Immunoregulation-based stem cell preparation as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a stem cell preparation based on immunoregulation and a preparation method and application thereof. The stem cell preparation based on immunoregulation is prepared from the following raw materials in parts by weight: 5 to 40 parts of freeze-dried powder, 40 to 120 parts of gastrodia elata polysaccharide and 2 to 6 parts of melatonin, the freeze-dried powder is obtained by mixing and freeze-drying a stem cell culture solution and curcumin lipidosome; the volume ratio of the stem cell culture solution to the curcumin liposome is 1: (10-32); the protein concentration in the stem cell culture solution is 1.2 mg / mL to 1.6 mg / mL. The stem cell preparation provided by the invention can utilize the immune regulation effect of the mesenchymal stem cells to realize the immune repair of adipose tissues.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a stem cell preparation based on immunoregulation and a preparation method and application thereof. Background Art

[0002] Obesity is a chronic metabolic disorder characterized by the expansion of adipose tissue and the storage of excessive triglycerides. It is the main cause of chronic diseases. It can induce a variety of metabolic dysfunctions related to cardiovascular diseases, increase the incidence and mortality of type II diabetes, coronary heart disease, stroke, congestive heart failure, hypertension, dyslipidemia, sleep apnea syndrome and certain cancers, such as ovarian cancer, thymic cancer and colon cancer, and even affect the normal functions of multiple organs such as the lungs, liver and kidneys. It can also cause bone and joint damage, intellectual decline and accelerate human aging. With the deepening of industrialization and urbanization in many countries in the world, the incidence and mortality of obesity are also increasing. Prevention and treatment of obesity has become an important health issue.

[0003] In recent years, more and more studies have found that fat and inflammation are inextricably linked. As an active endocrine organ, fat can secrete a variety of pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6) and C-reactive protein (CRP). These cytokines can induce and maintain a systemic low-grade inflammatory state, which can cause a series of metabolic-related diseases such as insulin resistance, leading to abnormal adipocyte function and further aggravating obesity. Correcting inflammation in adipose tissue and restoring its immune homeostasis have a positive effect on improving obesity. Mesenchymal stem cells (MSCs) have extremely strong immunoregulatory functions.

[0004] At present, some publicly reported stem cell preparations based on mesenchymal stem cells have been shown to have immunomodulatory functions. For example, the Chinese patent with patent number CN202410005936.7 discloses a combined preparation using salvia miltiorrhiza extract and umbilical cord mesenchymal stem cell extract to improve the composition of the coliform group, thereby improving obesity. The Chinese patent with patent number CN202210747420.0 discloses the use of DPP4-specific monoclonal antibodies in combination with adipose mesenchymal stem cells, which can have a pro-proliferation and anti-apoptosis effect on pancreatic beta cells, thereby further enhancing the weight loss effect of stem cell preparations on obese mice and the effect of lowering blood sugar. However, the existing stem cell preparations are less used in immunomodulation, and it is also necessary to develop a stem cell preparation for improving the immune balance of adipose tissue. Summary of the invention

[0005] In order to develop a stem cell preparation for improving the immune balance of adipose tissue, the present invention provides a stem cell preparation based on immunomodulation and its preparation method and application. To achieve the above purpose, the present invention adopts the following technical solution.

[0006] The present invention provides a stem cell preparation based on immunoregulation, which is composed of the following raw materials by weight: 5 to 40 parts of freeze-dried powder, 40 to 120 parts of gastrodia elata polysaccharide and 2 to 6 parts of melatonin.

[0007] The freeze-dried powder is obtained by mixing stem cell culture fluid and curcumin liposomes and then freeze-drying; the volume ratio of the stem cell culture fluid to the curcumin liposomes is 1:10-32.

[0008] The protein concentration in the stem cell culture medium is 1.2 mg / mL to 1.6 mg / mL. The stem cell culture medium contains a variety of stem cell factors.

[0009] The present invention provides a stem cell preparation based on immunoregulation, which is prepared by using a freeze-dried powder prepared by compounding a stem cell culture solution with curcumin liposomes as a raw material, and compounding with melatonin and gastrodia polysaccharide. The stem cell preparation based on immunoregulation has an immunoregulatory effect and can achieve immune repair of adipose tissue. The stem cell preparation based on immunoregulation provided by the present invention belongs to a stem cell preparation for improving the immune balance of adipose tissue. The development of a stem cell preparation for improving the immune balance of adipose tissue solves the technical gap in the field of stem cell preparations to a certain extent.

[0010] Preferably, the stem cell culture medium is a stem cell culture medium treated with a Sophora japonica ear extract.

[0011] Treated with Sophora japonica extract means that the Sophora japonica extract is added during the process of culturing stem cells to obtain stem cell culture fluid. The present invention has experimentally found that Sophora japonica extract can upregulate the expression of a miRNA in stem cell secretions. The miRNA here is miRNA-26b-5p. The literature reports that an increase in miRNA-26b-5p will inhibit NF-κB activation, thereby increasing the proportion of macrophage M2 polarization and inhibiting M1 polarization.

[0012] Preferably, the preparation method of the stem cell culture medium is as follows:

[0013] Stem cells were collected at a rate of 8×10 3 ~1×10 4 / cm 2The cell density was inoculated in a complete culture medium containing 0.2% to 1.0% by mass of Sophora japonica erithidea extract and cultured at 36° C. to 38° C. and 4% to 6% CO2.

[0014] When the stem cells have grown to 80% to 90% fusion, the cell supernatant is collected; the collected cell supernatant is centrifuged to remove dead cells and cell debris, and the supernatant is collected again and filtered; the filtrate obtained by filtration is separated and concentrated to obtain the stem cell culture medium.

[0015] Preferably, the separation and concentration method is: first centrifuge at 1500g to 3000g for 5min to 15min, discard the precipitate after centrifugation, and take the supernatant; then pass the supernatant through a 100KD ultrafiltration membrane to collect the dialysate, and pass the dialysate through a 3KD ultrafiltration membrane to collect the retentate. This step is because the concentration interval of 3KD-100KD can obtain more immune regulatory cytokines secreted by stem cells, such as leukemia inhibitory factor (LIF, 38-64KD), human leukocyte antigen G5 (HLA-G5, 34KD), transforming growth factor β (TGF-β, 44.3KD), and hepatocyte growth factor (HGF, 30-90KD).

[0016] Preferably, the stem cells are stem cells that have been passaged and expanded to generations P3 to P7. Within this generation, the genetic genes of stem cells are stable, the cell activity is optimal, and the proliferation rate is relatively fast.

[0017] Preferably, the stem cells are mesenchymal stem cells, which are derived from any one of the following tissues: bone marrow, umbilical cord blood, umbilical cord tissue, placental tissue and adipose tissue.

[0018] Preferably, the curcumin liposome is prepared by a thin film dispersion method using lecithin, cholesterol, CPP-AA-PEG-DSPE, Tween 80, vitamin E and curcumin as raw materials.

[0019] The mass ratio of the lecithin, the cholesterol, the CPP-AA-PEG-DSPE, the Tween 80, the vitamin E and the curcumin is 0.5-10: 0.1-0.25: 0.08-0.2: 0.005-0.02: 0.01-0.045: 0.002-0.01.

[0020] Preferably, the CPP-AA-PEG-DSPE is prepared from active ester-polyethylene glycol-phospholipid and CPP-AA peptide. The specific preparation method comprises the following steps:

[0021] The active ester-polyethylene glycol-phospholipid is dissolved in water to obtain an active ester-polyethylene glycol-phospholipid solution with a concentration of 18 mg / mL to 22 mg / mL.

[0022] The CPP-AA peptide is added to the active ester-polyethylene glycol-phospholipid solution and mixed evenly, and the mixture is reacted for 10 to 16 hours to obtain a reaction solution.

[0023] The reaction solution was placed in a dialysis bag with a cutoff of 3.5 KD and dialyzed for 36 h to 72 h, and the liquid in the dialysis bag was freeze-dried to obtain the CPP-AA-PEG-DSPE.

[0024] The present invention also provides a method for preparing the stem cell preparation based on immunoregulation, comprising the following steps:

[0025] The freeze-dried powder, the gastrodia elata polysaccharide and the melatonin are taken respectively according to the formula amounts.

[0026] The freeze-dried powder, the Gastrodia elata polysaccharide and the melatonin are uniformly mixed to obtain the stem cell preparation based on immunoregulation.

[0027] The present invention also provides the use of the stem cell preparation based on immunoregulation in weight loss. The stem cell preparation based on immunoregulation is used to prepare medicines with weight loss effects or functional foods with weight loss effects.

[0028] Preferably, the medicine with weight loss effect is prepared by using the stem cell preparation based on immunomodulation as an active ingredient and supplemented with pharmaceutically acceptable excipients. The medicine is an oral medicine.

[0029] The functional food with weight loss effect is prepared by using the stem cell preparation based on immune regulation as an active ingredient and supplemented with auxiliary materials acceptable in food science.

[0030] The dosage forms of the medicine with weight loss effect and the functional food with weight loss effect include any one or more of capsules, granules, tablets, powders or pills.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1. The present invention provides a stem cell preparation based on immunoregulation and its preparation method and application. The stem cell preparation based on immunoregulation provided by the present invention is a stem cell preparation for improving the immune balance of adipose tissue. The development of a stem cell preparation for improving the immune balance of adipose tissue solves the technical gap in the field of stem cell preparations to a certain extent.

[0033] 2. The present invention has found that the extract of Sophora japonica erulea acts on mesenchymal stem cells and collects the culture, which can affect the polarization ratio of macrophages, reduce M1 macrophages, and correspondingly, increase the ratio of M2 macrophages. Studies have found that macrophages are key factors affecting inflammation and obesity-related insulin resistance. They can be divided into pro-inflammatory macrophages M1 and anti-inflammatory macrophages M2. In the obese state, pro-inflammatory macrophages M1 proliferate in large quantities, exacerbating insulin resistance in adipose tissue and other organs. Although the anti-inflammatory macrophages M2 in adipose tissue can inhibit inflammation, their proportion relative to macrophages M1 is much lower in obesity. As anti-inflammatory immune cells, macrophages M2 play a significant anti-inflammatory role in obesity. The cells can correct the inflammation / immune homeostasis in adipose tissue and further improve lipid metabolism.

[0034] 3. The present invention uniquely uses liposomes modified with CPP-AA peptide. First, as a macrophage-targeted polypeptide with the function of crossing the cell membrane, CPP-AA can specifically target macrophages, inhibit the spontaneous differentiation of macrophages into inflammatory cell phenotypes, inhibit the release of inflammatory factors and tissue damage, and thus effectively inhibit inflammatory-related diseases. Secondly, the polypeptide molecule has a transmembrane structure, a clear target, and high efficiency; and the synthetic technology line is mature, easy to prepare in large quantities, with good stability and potential application prospects. Finally, based on curcumin liposomes, this polypeptide is modified onto curcumin liposomes, which further enhances the role of targeted regulation of macrophages and correction of adipose tissue inflammation.

[0035] 4. The present invention further compounds melatonin and Gastrodia elata polysaccharide. Melatonin is a powerful free radical scavenger that can reduce oxidative stress, regulate the body's immune balance, and improve energy metabolism. Gastrodia elata polysaccharide can regulate the balance of intestinal microbiota, promote the growth of beneficial bacteria, inhibit the reproduction of harmful bacteria, improve the intestinal defense against pathogenic microorganisms, maintain the integrity of the intestinal barrier, and further enhance the immunoregulatory ability of the preparation from the direction of enhancing intestinal immune function.

[0036] In addition, studies have confirmed that Gastrodia elata polysaccharides may help control body weight by affecting the activity of enzymes related to energy metabolism and regulating the metabolism of fat and carbohydrates. Some studies have shown that Gastrodia elata polysaccharides may inhibit the proliferation and differentiation of adipocytes, improve insulin sensitivity, and reduce the accumulation of fat in the body. Melatonin promotes the activity of brown fat, thereby helping to reduce the expansion of adipocytes and fat accumulation. All of the above can add icing on the cake to the application of this stem cell preparation in weight loss. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1The figure shows the changes in body weight of the control group and the model group 8 weeks after modeling in the present invention; the control group was on a normal diet; the model group was on a high-fat diet; **P<0.01.

[0038] Figure 2 The changes in blood lipids in the control group and the model group after 8 weeks of modeling in the present invention: Figure 2 Figure A shows the cholesterol content; Figure 2 Figure B shows the triglyceride content; the control group was a normal diet group; the model group was a high-fat diet group; *P<0.05, **P<0.01, ***P<0.001.

[0039] Figure 3 The figure is a statistical chart of the weight, body fat percentage, Lee's index and liver index of mice in the present invention; wherein, Figure 3 Figure A in the figure is a weight statistics chart; Figure 3 Figure B is a statistical chart of body fat percentage; Figure 3 Figure C is the Lee's index statistical chart; Figure 3 Figure D is a statistical graph of liver index; * represents compared with the Control group, and # represents compared with the Model group.

[0040] Figure 4 The statistical graphs of TC, TG, HDL-C and LDL-C in the serum of each group of mice in the present invention; Figure 4 Figure A in the figure is a TC statistical chart; Figure 4 Figure B is the TG statistical chart; Figure 4 Figure C in the figure is a statistical diagram of HDL-C; Figure 4 Figure D is the LDL-C statistical chart; among them, the Chinese name of TC is total cholesterol; the Chinese name of TG is total triglycerides; the Chinese name of HDL-C is high-density lipoprotein cholesterol; the Chinese name of LDL-C is low-density lipoprotein cholesterol.

[0041] Figure 5 This is a quantitative statistical diagram of flow cytometry detection of M1 macrophages in the present invention. DETAILED DESCRIPTION

[0042] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0043] Reagents and Materials The reagents and materials are as follows:

[0044] The umbilical cord tissue of full-term cesarean section newborns was collected from Xi'an Huxian Maternal and Child Health Hospital, donated with the consent of the mothers themselves, and passed the test.

[0045] Sophora japonica extract was purchased from Xi'an Sinuote Biotechnology Co., Ltd.

[0046] CPP-AA peptide was provided by Nanjing GenScript Biotechnology Co., Ltd.

[0047] Gastrodia elata polysaccharide was purchased from Xi'an Huike Biological Company.

[0048] Melatonin was purchased from Shaanxi Angxu Biotechnology Co., Ltd.

[0049] The double antibody in the physiological saline containing double antibodies is penicillin / streptomycin solution, penicillin / streptomycin solution: 100×, sterile, purchased from Dalian Meilun Biotechnology Co., Ltd.

[0050] 0.25% trypsin digestion solution containing EDTA was purchased from Dalian Meilun Biotechnology Co., Ltd.

[0051] The culture medium was α-MEM culture medium purchased from Gibco; fetal bovine serum (FBS) was purchased from Gibco; and the complete culture medium was α-MEM culture medium supplemented with 5% double antibody by mass fraction and 10% fetal bovine serum by mass fraction.

[0052] Example 1

[0053] A method for preparing a stem cell preparation based on immunomodulation comprises the following steps:

[0054] 1. Extraction and separation of umbilical cord mesenchymal stem cells

[0055] Umbilical cord tissue from full-term cesarean section newborns was collected and placed in umbilical cord preservation solution. It was transported to the GMP laboratory by a dedicated person and processed within 24 hours. The umbilical cord was repeatedly washed with saline containing 5% penicillin / streptomycin solution, blood stains on the surface were removed, and it was cut into 3 cm segments. The umbilical artery and umbilical vein were removed and cut into pieces with scissors to about 2 mm. 3 Small pieces were evenly spread in a 10 cm culture dish, placed in a 37 ° C, 5% CO2 incubator for culture, and 3 mL of complete culture medium was added. After 24 hours, complete culture medium was added again. Adding culture medium too early will cause the umbilical cord tissue to not adhere firmly to the wall, and adding culture medium later than 24 hours will be detrimental to the cells crawling out.

[0056] After 3 days, add 5 mL of complete medium again, shake the culture dish slowly to cover the tissue blocks, and continue to culture in an incubator at 37°C and 5% CO2. Change the medium every 3.5 days, and be sure to change the medium slowly to prevent the tissue blocks from floating.

[0057] Observe under an inverted microscope. When the cell fusion around most tissue blocks reaches 80%, primary cells can be harvested. Aspirate the culture medium and tissue blocks, transfer the tissue blocks to a new culture dish and repeat the above steps until no cells crawl out. After rinsing twice with saline, add 3 mL of 0.25% trypsin digestion solution containing EDTA, add complete culture medium to terminate digestion, resuspend the cells and count them. These cells are P0 generation cells. After being identified as qualified mesenchymal stem cells in accordance with the General Requirements for Stem Cells, the cells are divided into 8×10 3 / cm 2 The cells were subcultured, expanded, cultured, and cryopreserved until P5 umbilical cord mesenchymal stem cells were obtained for subsequent use.

[0058] 2. Cell culture collection

[0059] The above P5 umbilical cord mesenchymal stem cells were taken and cultured in complete medium containing 4 mg / mL Sophora japonica extract at a rate of 8×10 3 cm 2 The cell density was inoculated in a ten-layer cell factory, and then cultured in a saturated humidity incubator at 37°C and 5% CO2, and the cell status was observed under an inverted microscope. When the cell fusion growth reached 80%, the above cell supernatant was collected, and the collected cell supernatant was centrifuged at 300g and 4°C for 10 minutes to remove dead cells. After discarding the precipitate, the supernatant was collected and then centrifuged at 2000g and 4°C for 10 minutes to remove cell debris. The filtrate supernatant was collected and then separated and concentrated by Millipore ultrafiltration concentration in sequence. The ultrafiltration concentration membrane packages were 100KD and 3KD respectively to obtain stem cell culture medium.

[0060] After the protein concentration of the stem cell culture medium was determined by the BCA method, it was divided and stored in a -80°C refrigerator for use. At this time, the protein concentration of the stem cell culture medium was 1.2 mg / mL. Before use, it was sterilized by filtration with a 0.22μ filter membrane to obtain the stem cell culture medium for subsequent experiments.

[0061] 3. Preparation of Curcumin (CUR) Liposomes

[0062] (1) CPP-AA peptide

[0063] Dissolve 20 mg of active ester-polyethylene glycol-phospholipid in 1 mL of water, add 8 mg of CPP-AA peptide, stir and react for 12 h to obtain a reaction solution, put the reaction solution into a dialysis bag with a cutoff of 3.5 KDa, place the dialysis bag in an aqueous solution, dialyze for 48 h, and then freeze-dry the liquid in the dialysis bag to obtain CPP-AA-PEG-DSPE.

[0064] Among them, the amino acid sequence of CPP-AA peptide is shown in SEQ ID NO.1: KIKKVKKKGRKHAGVISVLAQQAAKLTSDPTDIP.

[0065] The English abbreviation of active ester-polyethylene glycol-phospholipid is NHS-PEG 2000 -DSPE was purchased from Shaanxi Xinyan Bomei Biotechnology Co., Ltd.

[0066] (2) Weigh 0.5 g of lecithin, 0.1 g of cholesterol, 0.07 g of CPP-AA-PEG-DSPE, 0.005 g of Tween 80, and 0.01 g of vitamin E, add 50 mL of ethanol, shake to fully dissolve, then add 2 mL of anhydrous ethanol solution containing 5 mg / mL curcumin, evaporate to dryness on a rotary evaporator at 55° C. and a vacuum degree of 0.08 MPa to form a film, then add 40 mL of a phosphate buffer solution of pH = 6.5, rotate at 65° C. and 12 r / min to dissolve the film. Place at room temperature for 6 hours to fully hydrate, and obtain a curcumin liposome solution.

[0067] Subsequently, the curcumin liposome solution was subjected to probe ultrasound under the following conditions to obtain a final curcumin liposome solution.

[0068] Wherein, the mass of curcumin in the anhydrous ethanol solution containing curcumin is 0.01 g.

[0069] The ultrasonic conditions were 50 W, 5 min, 3 s of ultrasonication, and 2 s of rest.

[0070] 4. Preparation of freeze-dried powder

[0071] The curcumin liposome solution and the stem cell culture medium were mixed evenly at a volume ratio of 1:3 to obtain a mixed solution; mannitol and trehalose were added to the mixed solution as freeze-drying protective agents to obtain a freeze-dried powder stock solution.

[0072] The above-mentioned lyophilized powder stock solution is sterilized by filtering with a 0.22 μm filter membrane and then dispensed into vials, and freeze-dried at a pre-freezing temperature of -70°C, a pre-freezing time of 8 hours, and a total drying time of 48 hours to obtain a stem cell culture solution lyophilized powder, which is stored at room temperature for later use. Stem cell culture solution lyophilized powder is referred to as lyophilized powder in the present invention.

[0073] The mass ratio of the added mannitol to the mixed solution is 8%.

[0074] The added trehalose accounts for 2% by mass of the mixed solution.

[0075] The lyoprotectant accounts for 10% by weight of the mixed solution.

[0076] 4. Stem cell preparations

[0077] Take 0.01g of freeze-dried powder, 0.1g of Gastrodia elata polysaccharide, and 0.003g of melatonin and mix them evenly to obtain the stem cell preparation.

[0078] In order to illustrate the effect of a stem cell preparation based on immunomodulation provided by the present invention, the following study was conducted:

[0079] (1) Obese mouse model

[0080] ①Modeling method

[0081] After 1 week of adaptive culture, 3-week-old C57BL / 6J male mice were randomly divided into 5 groups: control group and model group, with 10 mice in the control group and 40 mice in the model group. The control group was fed with a normal low-fat diet, and the model group was fed with a high-fat diet. After 5 weeks of continuous feeding, the mice were fasted but not watered for 12 hours, 0.25 mL of blood was collected from the orbits and placed in a 4°C environment for 4 hours, centrifuged at 4000 rpm for 10 minutes, and the upper serum was collected and packaged for testing. If the blood lipid level of the modeling mice was significantly higher than that of the normal group (P<0.05), the modeling was considered successful.

[0082] Among them, ordinary low-fat feed: H10010, the formula is 10% fat, 70% carbohydrates, and 20% protein.

[0083] High-fat feed: H10060, the formula is 60% fat, 20% carbohydrates, and 20% protein.

[0084] The above feeds were purchased from Beijing Huafukang Biotechnology Co., Ltd.

[0085] Blood lipid level refers to the content of total cholesterol TC and total triglycerides TG.

[0086] ②Modeling results

[0087] Compared with C57 mice fed with ordinary feed, C57 mice fed with high-fat feed had greasy hair and bloated bodies. Figure 1 It can be seen that after 8 weeks of obesity model treatment, the average weight of mice in the model group was higher than that in the control group, and the difference was significant. Figure 2 Compared with the normal diet group, the TC and TG contents of mice in the high-fat diet group were significantly increased, indicating that the obesity model was successfully established.

[0088] (2) Dosage method and index detection

[0089] The mice with successful modeling were taken as the experimental group, and the experimental groups were gavaged with 0.2 mL of the following substances according to the following doses: Orlistat was suspended in pure water containing 0.5% CMC-Na by mass to prepare an orlistat suspension with a drug content of 3 mg / mL for later use.

[0090] Positive control group: ORL, orlistat suspension 60 mg / kg / d.

[0091] Stem cell powder group: SC, 10 mg / kg / d.

[0092] Melatonin group: MT, 2 mg / kg / d.

[0093] Gastrodia elata polysaccharide group: GP, 100 mg / kg / d.

[0094] Stem cell powder + melatonin + Gastrodia elata polysaccharide group: SC / MT / GP, 5 mg / kg / d.

[0095] The model group (Model) and the control group (Control) were intragastrically administered with the same dose of 0.9% saline.

[0096] The mice were fed continuously for 10 weeks. The model group and the experimental group were fed with a high-fat diet, and the control group was fed with a normal diet. The mice were given the drug once every 2 days at the same time. During the experiment, the weight data of the mice in each treatment group were collected once a week, the weight changes of the animals were recorded, and the dosage volume was adjusted according to the weight changes. The mice were fasted the night before the sacrifice and dissection, and the last weighing was performed the next day. At the same time, after the 10-week feeding, all animals were fasted overnight but not water-free, weighed, and the length from the nose tip to the tail of the mice was measured to calculate the Lee's index.

[0097] in,

[0098] In the above calculation formula, the unit of body weight is g; the unit of body length is cm.

[0099] At the same time, the mouse abdomen was turned upward, and the liver of the mouse was collected by dissection, washed with cold saline, wiped dry, weighed and recorded, and the liver index was calculated. The calculation formula was: liver index = (liver weight / mouse body weight) × 100%.

[0100] Wherein, in the above calculation formula, the unit of liver mass and mouse body mass is g.

[0101] At the same time, make a small longitudinal incision in the inguinal area of ​​the mouse to expose the adipose tissue and peel off all the right lower inguinal adipose tissue; make a small incision in the scapular area to expose the interscapular area, and carefully peel off the brown adipose tissue located in the interscapular area, which is usually darker in color and harder in texture. Make a midline incision in the abdomen, then separate the skin and muscles to both sides to expose the testicles and epididymis, and separate the peritesticular adipose tissue; carefully expose both kidneys and separate the bilateral perirenal adipose tissue; similarly, make small incisions in multiple parts of the mouse, and directly separate the adipose tissue from under the skin to obtain subcutaneous adipose tissue. Among them, the right lower inguinal adipose tissue, peritesticular adipose tissue, bilateral perirenal adipose tissue, and subcutaneous adipose tissue are mainly white adipose tissue. Refer to the above method until all white fat and brown fat are separated, and the above adipose tissues are washed and dried with cold saline, weighed and recorded. Calculate the following indicators at the same time:

[0102] Body fat percentage = (perirenal and peri-epididymal fat weight + inguinal fat weight + bilateral perirenal fat weight + subcutaneous fat weight + brown fat weight) / body weight × 100%.

[0103] Among them, in the above calculation formula, the unit of perirenal and peri-epididymal fat weight, inguinal fat weight, bilateral perirenal fat weight, subcutaneous fat weight, brown fat weight and body weight is g.

[0104] In addition, the eyeballs of mice were removed to kill the mice and blood was collected. After being placed at room temperature for 30 minutes, the blood was centrifuged at 3000 rpm for 15 minutes, and the upper serum was collected for subsequent detection of the content of total triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C). The above kits were purchased from Nanjing Jiancheng Biological. Among them, the English abbreviation of total triglycerides is TG; the English abbreviation of total cholesterol is TC; the English abbreviation of low-density lipoprotein cholesterol is LDL-C; the English abbreviation of high-density lipoprotein cholesterol is HDL-C.

[0105] At the same time, the peritesticular adipose tissue was cut into 2 mm × 2 mm blocks with ophthalmic scissors, and an appropriate amount of type II collagenase was added. The adipose tissue was digested into chyme at 37°C for 30 min, and centrifuged at 1500 rpm for 5 min. The upper layer of adipose tissue was discarded, washed with PBS, and resuspended after filtering with a cell sieve. The resuspended cells of the adipose tissue after antibody staining were detected by flow cytometry.

[0106] Among them, the antibodies selected are as follows: allophycocyanin anti-mouse F4 / 80 antibody (APC-F4 / 80), Peridinium chlorophyll protein anti-mouse CD11c antibody (PerCP-CD11c), and fluorescein isothiocyanate anti-mouse CD11b antibody (FITC-CD11b).

[0107] Among them, allophycocyanin anti-mouse F4 / 80 antibody: APC-F4 / 80; Peridinium chlorophyll protein anti-mouse CD11c antibody: PerCP-CD11c; fluorescein isothiocyanate anti-mouse CD11b antibody: FITC-CD11b. All of the above antibodies were purchased from Abcam. F4 / 80+CD11c+CD11b+ is defined as M1 macrophages; F4 / 80+CD11c+CD11b- is defined as M2 macrophages. The infiltration of macrophages in adipose tissue was calculated based on the results of flow cytometry.

[0108] (2) Index evaluation results

[0109] ① Changes in body weight, body fat, and liver index

[0110] The final body weights of C57 mice in different treatment groups are shown in Figure 3 Figure A in Figure 2. The weight of the positive drug group was significantly lower than that of the model group and was closer to that of the normal group. The weight loss effect of the positive drug group was good. The weight of SC, MT and GP mice was lower than that of the model group, but the difference was not significant, indicating that the intervention effect on the weight of C57 mice induced by a high-fat diet was weak. The weight of the SC+MT+GP group was lower than that of the model group, and the weight difference was significant (P<0.05), indicating that the SC+MT+GP combination had a good control effect on the weight gain induced by a high-fat diet.

[0111] Lee's index and body fat percentage can both be used as indicators to evaluate the degree of obesity in obesity models. The higher the Lee's index and body fat percentage, the more severe the obesity. Figure 3 Figure B and Figure 3 The results of Figure C in Figure 2 show that the Lee's index and body fat percentage were lower than those of the model group, and the effects of the preparation group and the positive group were equivalent, both of which were significantly different from the model group (P < 0.05).

[0112] At the same time from Figure 3 As shown in Figure D, the liver weight of mice in the model group increased significantly compared with the control group. The livers of mice in the model group accumulated a large amount of fat, with a significant difference (P<0.01). After drug treatment in each group, the liver weight was significantly reduced compared with that in the model group. Except for the GP group, there was no significant difference, while the other groups had significant differences (P<0.01), indicating that each group was able to effectively alleviate the accumulation of fat in the liver to a certain extent.

[0113] ② Serological indicators

[0114] Figure 4 The statistical graphs of TC, TG, HDL-C and LDL-C in the serum of mice in each group; Figure 4 Figure A in the figure is a TC statistical chart; Figure 4 Figure B is the TG statistical chart; Figure 4Figure C in the figure is a statistical diagram of HDL-C; Figure 4 Figure D is a statistical chart of LDL-C.

[0115] Depend on Figure 4 It can be seen that the higher the TG, TG, and LDL-C levels in the blood, the higher the fat content in the blood, the easier it is to deposit in the blood vessels, and the higher the risk of cardiovascular disease; HDL-C helps to remove cholesterol from the blood and transport it to the liver for metabolism and excretion. It has a good effect on lowering blood lipoproteins. The higher the content, the lower the risk of cardiovascular disease. Figure 5 It can be seen that compared with the control group, the TG, TC and LDL-C levels in the serum of the model group were significantly increased, and the HDL-C content was significantly decreased (the difference was extremely significant P<0.01). After treatment in each experimental group, the TG, TC and LDL-C levels in the serum of mice were significantly decreased, and HDL-C was significantly increased.

[0116] The above results indicate that the preparation group can significantly improve lipid metabolism disorders caused by a high-fat diet and has the effect of improving abnormal blood lipid metabolism in obese mice.

[0117] ③ Macrophage fat infiltration

[0118] Because the para-epididymal adipose tissue is a typical adipose endocrine organ and is closely related to fat metabolism and cytokine secretion in various adipose tissues throughout the body, the para-epididymal adipose tissue was used as a representative to study the infiltration of macrophages in adipose tissue.

[0119] from Figure 5 The quantitative results of M1 macrophages in the flow cytometry test results show that each group can reduce the number of M1 macrophages, among which the positive drug ORL group significantly reduced the number of M1 macrophages and reduced adipose tissue inflammation, with the most significant effect. The intervention of the SC, MT, and GP groups can also resist the adipose inflammation induced by a high-fat diet, and the intervention effect of stem cell preparations is better.

[0120] From the above results, it can be seen that the immune-regulating stem cell preparation provided by the present invention can regulate the proportion of M1 macrophages in adipose tissue, regulate and restore the immune balance of adipose tissue, thereby reducing body weight and improving fat accumulation and dyslipidemia caused by obesity.

[0121] The immunoregulatory-based stem cell preparation provided by the present invention is a stem cell preparation for improving the immune balance of adipose tissue. The development of a stem cell preparation for improving the immune balance of adipose tissue solves the technical gap in the field of stem cell preparations to a certain extent.

[0122] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once the basic inventive concepts are known, and all such changes and modifications fall within the scope of the present invention.

Claims

1. A stem cell preparation based on immunomodulation, characterized in that: The preparation comprises the following raw materials by weight: 5 to 40 parts of freeze-dried powder, 40 to 120 parts of gastrodia elata polysaccharide and 2 to 6 parts of melatonin; The freeze-dried powder is obtained by mixing stem cell culture medium and curcumin liposomes and then freeze-drying; the volume ratio of the stem cell culture medium to the curcumin liposomes is 1:10-32; The protein concentration in the stem cell culture medium is 1.2 mg / mL to 1.6 mg / mL.

2. The stem cell preparation based on immunomodulation according to claim 1, characterized in that: The stem cell culture medium is a stem cell culture medium treated with Sophora japonica urn extract; Treated with Sophora japonica urn extract means that the Sophora japonica urn extract is added during the process of culturing stem cells to obtain stem cell culture medium.

3. The stem cell preparation based on immunomodulation according to claim 2, characterized in that: The preparation method of the stem cell culture medium is as follows: Stem cells were collected at a rate of 8×10 3 ~1×10 4 / cm 2 The cells are inoculated into a complete medium containing 0.2% to 1.0% by mass of a Sophora japonica erithidea extract and cultured at 36° C. to 38° C. and 4% to 6% CO2; When the stem cells have grown to 80% to 90% fusion, the cell supernatant is collected; the collected cell supernatant is centrifuged to remove dead cells and cell debris, and the supernatant is collected again and filtered; the filtrate obtained by filtration is separated and concentrated to obtain the stem cell culture medium.

4. The stem cell preparation based on immunomodulation according to claim 3, characterized in that: The separation and concentration method is: First, centrifuge at 1500g-3000g for 5min-15min, discard the precipitate after centrifugation, and take the supernatant; then pass the supernatant through a 100KD ultrafiltration membrane to collect the dialysate, and at the same time, pass the dialysate through a 3KD ultrafiltration membrane to collect the retentate.

5. The stem cell preparation based on immunomodulation according to claim 3, characterized in that: The stem cells are stem cells that have been passaged and expanded to generations P3 to P7.

6. The stem cell preparation based on immunomodulation according to claim 5, characterized in that: The stem cells are mesenchymal stem cells; the mesenchymal stem cells are derived from any one of the following tissues: Bone marrow, cord blood, umbilical cord tissue, placental tissue, and adipose tissue.

7. The stem cell preparation based on immunomodulation according to claim 1, characterized in that: The curcumin liposomes are prepared by thin film dispersion method using lecithin, cholesterol, CPP-AA-PEG-DSPE, Tween 80, vitamin E and curcumin as raw materials; The mass ratio of the lecithin, the cholesterol, the CPP-AA-PEG-DSPE, the Tween 80, the vitamin E and the curcumin is 0.5-10: 0.1-0.25: 0.08-0.2: 0.005-0.02: 0.01-0.045: 0.002-0.

01.

8. The method for preparing the stem cell preparation based on immunoregulation according to claim 1, characterized in that: The steps include: Take the freeze-dried powder, the gastrodia elata polysaccharide, and the melatonin respectively according to the formula amount; The freeze-dried powder, the Gastrodia elata polysaccharide and the melatonin are uniformly mixed to obtain the stem cell preparation based on immunoregulation.

9. The use of the stem cell preparation based on immunomodulation in weight loss according to claim 1, characterized in that: The stem cell preparation based on immunoregulation is used for preparing medicines with weight loss effect or functional foods with weight loss effect.

10. The use according to claim 9, characterized in that: The medicine with weight loss effect is prepared by using the stem cell preparation based on immunoregulation as an active ingredient and supplemented with pharmaceutically acceptable excipients; The functional food with weight loss effect is prepared by using the stem cell preparation based on immune regulation as an active ingredient and supplemented with auxiliary materials acceptable in food science.

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