燥。
6、相机拍照后，利用图像分析软件ImageJ进行分析处理，计算斑块面积所占整个血管的比例。
(三)主动脉根的冰冻切片及油红O染色
1、取小鼠心脏，切除心尖后，OCT包埋后，置于-80℃冰箱过夜。
2、采用连续切片的方式，收集从0μm-400μm的切片，厚度为5μm。
3、将冰冻切片置于含4％多聚甲醛的PBS溶液中固定10min，经ddH 2O清洗后，置于盛有浓度为60％异丙醇的染缸中媒染，5min。
4、再转移至含油红O工作液的染缸中，染色15min。
5、用浓度为60％异丙醇洗去多余的浮色，晾干后拍照，并用图像分析软件ImageJ分析处理。
实施例1人源性肌肉干细胞提取、扩增、鉴定及分化
(一)人源性肌肉干细胞的提取
1、配制试剂：洗涤液(含20％胎牛血清+1％双抗的DMEM LOW培养基)；粗消化液(将Ⅱ型胶原酶加入洗涤液中，使Ⅱ型胶原酶的终浓度为750U/mL)；精消化液(将Ⅱ型胶原酶和分散酶依次加入洗涤液，使其终浓度分别为1000U/mL、11U/mL)；肌肉干细胞生长完全培养基(20％胎牛血清+40％DMEM LOW+40％MCDB 131+胰岛素转铁蛋白硒(Insulin-transferrin-X)+1％双抗+10μmol p38抑制剂)。
2、经伦理审批，从医院获取人来源的肌肉组织块，放入10cm培养皿中，滴加2mL粗消化液，剪碎形成组织原浆液，再用13mL粗消化液充分冲洗培养皿，所有组织碎片均转移至50mL离心管中。
3、离心管水平放置在恒温摇床上，37℃孵育60min，转速为70rpm。
4、孵育消化结束后，用洗涤液中和，加至50mL，上下颠倒混匀，500g离心5min，吸去上清。
5、向沉淀的细胞团块中加入15ml的精消化液，充分混匀后，再次放入恒温摇床中，37℃孵育60min，转速为70rpm。
6、孵育结束后，加入15mL洗涤液进行中和，并用70μm的细胞筛网进行过滤。
7、过滤后，将所得液体500g离心5min，吸去上清，获得细胞沉淀。
8、用500μL完全培养基重悬细胞沉淀，用于流式分选。
9、吸取出10μL细胞悬液至含有190μL完全培养基的流式管中作阴性对照使用。
10、将剩余细胞悬液进行流式分选抗体染色，标记抗体分别为CD31-PE，CD34-PE，CD45-PE，CD29-APC，CD56-BV421,EGFR-BV421染色40min，每20min摇一次,四度存放。染色完成后，完全培养基清洗离心，并最终使用200μL完全培养基进行细胞重悬。
11、上机，进行流式分选。
(二)人源性肌肉干细胞的体外扩增培养
1、待全部细胞悬液分选完毕后，800rpm离心5min，去除洗涤培养基上清，加入完全培养基重悬后，转移至细胞外基质预包被过的培养皿中(Extra Cellular Matrix，ECM包被24小时)进行培养扩增。
2、每两天更换一次新鲜培养基直至细胞融合度达到60～70％。
3、使用胰蛋白酶对肌肉干细胞进行传代，随后将子代细胞种植于新的ECM包被的培养皿中继续培养扩增。
(三)人源性肌肉干细胞的鉴定
1、细胞生长至融合度达70％，可用胰酶消化获取所需细胞。
2、分别对人肌肉干细胞表面表达的标志蛋白进行流式鉴定、人肌肉干细胞特异性核因子PAX7的流式表达鉴定。
(四)人源性肌肉干细胞肌分化能力鉴定
1、将肌肉干细胞培养于胶原包被的培养板至细胞完全融合。
2、用无血清DMEM培养基洗涤细胞三次，而后换成肌肉干细胞分化培养基(DMEM培养基+2％马血清)继续培养。分化培养第1天，细胞开始伸长并发生细胞间的融合。第2～3天细胞融合达到峰值，大部分细胞已经完成向肌管的分化过程。此时进行免疫荧光染色人肌肉干细胞特异性分化成熟蛋白肌球蛋白重链(MyHC)，从而鉴定实验以确定分离的细胞具有干细胞的分化特性。
结果如图1所示。
图1表明：本发明分离的细胞高表达肌肉干细胞标志物PAX7。分离的细胞为内皮细胞标志物CD31阴性的细胞，是造血干细胞标志物CD34阴性的细胞，是白细胞标志物CD45阴性的细胞。该分离的细胞高表达干细胞标志物CD29和CD56，且表达比率为100％。且该细胞高表达表皮生长因子受体EGFR达90％。所以该分离出的细胞为CD31 -，CD34 -，CD45 -，CD29 +，CD56 +，EGFR +和PAX7 +的肌肉干细胞。体外培养扩增的肌肉干细胞能够很好地维持干细胞表面标志以及核内特异性因子PAX7的表达，且能分化为成熟且具有功能性的肌纤维结构，说明以上体系所培养的肌肉干细胞具有优良的干性和肌纤维生成能力。
目前，ApoE -/-小鼠和LDLR -/-小鼠是动脉粥样硬化中较常用的两种动物模型。故本实施例中，采用了上述的两种小鼠构建动脉粥样硬化模型。
实施例2人源性肌肉干细胞对ApoE -/-小鼠动脉粥样硬化进程的延缓作用
(一)小鼠模型建立
随机选取12-14周龄雄性ApoE -/-小鼠进行分组，分为模型组(高脂饮食组， PBS)和治疗组(人源性肌肉干细胞注射的高脂饮食小鼠组，hMuSC)，均投喂含量40％Kal脂肪含量的高脂饮食，持续6周。(见图2的A图左侧)
(二)人源性肌肉干细胞体内移植及实施的检测指标
ApoE -/-小鼠：在喂饲高脂饲料的第三周，采用尾静脉注射的方式，每周给予模型组注射无菌PBS，注射体系为200μL。给予治疗组注射人源性肌肉干细胞(Human MuSC，即hMuSC)，注射量为每只小鼠注射5×10 5个细胞，注射体系为200μL。注射周期为每周一次，共注射4次。在最后一次注射完成的第四天给予小鼠实施安乐死，进行解剖取材，并完成后续的血脂水平、血清中炎症因子、斑块大小或面积和组织病理学的检测指标(见图2的A图右侧)。
(三)原位主动脉弓及分支处斑块面积观察
实验结果如图2B，显示了对小鼠主动脉弓及其分支处斑块原位直观比较。
(四)主动脉的油红O染色
对主动脉进行油红O染色，打开血管后进行拍照，直观的比较斑块的面积大小，并利用imageJ对着色区域进行量化，计算斑块面积和斑块面积占整个主动脉面积的占比(见图2的C图)。
(五)主动脉根的冰冻切片及油红O染色
实验结果如图2的D图，对主动脉根进行冰冻切片后进行油红O染色并拍照，以及脂质沉积程度的比较。图2E显示了不同距离主动脉根瓣膜处斑块面积的比较图。
(六)小鼠附睾脂肪指数的检测
给小鼠称重后，处死小鼠并取小鼠的附睾脂肪，小鼠附睾脂肪指数＝附睾脂肪的重量/小鼠的体重(见图2的F图)。
(七)小鼠体内炎症水平的检测
取得小鼠外周血，静置30min后进行离心，离心条件设置为3000rpm，30min。离心后，吸取血清。根据ELISA试剂说明书检测血清中IL-6的含量(见图2的G图)。
结果如图2所示，本次实验在喂食高脂饮食的第三周即给予了肌肉干细胞治疗，故此，该实验可被视为肌肉干细胞对ApoE -/-小鼠动脉粥样硬化的预防作用。
结果表明，通过主动脉弓原位图片，主动脉根和主动脉的油红O染色，能够清楚的看到，6周的高脂饮食即可诱导ApoE -/-小鼠产生严重的动脉粥样硬化，主要集中在主动脉弓处及其分支斑块的蓄积和主动脉根瓣膜处脂质的沉积。而经hMuSC的治疗后，小鼠的斑块面积有着明显的改善。除此之外，小鼠的附睾脂肪指数及血清中炎症因子白介素-6的水平也有明显下降。
为进一步明确肌肉干细胞的抗动脉粥样硬化作用，采用了LDLR -/-小鼠作为诱导动脉粥样硬化的模型(PBS组和hMuSC组的血脂水平并无显著性差异，数据未展示)。
实施例3人源性肌肉干细胞对LDLR -/-小鼠动脉粥样硬化进程的延缓作用
(一)小鼠模型建立
随机选取12-14周龄雄性LDLR -/-小鼠进行分组，分为模型组(高脂饮食组，PBS)和治疗组(人源性肌肉干细胞注射的高脂饮食小鼠组，hMuSC)，均投喂含量40％Kal脂肪含量的高脂饮食，持续8周(见图3的A图上侧)。
(二)人源性肌肉干细胞体内移植及实施的检测指标
LDLR -/-小鼠：在喂饲高脂饲料的第5周，采用尾静脉注射的方式，每周给予模型组注射无菌PBS，注射体系为200μL。给予治疗组注射人源性肌肉干细胞(Human MuSC，即hMuSC)，注射量为每只小鼠注射5×10 5个细胞，注射体系为200μL。注射周期为每周一次，共注射4次。在最后一次注射完成的第四天给予小鼠实施安乐死，进行解剖取材，并完成后续的斑块大小或面积、血清中炎症因子、组织病理学和外周血中髓系细胞(主要是巨噬细胞和促炎型Ly6C hi单核细胞)的变化等检测指标(见图3的A图的下侧)。
(三)原位主动脉弓及分支处斑块面积观察
如图3B所示，对小鼠主动脉弓及其分支处斑块原位直观比较。
(四)主动脉根的冰冻切片及油红O染色
如图3的C图所示，对主动脉根进行冰冻切片后进行油红O染色并拍照，比较主动脉根瓣膜处脂质沉积程度。
如图3的D图所示，比较不同距离主动脉根瓣膜处斑块面积。
(五)主动脉的油红O染色
如图3的E图所示，对主动脉进行油红O染色，打开血管后进行拍照，直观的比较斑块的面积大小，并利用ImageJ对着色区域进行量化，计算斑块面积和斑块面积占整个主动脉面积的占比。
(六)小鼠附睾脂肪指数的检测
给小鼠称重后，处死小鼠并取小鼠的附睾脂肪，小鼠附睾脂肪指数＝附睾脂肪的重量/小鼠的体重(见图3的F图)。
(七)血清中白介素-6含量检测(见图3的G图)
以上结果表明，通过主动脉弓原位图片，主动脉根和主动脉的油红O染色，能够清楚的看到，6周的高脂饮食即可诱导LDLR -/-小鼠产生严重的动脉粥样硬化，主要集中在主动脉弓处及其分支斑块的蓄积和主动脉根瓣膜处脂质的沉积。而经hMuSC的治疗后，小鼠的斑块面积有着明显的改善。除此之外，小鼠的附睾脂肪指数及血清中炎症因子白介素-6的水平也有明显下降。
(八)主动脉根瓣膜处中巨噬细胞积累情况检测(采用免疫荧光法检测Mac-3的表达程度，从而衡量巨噬细胞的积累情况)
1.对主动脉根进行冰冻切片，将切片置于4％多聚甲醛溶液固定10min，PBS清洗两次(放置在摇床)，5min/次。
2.在室温条件下，对切片进行封闭破膜处理1h。封闭破膜液配制：0.06g BSA溶于含6mL的PBS中，再加入0.1mL的Triton-100。
3.PBS清洗2次，5min/次。
4.孵育一抗(Mac-3的抗体，稀释比例为1:100)，4℃过夜。
5.PBS清洗3次，5min/次，室温孵育荧光二抗1h。
6.PBS清洗3次,Hochest染色10min(稀释比例为1:2000)
7.PBS清洗3次后，滴加抗荧光淬灭剂后封片，激光共聚焦显微镜拍照，ImageJ分析图片(见图4的A图)。
该结果表明：与PBS组相比，hMuSC治疗显著的降低了主动脉根瓣膜处斑块中的Mac-3的表达，也反映了hMuSC治疗显著抑制了巨噬细胞在血管壁的积累。
(九)外周血中巨噬细胞和促炎性单核细胞的检测(流式细胞技术检测)
1.经眼缘静脉采取小鼠外周血(300uL)，置于抗凝管，加入五倍体积的 裂红液裂红8min后，加入5mL含2％FBS的PBS中和，4℃条件下，500g离心5min。
2.弃上清(根据沉淀的颜色判断裂红是否完全，若呈红色需再次裂红)，每个样品加入50uL提前混合好的流式抗体(CD45-FITC、CD11b-PE-Cy7、Ly6C-BV421、F4/80-APC-Cy7，用含2％FBS的PBS稀释，稀释比例为1:200)，充分混匀，4℃避光孵育30min。
3.加200uL含2％FBS的PBS，混匀后，4℃条件下，500g离心。
4.弃上清，加200uL含2％FBS的PBS，混匀后上机检测，统计出巨噬细胞和促炎性单核细胞的数量和百分比。(在CD45 +的群中圈出的CD11b +F4/80 +为巨噬细胞，Ly6C hiCD11b +为促炎性的单核细胞，见图4的B和C)
该结果表明：hMuSC治疗显著地降低了巨噬细胞和促炎型单核细胞的数量和百分比，从而进一步的降低疾病小鼠的机体炎症水平。由于已有大量的文献报道高脂饮食所有诱导的动脉粥样硬化小鼠，往往伴随着髓系细胞的增多(主要是巨噬细胞和促炎性的单核细胞)，因此本发明的hMuSC治疗通过抑制髓系细胞的增多能够延缓该疾病的进程。
(十)小鼠血清中趋化因子CCL2含量的检测(见图4的D图)
图4的D图的结果表明，hMuSC治疗显著降低了小鼠血清中CCL2的表达。
由于CCL2不仅可以衡量机体的炎症水平，还可以诱导免疫细胞的招募(尤其是单核-巨噬细胞的招募)。因此，hMuSC治疗显著降低了小鼠血清中CCL2水平的实验结果，更加佐证了hMuSC通过降低机体炎症水平发挥抗动脉粥样硬化的作用。
(十一)主动脉中抗炎型巨噬细胞和促炎性巨噬细胞相关基因的表达水平
1.取小鼠主动脉，仔细剥离附着的脂肪，然后置于EP管中，加入1mL Trizol。
2.使用组织匀浆机研磨组织块，并在研磨彻底后，将EP管直接放入-80℃冰箱，冻存。
3.将冻存的EP管取出，刮去上层白色脂肪块，待Trizol解冻后，提取样品中的RNA。
4.利用反转录试剂盒将收集的RNA进行反转录，并将目的基因进行QPCR并进行比较分析。
对小鼠主动脉中促炎的巨噬细胞Ccl2和Il1b表达的检测及抗炎的巨噬细胞 Arg1表达的检测，如图4的E图所示。
实验结果表明，与模型组相比，注射肌肉干细胞提高了主动脉中抗炎型巨噬细胞Arg1，降低了促炎型巨噬细胞Il1b的表达，使巨噬细胞向抗炎型的巨噬细胞极化。
综上所述，尾静脉注射人来源的肌肉干细胞(hMuSC)能够显著延缓高脂饲料喂养的LDLR -/-小鼠的动脉粥样硬化的进程。这一结果与实施例2中的结果是一致的，即人源性肌肉干细胞对小鼠动脉粥样硬化的进程有很好的延缓作用。
实施例4人源性肌肉干细胞对ApoE -/-小鼠动脉粥样硬化进程的治疗作用
(一)小鼠模型建立
随机选取6-8周龄雄性ApoE -/-小鼠进行分组，分为对照组(正常饮食)，模型组(高脂饮食组)和治疗组(人源性肌肉干细胞注射的高脂饮食小鼠组)均投喂含量40％Kal脂肪含量的高脂饮食，持续10周(见图5的A图)。
(二)人源性肌肉干细胞体内移植
ApoE -/-小鼠：在喂饲高脂饲料的第7周，采用尾静脉注射的方式，每周给予对照组和模型组注射无菌PBS，注射体系为200μL。给予治疗组注射人源性肌肉干细胞(Human MuSC，即hMuSC)，注射量为每只小鼠注射5×10 5个细胞，注射体系为200μL。注射周期为每周一次，共注射4次，本次实验可作为肌肉干细胞对于动脉粥样硬化的治疗作用。
(三)原位主动脉弓及分支处斑块面积观察
观察6周正常饮食小鼠、6周高脂饮食、10周正常饮食+PBS、10周高脂饮食+PBS、10周高脂饮食+hMuSC治疗的主动脉弓原位图片(见图5的B图)。
(四)主动脉的油红O染色
观察6周正常饮食小鼠、6周高脂饮食、10周正常饮食+PBS、10周高脂饮食+PBS、10周高脂饮食+hMuSC治疗的主动脉油红O染色图片(见图5的C图)；
对主动脉斑块面积所占主动脉面积的比例进行统计(见图5的E图)。
(五)主动脉根的冰冻切片及油红O染色
(六)小鼠附睾脂肪指数的检测
给与小鼠称重后，处死小鼠并取小鼠的附睾脂肪，小鼠附睾脂肪指数＝附 睾脂肪的重量/小鼠的体重(见图5的D图)。
如图5所示，结果表明：6周正常饮食组则无明显的斑块出现，而6周高脂饮食即可诱导较为严重的动脉粥样硬化。本次实验在第7周才给予hMuSC治疗，并且经过四次的hMuSC输注，显著减小了主动脉弓及其分支的斑块面积，而模型组则发生了更为严重的病情。与正常组相比，高脂饮食显著增加了附睾脂肪指数，经hMuSC治疗后，其附睾脂肪指数与正常饮食组相当。
综上所述，肌肉干细胞对于动脉粥样硬化的治疗也具有良好的作用。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (13)

  1. 一种肌肉干细胞的用途，其特征在于，所述肌肉干细胞用于制备药物组合物，所述药物组合物用于选自下组的一种或多种应用：
    (a)延缓动脉粥样硬化进程
    (b)减少主动脉弓及其分支处斑块面积；
    (c)减少主动脉瓣膜处脂质的沉积；
    (d)减少主动脉瓣膜处巨噬细胞的聚集；
    (e)减少外周血中促炎型单核细胞的数量和比例；
    (f)减少外周血中巨噬细胞的数量和比例；
    (g)减少白色脂肪组织重量；
    (h)缓解机体炎症反应；
    (i)降低血清中IL-6和CCL2的水平；
    (j)差异性调节斑块中巨噬细胞内促抗炎基因的表达水平；
    (k)提高抗炎型巨噬细胞相关基因的表达水平；
    (l)降低促炎型巨噬细胞相关基因的表达水平；
    (m)预防、缓解和/或治疗动脉粥样硬化；
    (n)预防、缓解和/或治疗心血管疾病。
  2. 如权利要求1所述的用途，其特征在于，所述药物组合物用于预防、缓解和/或治疗心血管疾病。
  3. 如权利要求1所述的用途，其特征在于，所述心血管疾病选自下组：动脉粥样硬化、心肌梗死、脑梗死、主动脉瘤。
  4. 如权利要求2所述的用途，其特征在于，所述心血管疾病选自下组：主动脉粥样硬化、冠状动脉粥样硬化、冠状动脉粥样硬化性心脏病、颈动脉和脑动脉粥样硬化、肾动脉粥样硬化和四肢动脉粥样硬化。
  5. 如权利要求1所述的用途，其特征在于，所述肌肉干细胞选自下组：鼠源性肌肉干细胞、人源性肌肉干细胞、猴源性肌肉干细胞、狗源性肌肉干细胞、猫源性肌肉干细胞、马源性肌肉干细胞、或其组合。
  6. 如权利要求1所述的用途，其特征在于，所述人源性肌肉干细胞为CD31 -，CD34 -，CD45 -，CD29 +，CD56 +，EGFR +和PAX7 +的细胞。
  7. 如权利要求1所述的用途，其特征在于，所述的肌肉干细胞可以是自体的， 也可以是同种异体的，或异种的。
  8. 如权利要求1所述的用途，其特征在于，所述的肌肉干细胞是从机体四肢或躯干肌肉中提取并通过细胞分选所获得。
  9. 一种用于人或动物的药物组合物，其特征在于，所述药物组合物含有：
    (i)肌肉干细胞作为第一活性成分；
    (ii)调节脂代谢的第二活性成分；和
    (iii)药学上可接受的载体；其中，所述肌肉干细胞为人源性肌肉干细胞，较佳地为CD31 -，CD34 -，CD45 -，CD29 +，CD56 +，EGFR +和PAX7 +的细胞。
  10. 如权利要求9所述的药物组合物，其特征在于，所述药物组合物用于选自下组的一种或多种应用：
    (a)延缓动脉粥样硬化进程
    (b)减少主动脉弓及其分支处斑块面积；
    (c)减少主动脉瓣膜处脂质的沉积；
    (d)减少主动脉瓣膜处巨噬细胞的聚集；
    (e)减少外周血中促炎型单核细胞的数量和比例；
    (f)减少外周血中巨噬细胞的数量和比例；
    (g)减少白色脂肪组织重量；
    (h)缓解机体炎症反应；
    (i)降低血清中IL-6和CCL2的水平；
    (j)差异性调节斑块中巨噬细胞内促抗炎基因的表达水平；
    (k)提高抗炎型巨噬细胞相关基因的表达水平；
    (l)降低促炎型巨噬细胞相关基因的表达水平；
    (m)预防、缓解和/或治疗动脉粥样硬化；
    (n)预防、缓解和/或治疗心血管疾病。
  11. 如权利要求9所述的药物组合物，其特征在于，所述的第二活性成分选自下组：他汀类药物、PCSK9抑制剂、贝特类药物、抗凝血类药物或溶栓类药物。
  12. 如权利要求9所述的药物组合物，其特征在于，所述药物组合物为预防、缓解和/或治疗主动脉粥样硬化、冠状动脉粥样硬化、颈动脉和脑动脉粥样硬化、冠状动脉粥样硬化性心脏病、肾动脉粥样硬化和四肢动脉粥样硬化的药物。
  13. 如权利要求9所述的药物组合物，其特征在于，所述药物组合物的剂型选 自下组：注射剂、冻干剂。
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