CN106929474B

CN106929474B - M2 macrophage inducer

Info

Publication number: CN106929474B
Application number: CN201710209865.2A
Authority: CN
Inventors: 郝好杰; 李梓源; 易军; 周严恒; 刘刚; 陈惠华
Original assignee: Beijing Rmbio Tech Co ltd
Current assignee: Beijing Rmbio Tech Co ltd
Priority date: 2017-03-31
Filing date: 2017-03-31
Publication date: 2021-09-14
Anticipated expiration: 2037-03-31
Also published as: CN106929474A

Abstract

The invention provides an inducer of M2 macrophage and a preparation method thereof, wherein the inducer of M2 macrophage is a mesenchymal stem cell conditioned medium induced by a collagen/chitosan sponge scaffold. The preparation method of the inducer for M2 macrophage comprises the following steps: a) inoculating the mesenchymal stem cells on the collagen/chitosan sponge scaffold; b) adopting a serum-free culture medium to culture the mesenchymal stem cell-collagen/chitosan sponge scaffold; c) collecting the mesenchymal stem cells and the culture medium; d) adding a fresh serum-free culture medium, and continuously culturing the mesenchymal stem cells; and e) collecting the conditioned medium of the mesenchymal stem cells. The M2 macrophage inducer provided by the invention can effectively promote the polarization of macrophages to M2. The M2 macrophage inducer of the invention can be used for treating obesity.

Description

M2 macrophage inducer

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to an inducer for M2 macrophages, a preparation method thereof, and application of the inducer to medicines for treating obesity.

Background

Obesity occurs due to abnormal or excessive fat accumulation. There is a great deal of evidence that obesity is associated with the pathophysiology of many diseases, such as insulin resistance, diabetes, cardiovascular disease and cancer. The world health organization recently reported that in 2014, adults 18 years old and older had over 6 billion obesity with a 13% rate of obesity. Global obesity rates have doubled more than between 1980 and 2014. Therefore, the problem of obesity is receiving increasing attention.

Obesity is accompanied by chronic low-grade Inflammation in adipose tissue (Hotamisigil, G.S.,2006.Inflammation and metabolic disorders. Nature 444,860E 867.; Ferrante, A.W., et al, 2007. Obesistance-induced Inflammation: a metabolic diol in The language of Inflammation. J.Intern.Med.262, 40E 414.; Stienstra, R., et al.,2012.The Inflammation disorders in The gene. cell. Metab.15,10e 18.). More and more studies have shown that improving inflammation can improve insulin sensitivity, reduce adipocyte size in obese mice and improve lipid metabolism. For example, high fat diet mice have increased insulin sensitivity by administration of antagonists of the IL-1 receptor or IL-1b (Larsen, C.M., et al, 2007.Interleukin-1-receptor antagonist in type 2 diabetes mellitis. N.Engl. J.Med.356, 1517e1526.; Ridker, P.M., et al.,2012.Effects of Interleukin-1beta inhibition with canakinumab on-hemoglobin A1C, lipids, C-reactive protein, Interleukin-6, and fibrinogens: alpha. Phab random, placebo-controlled trial. circular 126,2739e 2748.). Furthermore, CCR 2-/-mice, when unable to replenish inflammatory monocytes from blood into the tissue, showed less susceptibility to obesity-related metabolism than high-fat diet mice (Weisberg, s.p., et al, 2006.CCR2 models in-flight and metabolic effects of high-fat feeding.j.clin.invest 116,115e 124.). Therefore, the search for methods to reduce obesity-related inflammation has led to a desire to address obesity and obesity-related insulin resistance.

Macrophages, which are key factors affecting inflammation and obesity-related insulin resistance, can be classified into pro-inflammatory macrophages M1 and anti-inflammatory macrophages M2. In the obese state, the pro-inflammatory macrophage M1 proliferates extensively, exacerbating insulin resistance in adipose tissue and other organs. Although the anti-inflammatory macrophages M2 in adipose tissue can inhibit inflammation, in obesity their proportion relative to macrophage M1 is much lower. Overall, in obesity, macrophage M1 replaces macrophage M2 and controls inflammation and macrophage homeostasis, stabilizing at a higher pro-inflammatory "set point". These data indicate that correction of inflammation/immune homeostasis of adipose tissue is necessary. The macrophage of M2 can regulate the homeostasis of macrophage in vivo, so as to improve obesity.

Mesenchymal Stem Cells (MSCs) are one of the most important multipotent adult stem cells, have self-renewal and self-differentiation potential, and secrete a variety of factors to participate in the repair and regeneration of damaged tissues and organs, thereby being widely used in the treatment of various diseases. MSCs can differentiate into various tissue cells such as pancreatic islets, nerves, vascular endothelium, bone, cartilage, muscle, liver, cardiac muscle and the like under specific induction conditions in vivo or in vitro. An increasing number of studies have demonstrated that Mesenchymal Stem Cells (MSCs) can exert immunomodulatory and inflammation-reducing effects via secreted cytokines. It was found that mesenchymal stem cells could relieve inflammatory responses and improve insulin resistance by promoting M2 macrophage polarization, (Xie Z, Hao H, Tong C, et al, human, biological and-derived mesenchymal stem cells, anti-inflammatory-insulin resistance in type 2 diabetes rates [ J ].2016,34(3):627 639.). The therapeutic mechanism of the efficacy of MSCs mainly depends on their paracrine activity, MSCs secrete a large amount of growth factors, and current research suggests that these bioactive substances provide the functions of immunomodulation and inflammation reduction of mesenchymal stem cells, that all bioactive factors and cytokines in MSC secretions can be collected in conditioned medium, and that accumulated evidence indicates that MSC conditioned medium has similar therapeutic effects to MSCs.

It has been shown that the stem cells have enhanced activity and Repair effect after being seeded on CCSS (collagen/chitosan sponge) collagen-chitosan sponge scaffold (Tong C, Hao H, Xia L, et al. Hypoxia precursor cells sequenced in a collagen-chitosan sponge skin Wound healing in a collagen-chitosan biomaterial with binding anion, 2015,24(1): 45.).

Based on the above studies, the present invention cultures MSCs with CCSS to obtain conditioned medium of activated mesenchymal stem cells for promoting the polarization of macrophages to M2 for improving obesity and related insulin resistance.

Disclosure of Invention

The invention provides an inducer of M2 macrophage and a preparation method thereof, wherein the inducer of macrophage can effectively promote the polarization of macrophage to M2, and is used for improving obesity and related insulin resistance.

In order to realize the purpose of the invention, the invention is realized by adopting the following technical scheme:

an inducer of M2 macrophage, which is characterized in that the inducer is a mesenchymal stem cell conditioned medium induced by a collagen/chitosan sponge scaffold. The collagen/chitosan sponge scaffold is obtained by freezing and freeze-drying a mixture of collagen and chitosan, and then shaking the mixture in a solution of N- (3-dimethylaminopropyl) -N' -Ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS). The mesenchymal stem cells are selected from umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells or the combination thereof.

Further, the collagen/chitosan sponge scaffold is prepared by mixing collagen and chitosan in a ratio of 1:1, then freezing at-80 ℃ for 2 hours, and then freeze-drying for 24 hours; after drying, the mixture was immersed in a solution containing N- (3-dimethylaminopropyl) -N' -Ethylcarbodiimide (EDC) and 8mM N-hydroxysuccinimide (NHS) while slowly shaking at room temperature for 12 hours.

A method for preparing an inducer of M2 macrophage comprises the following steps: a) inoculating the mesenchymal stem cells on the collagen/chitosan sponge scaffold; b) adopting a serum-free culture medium to culture the mesenchymal stem cell-collagen/chitosan sponge scaffold; c) collecting the mesenchymal stem cells and the culture medium; d) adding a fresh serum-free culture medium, and continuously culturing the mesenchymal stem cells; and e) collecting the conditioned medium of the mesenchymal stem cells.

Further, the step of seeding the mesenchymal stem cells on the collagen/chitosan sponge scaffold in the step a) comprises: dripping the mesenchymal stem cell single cell suspension liquid onto the top surface of the collagen/chitosan sponge scaffold for incubation and culture; then, equal amount of mesenchymal stem cells are inoculated on the mesenchymal stem cell-collagen/chitosan sponge scaffold construct for incubation and culture.

The step c) of collecting the mesenchymal stem cells and the culture medium comprises: removing the culture medium, digesting the mesenchymal stem cells by adopting trypsin-ETDA (ethylene-tetra-ethyl-DA) to shrink the mesenchymal stem cells to separate from the collagen/chitosan sponge scaffold; adding said removed medium to neutralize trypsin-EDTA; and gently pipetting the mesenchymal stem cells.

The mesenchymal stem cells are continuously cultured in the serum-free medium for 12 +/-1 hours in the step d).

Centrifuging and collecting the mesenchymal stem cells and the culture medium in the step e) to remove cell debris; the conditioned medium was then collected using a 7-kDa molecular weight cut-off bag.

The invention provides an application of an inducer of M2 macrophage in treating obesity.

The invention has the beneficial effects that:

the invention obtains the conditioned medium by culturing MSC through CCSS, and the conditioned medium is used as an inducer to induce macrophage polarization, thereby obviously enhancing the polarization efficiency of the macrophage and improving the effects of obesity and insulin resistance. The activated mesenchymal stem cell conditioned medium prepared by the invention is easy to control quality and industrialize through simple pretreatment, and the prepared conditioned medium can be prepared into freeze-dried powder and is easy to store.

Description of the drawings:

FIG. 1: BCA protein assay kit total protein concentration in purified MSC-CM was evaluated.

FIG. 2: relative expression levels of proinflammatory cytokines (IL-1, IL-6, TNF-alpha), anti-inflammatory cytokines (IL-10, TGF-beta) and CD163 in supernatants 24h and 48h after co-culture of mouse bone marrow macrophages with different MSCs by RT-PCR. Con for control, HG for high sugar, HG + CCSS-MSC-CM for high sugar + collagen chitosan scaffold cultured MSC conditioned medium.

FIG. 3: mouse food intake, inguinal adipose tissue (INAT) weight, and Epididymal Adipose Tissue (EAT) weight after a second macrophage infusion. A: mouse food intake; b: INAT weight; c: EAT weight. M0 is bone marrow macrophage without any treatment, un-M2 is untreated MSC-CM induced M2 macrophage; CCSS-M2 is CCSS cultured MSC-CM induced M2 macrophages.

FIG. 4: comparative plots of INAT and EAT adipocyte volume changes in mice from M0, un-M2 and CCSS-M2 macrophage treated groups were examined by hematoxylin-eosin (HE) staining.

FIG. 5A is a graph of the amount of p-AKT in EAT detected with a western blot; b: results of intraperitoneal glucose tolerance (IPGTT) test in mice.

FIG. 6: fat metabolism in different groups of mice. A: triglyceride levels of blood; b: low density lipoprotein levels; c high density lipoprotein/low density lipoprotein ratio in blood.

Detailed Description

The present invention will be described in further detail with reference to the following embodiments

The present invention has been described with reference to specific embodiments, but it will be apparent that various modifications and changes may be made without departing from the spirit and scope of the invention. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense.

Example 1 preparation of mesenchymal Stem cells

Preparation of umbilical cord mesenchymal stem cells

Operating in an ultra-clean workbench, taking fresh healthy fetal umbilical cords in term, washing with physiological saline containing 2 times of 100U/mL penicillin and streptomycin to remove blood stains, peeling off Walton gel, cutting into blocks with the size of less than 1 cubic millimeter, uniformly coating, slowly adding 10mL serum-free culture medium, and culturing in a carbon dioxide and saturated humidity incubator with the volume fraction of 5% at 37 ℃. After culturing the primary cells for 7 days, the serum-free culture solution is replaced, the cells are cultured until 70% fusion is achieved, the serum-free culture solution is removed, trypsin-sodium Ethylenediaminetetraacetate (EDTA) with the concentration of 0.25% and 0.1% respectively is added for digestion for 1min, the umbilical cord MSC is contracted and separated from the bottle wall, at the moment, the culture supernatant removed previously is added, the trypsin-EDTA solution is neutralized, and the umbilical cord MSC suspension is gently blown by using a pipette and transferred into a 50mL centrifuge tube. Centrifuging at 1000rpm for 8min, and removing supernatant; the MSCs were resuspended by re-addition of serum-free complete medium and counted. 10cm dishes were inoculated according to 1-pass 1.5. When about 70% of the cells were cultured and fused in MSC, subculture was performed according to the method described above.

Selecting MSC cultured in the third generation, digesting, neutralizing, washing and collecting cells according to the requirement of passage when the cell fusion degree is not more than 80% in the logarithmic growth phase, adopting cell freezing solution (90% fetal calf serum and 10% dimethyl sulfoxide (DMSO)) to resuspend MSC, fully mixing uniformly, and subpackaging 1mL of each tube in a freezing tube for sealing. Placing the freezing tube in a freezing box filled with isopropanol, refrigerating at-80 deg.C overnight, and transferring into a liquid nitrogen tank for storage the next day.

The MSC resuscitation steps are as follows: taking out the MSC cryopreservation tube, and immediately immersing the MSC cryopreservation tube into warm water at 37 ℃ for dissolution; taking a proper amount of serum-free culture medium suspension cells, centrifuging, and removing a supernatant; the cells were resuspended in serum-free medium and seeded in a petri dish and cultured in a 5% volume fraction carbon dioxide, saturated humidity incubator at 37 ℃.

Preparation of mesenchymal stem cells

Extracting 50mL of marrow liquid from the puncture point of the anterior superior iliac spine by using a conventional method, and injecting the marrow liquid into a 50mL sterile centrifuge tube; diluting with an equal amount of PBS containing 20u/mL heparin, and adding an equal volume of lymphocyte separation solution with specific gravity of 1.073g/mL along the tube wall; centrifuging at 2000rmp for 30min, and sucking the mononuclear cell layer; centrifuging and washing with sterile physiological saline for 2 times, centrifuging at 1000rpm for 5min each time, discarding supernatant, and collecting precipitate; suspending the bone marrow MSC obtained above with serum-free medium, counting cells, and culturing at 5 × 10⁶Each cell/mL was inoculated into a flask and cultured in an incubator containing 5% by volume of carbon dioxide at 37 ℃ and saturated humidity. After 3 days of culture, replacing serum-free culture solution, discarding non-adherent cells, and replacing the culture solution 1 time every 3 days later; 7-9 days later, when the bone marrow MSC fusion reaches 50% -60%, the first step is carried outAnd (5) carrying out one-time passage. Serum-free medium was removed, digested with trypsin-EDTA 37 at concentrations of 0.25% and 0.1%, respectively, for 1min at 37 ℃, bone marrow MSCs were contracted away from the vial wall, the previously removed culture supernatant was added, trypsin-EDTA was neutralized, and the mesenchymal cell suspension was pipetted gently into a 50mL sterile centrifuge tube. Centrifuging at 1000rpm for 8min, and discarding supernatant; serum-free complete medium was added again for resuspension, counted and the 10cm dishes were inoculated according to 1-1.5. Bone marrow MSC was cultured and fused at about 70% by subculture as described above.

Preparation of adipose-derived mesenchymal stem cells

Separating white adipose tissue of abdomen according to conventional method, removing visible microvessels and muscle tissue, washing with sterile PBS for three times, cutting adipose tissue with sterile scissors into paste with size below 1 cubic mm, adding sterile digestive juice (serum-free DME culture medium containing 0.1% collagenase type I and 0.05% trypsin), stirring at constant speed in water bath at 37 deg.C for 45-50min until tissue blocks are completely digested; filtering with 200 mesh screen to collect filtrate, neutralizing with low-sugar DMEM medium containing 10% Fetal Bovine Serum (FBS) in equal amount, centrifuging at 1500rpm for 10min, and discarding supernatant; at 1 × 10⁶Cell density of individual/mL, adipose MSCs resuspended in serum-free medium and seeded in culture flasks, cultured at 37 ℃, 5% volume fraction carbon dioxide, saturated humidity incubator, when adipose MSCs grow to near 80% confluency, MSC cells were digested with 0.25% trypsin solution at 1: 3 into a new culture flask for subculture. And fusing fat MSC to be not more than 80%, centrifuging at 1000rpm for 8min, discarding supernatant, adding serum-free complete medium again to resuspend MSC, counting, and inoculating to a 10cm culture dish according to 1-1.5. When about 70% of the adipose-derived MSCs are cultured and fused, subculture is carried out as described above.

EXAMPLE 2 preparation of collagen/Chitosan sponge scaffolds

Mixing collagen (5mg/mL) and chitosan (5mg/mL in 0.5M acetic acid) at a ratio of 1:1, stirring well, followed by freezing at-80 ℃ for 2 hours, and then freeze-drying for 24 hours; after drying, the mixture is immersed in a solution containing N- (3-dimethylaminopropyl) -N' -ethylcarbodiimideAmine (EDC) and 8mM N-hydroxysuccinimide (NHS) while shaking slowly at room temperature for 12 hours. The matrix was washed with double distilled water until the pH returned to the physiological range (7.0-7.4), and then lyophilized to obtain a collagen-chitosan sponge scaffold (CCSS). Co for CCSS⁶⁰And (5) sterilizing.

Example 3 preparation of collagen/Chitosan sponge scaffold MSC conditioned Medium

Grafting of MSCs onto collagen/chitosan sponge scaffolds

CCSS was soaked with PBS and placed in 6-well plates, 100. mu.L of a single cell suspension of third generation MSC culture medium, in which the number of MSC cells was 1X 10, was added dropwise to the top surface of CCSS⁶And 5% CO at 37 ℃₂And incubated for 4 hours to allow the cells to penetrate into the scaffold and adhere. Equal amounts of MSCs were seeded into cell-CCSS constructs and incubated for 4 hours. After the cells were adherent, 2mL serum-free medium was added to each well at 37 ℃ with 5% CO₂And culturing for 48 +/-2 hours. MSCs were cultured in blank wells as controls.

Preparation of collagen/chitosan sponge scaffold MSC conditioned medium

After a certain time of culturing of the MSCs on CCSS, the culture was removed, the inoculated CCSS was washed three times with PBS, digested with 0.25% trypsin ETDA for 1min, and the MSCs were contracted away from CCSS, at which time the previously removed supernatant was added, neutralized the trypsin EDTA, and gently pipetted. The MSC suspension was collected in a centrifuge tube, washed thoroughly 2 times with PBS, resuspended in 15mL serum-free DMEM medium, transferred to a T150 flask and cultured for 12 ± 1 hour. Transferring the MSC suspension into 50mL centrifuge tube, centrifuging at 3000rpm for 10min, collecting supernatant, dialyzing with 7-kDa molecular weight cut-off bag filter at PEG20000 and 4 deg.C, and concentrating the culture medium by 20 times to obtain conditioned medium, i.e. CCSS-MSC-CM.

Freeze drying CCSS-MSC-CM to obtain dry powder

Freezing the supernatant at-80 deg.C, vacuumizing, sublimation drying, removing ice crystal, and storing at-20 deg.C.

The growth factors secreted by the MSCs cultured by the CCSS are obviously increased

Growth ofThe factor is a high efficiency protein, enzyme combination, and the change of the concentration of the growth factor in the MSC-CM can be measured by evaluating the protein concentration of the purified MCS-CM through the BCA protein detection kit. 5X 10 cultured from 80mL CCSS⁷About 410. mu.g of protein was isolated from the MSC supernatant. As shown in FIG. 1, the total protein amount in MSC-CM cultured with CCSS was about 95. mu.g/million cells, while the total protein amount in MSC-CM was about 55. mu.g/million cells, and the total protein amount in level in MSC-CM cultured with CCSS was significantly higher than that in untreated MSC-CM, confirming that MSC cultured with CCSS can promote secretion of MSC growth factor.

Example 4 functional validation of collagen/chitosan sponge scaffold MSC conditioned Medium

(1) Collagen/chitosan sponge scaffold MSC-CM promotes the polarization of inflammatory mouse bone marrow macrophages to M2 macrophages

Preparation and induction of bone marrow macrophages

The mouse was sacrificed by introducing the neck, the fur of the hind limb of the mouse was peeled off at the foot, the foot was cut off together with the fallen fur, the hind leg was cut off, and the rat was placed in a petri dish containing sterile PBS. After the forceps are used for clamping one end of the leg bone, the muscle is removed by the scissors, and the leg bone is cut at the joint. Sucking serum-free 1640 medium with a 2mL syringe, puncturing the needle into the bone marrow cavity, repeatedly washing the bone marrow until the bone marrow turns white, adding the bone marrow washing liquid into a 50mL centrifuge tube, and centrifuging at 1000rpm for 5 min. The bone marrow cells obtained by centrifugation were resuspended in 10mL of a 1640 medium containing Macrophage Colony Stimulating Factor (MCSF) (50ng/mL) and serum, and then divided into two 25cm portions²In a cell culture flask of (1), placed at 37 ℃ and 5% CO₂The culture was carried out in an incubator with a moderate gas concentration for 3 days. The supernatant was discarded, and 5mL of the medium containing MCSF (50ng/mL) and serum-containing 1640 was replaced to continue the culture for 4 days. Then, in a 6-well plate, mouse bone marrow macrophages were cultured in 30mM glucose solution and treated with phorbol 12-myristate 13-acetate (PMA, 160ng/mL) to induce differentiation. After 3 days, non-adherent cells were removed by three rinses with PBS.

MSC-CM induction of bone marrow macrophages

Treatment of the above-described induced differentiated adherent cells with CCSS-MSC-CM (20. mu.g/mL) and untreated MSC-CM (20. mu.g/mL)The MSC-CMC treated bone marrow macrophages obtained in 48 hours, which are designated CCSS-M2 and un-M2, respectively, in this application. The bone marrow macrophages without any treatment were designated as M0. In preparation for injection, macrophages were first removed from the culture dish with a spatula and centrifuged at 1500rpm for 10 minutes. Then, the cells were diluted to 2.5X 10⁶/mL。

As a result:

collagen/chitosan sponge scaffold MSC-CM promoted the polarization of inflammatory mouse bone marrow macrophages to M2 macrophages

IL-1, IL-6 and TNF alpha are proinflammatory cytokines secreted by macrophages, IL-10, TGF-beta are anti-inflammatory cytokines produced by macrophages, and CD163 is a marker on the surface of M2 macrophages. The test adopts RT-PCR to detect the expression level of the cell factors. As shown in FIG. 2, the treatment of glucose and PMA increased the expression of proinflammatory cytokines in macrophages, and the expression was further increased with time, while the treatment of MSC-CM suppressed the expression of proinflammatory factors and maintained the expression of proinflammatory factors at a certain level; CCSS-MSC-CM significantly and significantly inhibited the expression of proinflammatory cytokines IL-1, IL-6 and TNF α relative to MSC-CM treated without collagen/chitosan sponge scaffold. The treatment of glucose and PMA reduces the expression of anti-inflammatory cytokines in macrophages, and the expression amount is further reduced along with the prolonging of time; the data that CCSS-MSC-CM significantly promoted IL-10 and TGF- β expression in mouse bone marrow macrophages and significantly promoted the expression of the M2 macrophage surface marker CD163 indicates that CCSS-MSC-CM promoted macrophage polarization to M2. MSC cultured by CCSS can secrete more growth factors, and the efficiency of inducing macrophage into M2 macrophage is improved.

(2) Collagen/chitosan sponge scaffold MSC-CM induced improvement in lipid metabolism by bone marrow macrophages

Induction and treatment of obese mice

Male C57BL/6N mice, 8 weeks old, were housed in separate ventilated cages at 22 ℃ under a 12 hour light/dark cycle. One week after acclimatization, the mice were divided into two groups, one group consisting of 6 mice, a standard diet group (normal group), the other group consisting of 24 mice, and a high fat diet (HFD, 60% fat) group, and the HFD mice were fed for 12 weeks to meet the obesity model.

Mice in the HFD group were subdivided into four groups of 6 mice each: HFD and PBS infusion group (obese group), HFD and M0 macrophage infusion group (M0 infusion group, experimental control), HFD and CCSS-MSC-CM induced M2 macrophage infusion group (CCSS-M2 infusion group) and HFD and untreated MSC-CM induced M2 macrophage infusion group (un-M2 infusion group). Then, 5X 10 in 0.2mL of PBS⁵M0, CCSS-MSC-CM induced M2 macrophages or untreated MSC-CM induced M2 macrophages were infused into mice via tail vein. One week later, a second injection of the same composition was made and all mice were observed for one week prior to sacrifice. Throughout the process, all mice maintained their previous diet and food intake was recorded throughout the week after the second injection. Animals were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (60mg/kg) prior to sacrifice. Taking blood from mice for lipid metabolism analysis for measuring Triglyceride (TG), high density lipoprotein cholesterol (HDLc) and low density lipoprotein cholesterol (LDLc); the weight of INAT (inguinal adipose tissue ) and EAT (epididymal adipose tissue) was recorded. Adipose tissue was fixed in formalin for pathological analysis.

As a result:

infusion of M2 macrophages reduced INAT weight

As shown in fig. 3A, the food intake of the obese group of mice did not change significantly after macrophage infusion treatment. Whereas infusion of macrophages reduced the weight of INAT and the weight of EAT in obese mice compared to obese mice. Mice infused with CCSS-M2 macrophages had a significant decrease in INAT weight (fig. 3B) and a greater decrease in EAT weight (fig. 3C) compared to the M0-treated and un-M2-treated groups.

Infusion of M2 macrophages reduced mouse adipocyte volume

Obesity causes the mouse adipocytes to become larger, and infusion of macrophages causes the mouse adipocytes to decrease in volume. As shown in FIG. 4, hematoxylin-eosin (HE) staining revealed a significant decrease in the volume of INAT and EAT (epididymal adipose tissue) adipocytes in mice of CCSS-M2 macrophage-infused group compared to M0-treated group and un-M2-treated group.

Infusion of M2 macrophages improves insulin resistance

p-AKT is a marker that is widely used to assess insulin sensitivity. p-AKT was detected in EAT by western blot and found to be significantly increased in the CCSS-M2 infused group compared to the M0 treated group and the un-M2 treated group (FIG. 5A). In addition, the results of the intraperitoneal glucose tolerance test (IPGTT) in FIG. 5B show a significant improvement in insulin resistance in the CCSS-M2 infused group. These results indicate that CCSS-M2 macrophage infusion improved insulin resistance in obese mice, and stimulated weight loss to some extent.

Infusion of M2 macrophages improved lipid metabolism

Obesity was found to increase the concentration of Triglyceride (TG) and plasma low density lipoprotein (LDL-c) in mice by measuring the lipid level of blood. Compared with the M0 treatment group and the un-M2 treatment group, the CCSS-M2 treatment group has the advantages that TG and LDL-c are obviously reduced, and HDL-c (high density lipoprotein)/LDL-c (low density lipoprotein) is obviously increased. These findings indicate that CCSS-M2 macrophage infusion can improve lipid metabolism.

MSC cultured by CCSS can secrete more growth factors, and the efficiency of inducing macrophage into M2 macrophage is improved. M2 macrophage is an anti-inflammatory immune cell that further improves insulin resistance and lipid metabolism by restoring inflammation/macrophage homeostasis to EAT. M2 macrophage induced by MSC-CM cultured by CCSS has more obvious treatment effect, treats obesity and obesity-related insulin resistance by restoring macrophage homeostasis, and brings new treatment hope for obese patients.

Claims

1. An inducer of M2 macrophage, which is characterized in that the inducer is a mesenchymal stem cell conditioned medium induced by a collagen/chitosan sponge scaffold; the collagen/chitosan sponge scaffold is prepared by mixing collagen and chitosan at a ratio of 1:1, freezing at-80 deg.C for 2 hr,then freeze-drying for 24 hours; after drying, the mixture was immersed in a solution containing N- (3-dimethylaminopropyl) -N' -Ethylcarbodiimide (EDC) and 8mM N-hydroxysuccinimide (NHS) while slowly shaking at room temperature for 12 hours; the mesenchymal stem cells are selected from umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells and bone marrow mesenchymal stem cells; the inducer for M2 macrophage is prepared by the following steps: a) will be 1 × 10⁶Dripping the suspension of mesenchymal stem cells into the top surface of the collagen/chitosan sponge scaffold at 37 ℃ and 5% CO₂Culturing for 4 hours, then inoculating equivalent mesenchymal stem cells on the mesenchymal stem cell-collagen/chitosan sponge scaffold construct and incubating for 4 hours; b) 5% CO at 37 ℃ in serum-free medium₂Culturing the mesenchymal stem cell-collagen/chitosan sponge scaffold for 48 +/-2 hours; c) removing the culture medium, digesting the mesenchymal stem cells with 0.25% trypsin-ETDA for 1min to shrink the mesenchymal stem cells away from the collagen/chitosan sponge scaffold, adding the removed culture medium, neutralizing trypsin-EDTA, and gently pipetting the mesenchymal stem cells to collect the mesenchymal stem cells and the culture medium; d) adding a fresh serum-free culture medium, and continuously culturing the mesenchymal stem cells for 12 +/-1 hours; and e) centrifuging at 3000rpm for 10min to collect the mesenchymal stem cells and the culture medium to remove cell debris, and then collecting the conditioned medium with a 7-kDa molecular weight cut-off bag.

2. A method for preparing an inducer of M2 macrophage comprises the following steps: a) mixing collagen and chitosan at a ratio of 1:1, freezing at-80 deg.C for 2 hr, and lyophilizing for 24 hr; after drying, the mixture was immersed in a solution containing 95% N- (3-dimethylaminopropyl) -N' -Ethylcarbodiimide (EDC) and 8mM N-hydroxysuccinimide (NHS) while slowly shaking at room temperature for 12 hours to obtain a collagen/chitosan sponge scaffold; b) will be 1 × 10⁶Dripping the suspension of mesenchymal stem cells into the top surface of the collagen/chitosan sponge scaffold at 37 ℃ and 5% CO₂Incubating for 4 hours for culture; then, equal amount of mesenchymal stem cells are inoculated on the mesenchymal stem cells-collagen/chitosan sponge branchIncubating on the architectural construct for 4 hours; c) 5% CO at 37 ℃ in serum-free medium₂Culturing the mesenchymal stem cell-collagen/chitosan sponge scaffold for 48 +/-2 hours; d) removing the culture medium, and digesting the mesenchymal stem cells for 1min by using 0.25% trypsin-ETDA (ethylene-tetra-ethyl-DA) to shrink the mesenchymal stem cells to separate from the collagen/chitosan sponge scaffold; adding said removed medium to neutralize trypsin-EDTA; and gently pumping the mesenchymal stem cells to collect the mesenchymal stem cells and the culture medium; e) adding a fresh serum-free culture medium, and continuously culturing the mesenchymal stem cells for 12 +/-1 hours; and f) centrifuging at 3000rpm for 10min to collect the conditioned medium of the mesenchymal stem cells to remove cell debris; then collecting the conditioned medium with a 7-kDa molecular weight cut-off bag; the mesenchymal stem cells are selected from umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells and bone marrow mesenchymal stem cells.