CN110917217A

CN110917217A - Application of muscle stem cells in preparation of anti-inflammatory drugs

Info

Publication number: CN110917217A
Application number: CN201911358302.5A
Authority: CN
Inventors: 时玉舫; 房建凯; 陈永井; 张胜超; 邵常顺
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2019-12-25
Filing date: 2019-12-25
Publication date: 2020-03-27
Anticipated expiration: 2039-12-25
Also published as: CN110917217B

Abstract

The invention discloses an application of muscle stem cells in preparing anti-inflammatory drugs. The muscle stem cells or the supernatant liquid of the muscle stem cells are mediated by anti-inflammatory macrophages, and the anti-inflammatory characteristics of the muscle stem cells or the supernatant liquid are shaped in the maturation process of the macrophages, so that inflammatory diseases with the inflammatory macrophages as main pathogenic cell populations can be effectively treated. For example, treatment of colitis mice with muscle stem cells or supernatants thereof according to the present invention may be effective in reducing disease symptoms of weight loss, diarrhea, hematochezia, bowel tissue thickening edema, structural damage, and immune cell infiltration.

Description

Application of muscle stem cells in preparation of anti-inflammatory drugs

Technical Field

The invention relates to an application of muscle stem cells in preparing anti-inflammatory drugs, and belongs to the technical field of immunoregulation.

Background

Skeletal muscle is the largest tissue organ of the human body, and accounts for about 35% of the body weight. It is involved in glycolipid metabolism of the body in addition to providing the energy necessary for individual exerciseAnd precise regulation of body temperature. In addition to myogenic cells, a large number of immune cells are also present in healthy skeletal muscle tissue. Per mm of adult organism³The muscle tissue contains 500-2000 white blood cells, which is equivalent to about 10 per liter of muscle tissue⁹And (4) white blood cells. Despite the large variety of intramuscular leukocytes, the vast majority of immune cell populations are monocytes and macrophages. They surround skeletal Muscle stem cells (MuSC). The synchronous functional transformation and phenotypic transformation of macrophages during muscle repair and regeneration directly determine the fate of muscle stem cells, the repair of injury and the outcome of regeneration. However, there is no report on how muscle stem cells reversely regulate the functional fate of immune cells at home and abroad.

In addition to being an important endocrine tissue, skeletal muscle is also a major immunoregulatory organ of the body, which can participate in the regulation of body homeostasis and physiological functions by producing various immune molecules. The inflammatory cytokine interleukin-6 (IL-6) released by motor muscles is capable of inactivating endotoxin-induced inflammatory responses. Skeletal muscle produced meteoroid protein (MTRNL) induces "browning" of white adipose tissue and induces polarization of IL-4 dependent anti-inflammatory macrophages. The IL-15 derived from muscle can promote the proliferation of naive T cells, enhance the development and cytotoxicity of natural killer cells, reduce lipid deposition, and prevent the lipopathy of internal organs. However, whether muscle stem cells are the core cellular component of skeletal muscle, are the cellular source of these immunoregulatory molecules, is not well defined by the scientific community.

Therefore, the research on the immunoregulatory function of the muscle stem cells and the development of a novel stem cell treatment means provide a firm theoretical basis for the expansion of the application field of the muscle stem cells.

Disclosure of Invention

In order to solve the technical problems, the invention provides an application of muscle stem cells in preparing anti-inflammatory drugs. The muscle stem cells amplified in vitro show stronger anti-inflammatory action in DSS-induced colitis mice.

The invention aims to provide an application of muscle stem cells in preparing anti-inflammatory drugs.

Further, the anti-inflammatory drug is muscle stem cells or cell supernatant extracted after the muscle stem cells are cultured.

Further, the cell supernatant is obtained by culturing the muscle stem cells until the fusion degree reaches 70-80%, washing with a buffer solution, continuously culturing for 20-25 hours by using a serum-free culture medium, collecting the culture supernatant, centrifuging and filtering.

Furthermore, the muscle stem cells are obtained by subjecting skeletal muscle tissue to enzymatic digestion and then flow sorting.

Further, the muscle stem cells are obtained from human, murine, porcine or rabbit sources.

Further, the preparation form of the medicine is injection.

Further, the drug is administered by intravenous injection.

Further, the anti-inflammatory agent is an agent for treating inflammatory diseases in which inflammatory macrophages are the main pathogenic cell population.

Further, the anti-inflammatory drug is a drug for treating colitis.

The invention has the beneficial effects that: the muscle stem cells or the supernatant liquid of the muscle stem cells are mediated by anti-inflammatory macrophages, and the anti-inflammatory characteristics of the muscle stem cells or the supernatant liquid are shaped in the maturation process of the macrophages, so that inflammatory diseases with the inflammatory macrophages as main pathogenic cell populations can be effectively treated. For example, treatment of colitis mice with muscle stem cells or supernatants thereof according to the present invention may be effective in reducing disease symptoms of weight loss, diarrhea, hematochezia, bowel tissue thickening edema, structural damage, and immune cell infiltration.

Drawings

FIG. 1 is a graph of phenotypic identification and differentiation potency assay of MuSC. (A) Detecting the expression of the cultured MuSC surface marker by flow cytometry; (B) detecting the expression of a nuclear factor PAX7 characteristic to MuSC by flow cytometry; (C) the differentiation potential of MuSC was examined by cellular immunofluorescence. MyHC: myosin heavy chain; hoechst: and (4) cell nucleus.

Scale bar: 50 μm.

FIG. 2 is a drawing of MuSC for alleviating colitis. (a-C) body weight, disease activity index and colon length of PBS and MuSC treated colitis mice on day 2 post disease induction; (D) histological scoring of representative colon H & E stained sections and MuSC-treated colitis mice, scale bar: 50 μm; (E) measuring the content of IL-6 in the serum of a MuSC-treated colitis mouse by adopting an ELISA method; (F-H) mice with MuSC supernatant treated colitis administered daily after induction of colitis body weight, disease activity index and colon length; (I) representative colon H & E stained sections and histological scoring, scale: 75 μm; (J) determining the content of IL-6 in the blood serum of the colitis mouse treated by the MuSC supernatant by adopting an ELISA method; data are presented as mean ± SEM. P <0.05, p <0.01, p < 0.001.

FIG. 3 shows the expression levels of anti-inflammatory genes of macrophages after MuSC supernatant treatment by qRT-PCR, (B) shows the changes in gene expression of IL-1 β and IL-6 after stimulation by LPS (50ng/mL) and IFN- γ (20ng/mL) by the macrophages after MuSC supernatant treatment by qRT-PCR, (C) shows the changes in the concentrations of IL-1 β and IL-6 in the culture fluid after stimulation by LPS (50ng/mL) and IFN- γ (20ng/mL) by ELISA, (D) shows the changes in gene and protein expression of macrophages after MuSC supernatant treatment by LPS (50ng/mL) and IFN- γ (20ng/mL) by qRT-PCR and flow cytometry, (E) shows the changes in the surface expression of immune cells infiltrated into tissue of mice treated with MuSC by flow cytometry, (F) shows the changes in the surface expression of macrophages after stimulation by LPS (50ng/mL) and IFN- γ (20ng/mL) shows the changes in the mean cell infiltration of MFI 0.001.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

1 colitis animal model establishment

1.1 Experimental animals

The mice of strain C57BL/6 used in the experiment were purchased from Experimental animals technology, Inc. of Weitongli, Beijing and were bred in strict compliance with the SPF barrier system standard. The animal experiment operation related to the subject is approved by animal experiment ethics committee of the university of suzhou.

1.2 colitis model establishment

On the day of experiment start, different mice were randomly assigned and given normal water diet and diet containing 4% DSS water, with the mice receiving water every two days for a total of 7 days. The disease mice were given different treatment treatments according to different experimental objectives and the body weight of the colitis mice was recorded daily.

1.3 colitis model treatment

Muscle stem cell therapy: on day 2 of induction in the mouse colitis model, mice were given tail vein injections of 2.5 x 10⁵The MuSC is used for disease treatment.

Muscle stem cell culture supernatant treatment: mice were given 200 μ l of concentrated MuSC culture supernatant per day for disease treatment starting from colitis model induction.

1.4 colitis disease Activity index

A. Weight loss (0 to 4 points)

0 minute: no weight loss occurred;

1 minute: lose less than 10% of the original body weight;

and 2, dividing: losing 10% -15% of the initial body weight;

and 3, dividing: losing 15% -20% of the initial body weight;

and 4, dividing: more than 20% of the initial body weight is lost.

B. Degree of diarrhea (0 to 2 points)

0 minute: no diarrhea;

1 minute: mild diarrhea;

and 2, dividing: moderate to severe diarrhea.

C. Rectal bleeding degree (0 to 2 points)

0 minute: no bleeding;

1 minute: mild bleeding;

and 2, dividing: moderate to severe bleeding.

D. Organism activity (0 to 2 minutes)

0 minute: is normal

1 minute: mild depression;

and 2, dividing: moderate to severe depression.

1.5 colitis histology score

A. Degree of thickening of intestinal wall (0 to 3 points)

0 minute: no thickening;

1 minute: thickening the mucosa;

and 2, dividing: thickening of mucosa and submucosa;

and 3, dividing: penetrating the intestinal wall.

B. Extent of crypt damage (0 to 3 points)

0 minute: no damage is caused;

1 minute: loss of goblet cells;

and 2, dividing: only the surface epithelium is intact;

and 3, dividing: the entire crypt and epithelial cells are lost.

C. Inflammatory cell infiltration status (0 to 2 points)

0 minute: no inflammatory cell infiltration;

1 minute: mild to moderate infiltration;

and 2, dividing: and (5) heavily infiltrating.

2 MuSC in vitro culture and functional identification

2.1 MuSC isolation and extraction and in vitro amplification

Dissection of hind limb muscles

(1) A10 cm dish containing 10ml of a washing medium (F10 medium containing 10% fetal bovine serum) was prepared.

(2) After the mouse is killed by cervical dislocation, the whole body of the mouse is soaked in 75% alcohol for about 10min to remove bacteria and various microorganisms attached to the surface of the animal, and then the mouse is fixed on a horizontal dissecting table for subsequent dissecting treatment.

(3) The ophthalmic forceps lift the skin of the lower abdomen of the mouse and a gap is cut by the ophthalmic scissors.

(4) Starting from the abdominal notch, the skin is cut to expose the entire lower limb muscles, extending to the achilles tendon of the lower limb.

(5) Collect hind limb total muscle in petri dish:

a) all tendons at the ankle of the lower limb are cut off, and then the tibialis anterior, extensor digitorum longus, gastrocnemius and soleus muscles are separated from the bottom to the top. At the junction of the end of the knee, all muscles of the lower leg were cut and placed in a petri dish.

b) The femoral quadriceps root above the knee is lifted using the ophthalmic forceps and then pulled upward to detach it from the femur. At the end of the femoral head, the quadriceps femoris muscle was cut and placed in a petri dish.

c) The remaining muscles of the thigh were trimmed along the femur and then placed all in a petri dish to remove any visible adipose tissue and tendons.

d) According to steps a-c, all muscle tissue of the opposite lower limb was collected.

(6) The washing medium which was originally used for soaking the wet muscles in the culture dish was aspirated off, all the muscles were collected to one end of the culture dish, 2ml of collagenase digest (750U/ml) was added dropwise to the muscle mass, and then all the muscles were minced into a muddy flesh puree with scissors.

(II) extraction of mononuclear cells

(1) The muscle tissue puree was transferred to a 50ml centrifuge tube containing 10ml collagenase digest, followed by 8ml fresh collagenase digest to wash and wash the residual tissue debris from the petri dish, ensuring that all tissue was transferred to the centrifuge tube.

(2) Placing the centrifuge tube containing the tissue puree on a preheated horizontal shaker, and incubating and digesting for 60min at 37 ℃ at a rotation speed of 70 r.p.m.

(3) After incubation and digestion, the washing medium was added to 50ml, the tube cap was tightened and inverted several times to mix the suspension thoroughly.

(4) Centrifuge at 500g for 5min, and then aspirate suspended adipose tissue and supernatant fluid from top to bottom with a wash pump with minimal suction to a level of 16ml fluid volume. The sediment at the bottom of the tube is not sucked away.

(5) 2ml collagenase stock (1000U/ml) and 2ml dispase stock (11U/ml) were added to the centrifuge tube.

(6) The cell pellet was resuspended using a 10ml pipette and blown up and down to avoid air bubbles until no cell pellet blocked the pipette.

(7) The tube containing 20ml of cell suspension was placed on a preheated horizontal shaker and digestion was continued at 37 ℃ for 30min at 70 r.p.m.

(8) After incubation and digestion, the washing medium was added to 50ml, the tube cap was tightened and inverted several times to mix the suspension thoroughly.

(9) Centrifuge at 500g for 5min, and then aspirate the supernatant with a wash pump from top to bottom with minimal suction to the level of 10ml liquid volume. The sediment at the bottom of the tube is not sucked away.

(10) The cell pellet was resuspended by adding 10ml of wash medium and then transferred to a 70 μm cell screen for filtration, allowing the cell suspension to freely pass through the screen by gravity and drip into a new 50ml centrifuge tube.

(11) 10ml of washing medium was aspirated to wash the original centrifuge tube, and then the liquid was transferred to a 70 μm cell screen for filtration.

(12) 10ml of washing culture medium is sucked to wash the 70 mu m cell screen, so that all cells can smoothly pass through the screen and enter a centrifuge tube below. After the filtration is finished, sucking the cell suspension adhered to the lower layer of the screen into the filtered cell suspension by using a 1ml gun head so as to ensure the maximized cell yield.

(13) Centrifuging for 5min at 500g, sucking the supernatant liquid to 10ml liquid volume level from top to bottom by using a washing liquid pump with minimum suction force, transferring the supernatant liquid to a 15ml centrifuge tube after heavy suspension cell precipitation, and simultaneously sucking 5ml washing culture medium to wash and wash the original centrifuge tube and transferring the washed supernatant liquid to the 15ml centrifuge tube.

(14) Centrifuge at 500g for 5min, pour out the supernatant, resuspend the cell pellet using wash medium rationally according to cell pellet size.

(III) cell staining

(1) And a small part of cells are sucked to be used as isotype control test tubes of antibodies with different colors so as to ensure the accuracy of a subsequent flow type sorting and drawing strategy.

(2) Different color-labeled antibodies (CD31, CD45, Sca1, and VCAM1) were added to the remaining cell suspension (antibody usage was every 10 th)⁷1. mu.g of each cell was used).

(3) The cell suspension was resuspended and then incubated on ice at 4 ℃ for 40 min. During this period the cell suspension was shaken every 10min to ensure adequate antibody staining.

(4) After incubation, add wash medium to 15ml, tighten the tube cap, invert several times upside down to mix the suspension well.

(5) Centrifuging for 5min at 250g, pouring out supernatant, re-suspending the cell suspension by using 1ml of washing culture medium, then adding PI staining solution with the final concentration of 0.3 mu g/ml, continuously mixing the cell suspension uniformly, and preparing for upper flow type sorting.

(IV) flow sorting

(1) And (4) loading the isotype control sample to prepare a proper gating strategy and circle out the correct muscle stem cell population.

(2) Excluding cell debris and dead cell populations, selecting CD31^-CD45^-Sca1^-VCAM1⁺The cell suspension is aspirated at a minimum flow rate for flow sorting.

(3) After all cell suspensions are sorted, centrifuging for 5min at 250g, removing supernatant of a washing culture medium, adding a muscle stem cell growth culture medium for resuspension, and transferring to a new culture dish for culture and amplification.

(V) expansion culture of muscle stem cells

(1) After centrifugation of freshly extracted muscle stem cells was completed, the cells were resuspended in muscle stem cell growth medium (F10 medium + 20% fetal bovine serum +2.5ng/ml bFGF), and then plated on collagen-coated culture dishes for expansion culture.

(2) And replacing the fresh culture medium every two days until the cell fusion degree reaches 60-70%.

(3) The muscle stem cells are passaged using trypsin, and then the daughter cells are seeded on new collagen-coated culture dishes for further culture and expansion.

2.2 MuSC muscle differentiation ability identification

MuSC was cultured on ECM coated plates until the cells were fully confluent. 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) for further culture. On day 1 of differentiation culture, cells began to elongate and cell-cell fusion occurred. Cell fusion reaches a peak value at days 2-3, and most cells have already completed the differentiation process to myotubes. At this time, immunofluorescent staining identification experiments are immediately carried out to determine the differentiation state of the muscle stem cells.

2.3 MuSC culture supernatant preparation

And (3) when the MuSC grows until the fusion degree reaches 70-80%, washing the cells twice by using PBS, changing the cells into a serum-free F-10 culture medium, continuously culturing for 24 hours, collecting cell culture supernatant, centrifuging for 10min by using 500g, filtering by using a 0.22 mu m filter membrane to remove necrotic fragments, concentrating the culture supernatant by using a 3KD ultrafiltration tube, and storing at-80 ℃ for later use.

3. Construction of bone marrow macrophage maturation System

(1) After the mice were sacrificed by cervical dislocation, the whole body was soaked in 75% alcohol for about 10min, and all the muscle tissues of femur and tibia were removed by the above-mentioned MuSC separation and extraction method.

(2) After removing the femoral calculus at two ends of the femur and tibia from the ophthalmic scissors, repeatedly flushing the marrow tissue in the bone by using a syringe containing sterile PBS, and centrifuging 400g of all collected marrow cells for 5min after the bone color is changed from red to white.

(3) The cell suspension was resuspended in DME/F12 medium containing 20% L929 supernatant and then plated on 10cm uncoated cell culture dishes for culture.

(4) On day 4 of culture, the macrophages in the dish were replated with DME/F12 medium containing 20% L929 supernatant. The cells obtained on day 7 of culture were bone marrow-derived mature macrophages.

4. Histopathology

4.1 colitis mouse intestinal tissue sample preparation

After the experimental treatment of colitis is finished, the large intestine tissues of each group of mice are collected and the length of the large intestine tissues is measured. The intestinal lumen is dissected and stool and mucus are removed. After being rinsed twice by PBS, the membrane is soaked in 4 percent paraformaldehyde solution for fixation for 48 hours, and then corresponding pathological technical treatment and detection are carried out.

4.2 preparation of Paraffin section of intestinal tissue

And dehydrating, transparentizing, waxing and embedding the fixed intestinal tissues to prepare tissue wax blocks for the next H & E dyeing.

TABLE 1 Paraffin-embedded tissue preparation procedure Table

4.3H & E staining

(1) Xylene dewaxing was done for 10min and repeated 3 times.

(2) Anhydrous ethanol for 4 min.

(3) 95% ethanol for 1 min.

(4) And (5) adding 85% ethanol for 1 min.

(5) 75% ethanol for 1 min.

(6) Washing with tap water for 1 min.

(7) Hematoxylin staining for 10 min.

(8) And washing with tap water to recover blue.

(9) Eosin staining for 5 min.

(10) Dehydrating with 75% ethanol for 10 s.

(11) Dehydrating with 85% ethanol for 10 s.

(12) Dehydrating with 95% ethanol for 1 min.

(13) Dehydrating with anhydrous ethanol for 4 min.

(14) The xylene was clear for 3 min.

(15) And (4) sealing the center gum.

RNA extraction and Gene expression detection

5.1 Total RNA extraction of cells

(1) Cell samples were harvested, cells were washed 2 times with PBS, and l ml of pre-chilled TRIzol reagent was added and blown repeatedly until cells were completely lysed.

(2) Adding 200 μ l chloroform, mixing, and standing at room temperature for 5 min. Centrifuge at 12,000g for 15min at 4 ℃.

(3) After centrifugation, the mixture was stratified. Carefully suck the uppermost layer of liquid out to a new 1.5ml EP tube, add 1ml isopropanol, mix well and then stand at room temperature for 10 min. Centrifuge at 12,000g for 10min at 4 ℃ and discard the supernatant.

(4) The precipitate was washed by adding 1ml of 75% ethanol in DEPC water and centrifuged at 12,000g for 5min at 4 ℃. The washing and centrifugation were repeated once.

(5) And (4) discarding the supernatant, drying at room temperature until no liquid remains in the EP tube, and obtaining the total RNA of the obtained cells as white sediment at the bottom of the tube. Mu.l DEPC water was added until the RNA was completely dissolved, and the concentration and purity of the resulting RNA were determined spectrophotometrically.

5.2 Synthesis of reverse transcription cDNA

1ng of total RNA was aspirated and PrimeScript was used^TMThe RT Master Mix kit reversely transcribes mRNA into cDNA, and the obtained cDNA is reserved for real-time fluorescent quantitative PCR detection.

The specific reaction system is as follows:

TABLE 2

Reaction conditions are as follows: 15min at 37 ℃; 5s at 85 ℃; keeping at 4 ℃.

5.3 real-time fluorescent quantitative PCR

The cDNA was used as a template, and a real-time fluorescent quantitative PCR reaction was performed using the primer sequences shown in Table 2 to detect the expression level of mRNA in each cell.

The specific reaction system is as follows:

TABLE 3

Reaction conditions are as follows:

TABLE 4

TABLE 5 sequence Listing of specific real-time fluorescent quantitative PCR primers

6. Enzyme-linked immunosorbent assay (mouse IL-1 β and IL-6ELISA assay)

(1) The experimental mouse is anesthetized, and after one eyeball is picked, whole blood of the mouse is collected. Standing at room temperature for 30min, centrifuging at 2400rpm for 30min, and sucking the supernatant.

(2) And calculating the number of pre-coated plates required by the experiment, uniformly mixing 50 mu l of serum sample or standard solution with 50 mu l of sample analysis buffer solution in the kit, sealing the reaction hole by using a sealing plate film (transparent), and incubating at room temperature for 120 min. Where the experimental blank wells were set with addition of only TMB solution and stop solution.

(3) The plate was washed 5 times and the last time was patted dry on thick absorbent paper.

(4) Add 100. mu.l/well of biotinylated antibody, seal the reaction well with a sealing membrane (clear) and incubate at room temperature for 60 min.

(5) The plate was washed 5 times and the last time was patted dry on thick absorbent paper.

(6) 100. mu.l/well of Streptavidin was labeled with horseradish peroxidase. The reaction wells were sealed with a sealing plate (white) and incubated at room temperature in the dark for 20 min.

(7) The plate was washed 5 times and the last time was patted dry on thick absorbent paper.

(8) Adding 100 μ l/well of color reagent TMB solution, sealing the reaction well with sealing plate (white), and incubating at room temperature in dark for 20 min. (9) Adding 50 mul/hole of stop solution, mixing evenly, and immediately measuring A450 value by using a microplate reader.

7. Statistical analysis

Statistical analysis of all experiments was processed using Prism software (version 8.0, La Jolla, CA, USA). The test type is unpaired two-tailed student t test. All data are expressed as mean and Standard Error (SEM). A P value of <0.05 indicates that the difference is statistically significant.

Example 1: misc for relief of DSS-induced colitis

To explore whether muscs have anti-inflammatory therapeutic effects, we injected the expanded muscs intravenously into mice with Dextran Sodium Sulfate (DSS) -induced colitis. The results show that: a single intravenous injection of MuSC significantly improved disease symptoms of colitis compared to PBS-treated control colitis mice. The weight loss of the mice slowed and the disease activity index decreased (fig. 2A and 2B). In addition, a reduction in colonic tissue atrophy in mice is also indicative of a beneficial therapeutic effect of MuSC (fig. 2C). Meanwhile, the mice infused with muscs intravenously showed various degrees of remission of the thickening of the intestinal wall, the damage to crypts and the infiltration of inflammatory cells into the colon as detected by histopathology (fig. 2D). Serum IL-6 concentrations, which also appeared to decrease in blood of MuSC-infused mice, served as an important indicator for monitoring the disease progression in colitis (fig. 2E).

To explore the possibility of a "decellularization" treatment of muscs, it was examined whether the MuSC culture supernatants had similar therapeutic effects. We intraperitoneally injected cultured cell supernatants into colitis mice and monitored disease progression for colitis. The results show that: MuSC supernatants were indicated to have a colitis therapeutic effect similar to cells, whether in disease symptoms, histopathological features, and immunological indices (FIGS. 2F-2J). In conclusion, the MuSC cells or MuSC supernatant have a beneficial therapeutic effect in alleviating colitis.

Example 2: MuSC confers to macrophages an anti-inflammatory phenotypic trait

The results show that macrophages treated with MuSC supernatant exhibit a significant increase in anti-inflammatory genes (Arg-1, Chil3, CD206 and TGF- β) (fig. 3A), that macrophages treated with MuSC supernatant exhibit no expression of inflammatory pathogenic factors (IL-1 β and IL-6) at high levels (fig. 3B and 3C) even under stimulation of inflammatory factors (lipopolysaccharide and interferon γ), that increased expression of macrophage PD-L1 is critical for the outcome of disease inflammation and the restoration of immune homeostasis.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

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Claims

1. An application of muscle stem cell in preparing antiinflammatory medicine is provided.

2. The use of claim 1, wherein the anti-inflammatory agent is a muscle stem cell or a cell supernatant extracted from a cultured muscle stem cell.

3. The use according to claim 2, wherein the cell supernatant is obtained by culturing the muscle stem cells until the confluency reaches 70-80%, washing with a buffer solution, culturing for 20-25 hours in a serum-free medium, collecting the culture supernatant, and centrifuging and filtering.

4. The use of claim 1, wherein said muscle stem cells are obtained by flow sorting after enzymatic digestion of skeletal muscle tissue.

5. The use of claim 1, wherein said muscle stem cells are obtained from human, murine, porcine, or rabbit sources.

6. The use according to claim 1, wherein the medicament is formulated as an injection.

7. The use of claim 6, wherein said medicament is administered by intravenous injection.

8. The use according to claim 1, wherein the anti-inflammatory agent is an agent for the treatment of inflammatory diseases with inflammatory macrophages as the predominant pathogenic cell population.

9. The use of claim 8, wherein the anti-inflammatory agent is a colitis treatment agent.