CN117899112A - Application of probiotics combined with mesenchymal stem cells in preparation of ovarian anti-aging drugs - Google Patents
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Abstract
The invention discloses an application of probiotic combined mesenchymal stem cells in preparing an ovarian anti-aging medicament, which is characterized in that the concentration of a bifidobacterium triple viable bacteria capsule or a composite lactobacillus enteric capsule in a probiotic preparation is 1g/ml; the final concentration of the mesenchymal stem cells in the cell slow-release liquid is 5 multiplied by 10 6/ml. Based on the double effects of regulating immune and endocrine systems of mesenchymal stem cells and probiotics, the adipose tissue mesenchymal stem cells are easy to obtain, easy to realize quantitative production, low in ethical dispute and low in immunogenicity, are ideal cell therapeutic preparations, are combined with probiotics for application, and have no toxic or side effect basically compared with other hormone drugs and immunosuppressants. Compared with the existing intravenous infusion intervention way, the ovarian targeting mesenchymal stem cell injection method avoids intravenous thrombolysis, anaphylactic reaction and the like, and abdominal and local wounds caused by intervention treatment, and can effectively prevent side effects and damage caused by treatment of patients.
Description
Technical Field
The invention relates to the technical field of biology, in particular to application of probiotics combined with mesenchymal stem cells in preparation of an ovarian anti-aging medicament.
Background
Premature ovarian failure (Premature Ovarian Failure, POF) is a relatively common gynaecological disease caused by premature ovarian failure that can lead to amenorrhea in women before 40 years of age with an increase in gonadotrophin and a decrease in estrogen levels. The prevalence of POF increases year by year, with 1% in women under 40 years old, 0.1% in women under 30 years old, and even in ages under 20 years old, and has developed into a disease that seriously jeopardizes the physical and mental health of women. The disease not only results in infertility but also increases the risk of occurrence of cardiovascular diseases, osteoporosis and sexual dysfunction. The main factors causing POF include hereditary factors, autoimmune factors, iatrogenic factors, environmental factors and the like, and the incidence of iatrogenic secondary premature ovarian failure is gradually rising along with the wide application of chemotherapy in gynecological tumors. Because of its complex etiology, the treatment is troublesome, and how to develop reliable POF treatment means is becoming important.
The main measures for treating POF at present comprise hormone replacement therapy, immunoregulation, ovarian tissue cryopreservation transplantation, embryo cryopreservation transplantation and the like, and most clinical treatment methods can improve POF symptoms and promote the fertility of patients, but have little effect, can not fundamentally recover the ovarian function, and have ethical disputes and adverse reactions. In recent years, stem cell transplantation becomes one of research hotspots for POF treatment, wherein human mesenchymal stem cell transplantation is probably the most effective method for treating POF, and a relatively good treatment effect is shown. The specific mechanism is that the mesenchymal stem cell transplantation promotes the proliferation of granulosa cells, inhibits the apoptosis, regulates the immunity, secretes various cytokines, regulates the ovarian microenvironment and the like, thereby improving and enhancing the regulation of the ovarian function. It has now been found that the ovaries contain proliferating stem cells, and that persons suffering from POF or perimenopause can indirectly provide a suitable microenvironment for ovarian aging by diet, exercise, improving immune and circulatory functions, helping the ovarian germ stem cells to remain active, and continuing to replenish the follicular pool. This suggests that restoration of ovarian function can be achieved by activating the viability of endogenous stem cells in POF patients. However, the production of endogenous stem cells decreases as the human body ages. Studies have shown that stem cells in humans at age 35 "back up" less than half of the time younger, and that physical personality declines, suggesting that imbalance in stem cell function may be an important mechanism for a female to have a "cliff-like" decline in fertility after age 35.
The immunoregulation and immune response of the premature ovarian failure patients are all in a senescent state. How to delay aging by effective means so as to prevent the 'loss' of endogenous stem cells as much as possible and reasonably regulate and improve the ovarian microenvironment provides reliable scientific basis for preventing and treating POF. In recent years, several studies have reported an important role for the intestinal flora in regulating health and longevity. The gut flora promotes maturation of the immune response during early development, contributing significantly in protecting against pathogens, providing energy and nutrition, and maintaining host health. There is growing evidence that dysregulation of intestinal flora not only affects B lymphocyte activation and autoantibody production, but also induces abnormal activation of innate immune cells, resulting in upregulation of pro-inflammatory cytokines; ovarian damage may also be caused by altering T cell subsets, T cell mediated damage, and natural killer cell depletion. This suggests that there is a correlation between intestinal flora and the occurrence and development of POF, and maintaining good intestinal flora homeostasis may become a key point for controlling POF. Significant changes in intestinal flora occur in the transition from adult to elderly, manifested by gradual decline in bifidobacteria, lactobacilli, bacteroides and enterobacteria abundance. These probiotics are dominant species in the intestinal tract of healthy people, the number of which is dynamic and has advantages with age, and the probiotics have close relation with a plurality of physiological and pathological changes in the organism. Research shows that they have the functions of regulating intestinal flora, improving immunity, reducing cholesterol, resisting tumor, delaying senility, promoting digestion and absorption of calcium, iron, magnesium, zinc and other mineral matters and in-vivo nutrient substances, etc., and can reverse the change of cell cycle regulating factors, strengthen the antioxidant capacity of cells and delay the aging of body cells. At present, the probiotic tablets and capsules which are approved are widely applied to the fields of food, health care, medical treatment and the like.
However, there are few drugs for treating premature ovarian failure by combining probiotics with mesenchymal stem cells, and urgent demands exist in the market.
Disclosure of Invention
According to the requirements of the prior art, the invention provides application of probiotics combined with mesenchymal stem cells in preparation of an ovarian anti-aging medicament.
In order to achieve the above purpose, the present invention is realized by the following technical scheme:
The application of the probiotic and the mesenchymal stem cells in preparing the ovarian anti-aging medicament is characterized in that the concentration of the bifidobacterium triple viable bacteria capsule or the composite lactobacillus enteric capsule in the probiotic preparation is 1g/ml; the final concentration of the mesenchymal stem cells in the cell slow-release liquid is 5 multiplied by 10 6/ml.
In the present invention, further, the pH of the probiotic preparation is in the range of 6.5 to 7.0.
In the invention, the preparation method of the probiotic preparation further comprises the following steps: under the aseptic condition, weighing 100mg of sodium hyaluronate and 4g of human serum albumin, adding into 200ml of aseptic medical physiological saline to obtain a preparation slow-release liquid, sterilizing a matrix, and filtering and sterilizing by adopting a 0.22 mu m filter head; removing the bifidobacterium triple viable bacteria capsule or the composite lactobacillus enteric capsule respectively, taking out the content, suspending in the preparation slow release liquid, and subpackaging into a disposable prefilled syringe.
In the present invention, the probiotic preparation is administered orally in an amount of 0.5g/10g of body weight for a period of 14 days or longer.
In the present invention, further, the mesenchymal stem cells are adipose tissue mesenchymal stem cells or dermal mesenchymal stem cells.
In the invention, in the process of separating and culturing the mesenchymal stem cells, the culture medium is low-sugar DMEM containing 15% of fetal bovine serum, the inoculation density is 5×10 3 pieces/cm 2, and the ratio of DMEM, inactivated fetal bovine serum and DMSO in the frozen stock solution is 5:4:1.
In the invention, further, in the isolated culture process of the mesenchymal stem cells, the tissue washing liquid is DPBS, the tissue washing liquid contains 1% of double antibodies (streptomycin and gentamicin), the tissue digestion liquid is 1x DPBS containing 0.075% of collagenase (type I), and the cell digestion liquid is 1x DPBS containing 0.05% of trypsin.
In the present invention, further, the mesenchymal stem cells were transferred to passage 4 in the stem cell medium, and FACS buffer was 1x PBS containing 2% fbs, 0.5% paraformaldehyde and 0.1% nan 3.
In the present invention, further, the adipogenic differentiation induction medium composition of the mesenchymal stem cells is low-sugar DMEM, 10% dbs, 1 μΜ dexamethasone, 0.5mm 3-isobutyl-1-methylxanthine, 10 μΜ bovine insulin, 60 μΜ indomethacin and 1% glutamine; or the osteogenic differentiation induction medium is composed of low sugar DMEM, 10% dbs, 1 μm dexamethasone, 50 μm ascorbic acid, 10mM sodium beta-glycerophosphate and 1% glutamine.
In the present invention, further, the adipose tissue-derived mesenchymal stem cells are derived from the groin and/or abdomen of an animal; the dermal tissue is derived from the skin of the back of an animal.
In summary, the invention has the following beneficial effects:
The invention solves the problem that the effect of independently using the mesenchymal stem cells to treat climacteric symptoms such as premature ovarian failure is limited by applying the probiotics combined with the mesenchymal stem cells to prepare the ovarian anti-aging medicament, and creatively combines the probiotic and stem cell treatment effects, thereby breaking through the limitation and constraint of the conventional scheme. Based on the double effects of regulating immune and endocrine systems of mesenchymal stem cells and probiotics, the adipose tissue mesenchymal stem cells are easy to obtain, easy to realize quantitative production, low in ethical dispute and low in immunogenicity, are ideal cell therapeutic preparations, are combined with probiotics for application, and have no toxic or side effect basically compared with other hormone drugs and immunosuppressants. Compared with the existing intravenous infusion intervention way, the ovarian targeting mesenchymal stem cell injection method avoids intravenous thrombolysis, anaphylactic reaction and the like, and abdominal and local wounds caused by intervention treatment, and can effectively prevent side effects and damage caused by treatment of patients.
Drawings
FIG. 1 shows the pathological staining result of the ovary of a mouse after the treatment of the bifidobacterium triple viable bacteria and the compound lactobacillus preparation;
Figure 2 effect of bifidobacterium triple viable and complex lactobacillus formulations on the serum hormone E2, FSH and LH levels in mice, compared to model group: * p <0.05;
Fig. 3. Bifidobacterium triple viable bacteria preparation continuously complemented changes in serum hormone E2, FSH and LH levels in mice, compared to model group: * p <0.05, < p <0.05;
Fig. 4. Morphological observations of adipose mesenchymal stem cells (3 rd generation);
FIG. 5 shows a graph of adipose-derived mesenchymal stem cell growth;
FIG. 6 shows the results of adipose-derived mesenchymal stem cells induced differentiation;
FIG. 7 shows the results of the surface marker flow assay for adipose tissue-derived stem cells;
FIG. 8 shows the results of induced differentiation of mouse dermal mesenchymal stem cells;
FIG. 9 shows the results of the logistics of the mouse dermal mesenchymal stem cell surface markers;
Fig. 10, adipose tissue-derived stem cells and bifidobacterium triple viable bacteria preparation combined treatment of mice serum hormone E2, FSH and LH level changes, compared to model group: * p <0.05, < p <0.05; compared to the control group (stem cell transplantation only): #p <0.05;
Fig. 11, adipose tissue-derived stem cells and bifidobacterium triple viable bacteria preparation combined treatment mice follicular count, ratio to model group: * p <0.05, < p <0.05; compared to the control group (stem cell transplantation only): #p <0.05.
Detailed Description
The raw materials and equipment used in the invention are common raw materials and equipment in the field unless specified otherwise; the methods used in the present invention are conventional in the art unless otherwise specified.
Unless otherwise defined, all terms used in the specification have the same meaning as commonly understood by one of ordinary skill in the art, but are defined in the specification to be used in the event of a conflict.
The invention provides an application of probiotic combined mesenchymal stem cells in preparation of an ovarian anti-aging medicament, wherein the concentration of a bifidobacterium triple viable bacteria capsule or a composite lactobacillus enteric capsule in a probiotic preparation is 1g/ml; the final concentration of the mesenchymal stem cells in the cell slow-release liquid is 5 multiplied by 10 6/ml.
The preparation method of the probiotic preparation comprises the following steps: under the aseptic condition, weighing 100mg of sodium hyaluronate and 4g of human serum albumin, adding into 200ml of aseptic medical physiological saline to obtain a preparation slow-release liquid, sterilizing a matrix, and filtering and sterilizing by adopting a 0.22 mu m filter head; removing the bifidobacterium triple viable bacteria capsule or the composite lactobacillus enteric capsule respectively, taking out the content, suspending in the preparation slow release liquid, and subpackaging into a disposable prefilled syringe.
The hyaluronate has slow release and anti-inflammatory effects, can provide weak acid environment, and is beneficial to the treatment effect of probiotics. The bifidobacterium triple viable bacteria capsule (3 strains of bifidobacterium, lactobacillus acidophilus and enterococcus faecalis) is produced by Shanghai pharmaceutical friendship pharmaceutical factory Co., ltd, chinese medicine standard S10950032, is prepared into 1g/ml liquid medicine by using physiological saline containing 0.05% of sodium hyaluronate and 2% of human serum albumin, and is stored in a refrigerator at 4 ℃ in a dark place. The composite lactobacillus enteric capsule is a poly gram composite lactobacillus enteric capsule, chinese medicine standard character H19980184 is purchased from Guangdong medical care pharmacy chain limited company, and the used slow release liquid is physiological saline containing 0.05% of sodium hyaluronate and 2% of human serum albumin, so as to prepare 1g/ml liquid medicine.
In the technical scheme, the molecular weight of the sodium hyaluronate is 30-300 ten thousand daltons.
The pH value of the bifidobacterium triple viable bacteria preparation ranges from 6.5 to 7.0, and the pH value of the bifidobacterium triple viable bacteria preparation is 6.8, and the bifidobacterium triple viable bacteria preparation is taken orally.
The preparation sustained-release liquid for probiotics provided by the invention is normal saline containing 0.05% of sodium hyaluronate and 2% of human serum albumin, but other sustained-release liquid schemes are also provided, including normal saline, normal saline containing 0.05% of sodium hyaluronate and normal saline containing 2% of human serum albumin.
The probiotic preparation of the invention is orally taken, and various antibacterial agents such as penicillins, cephalosporins, macrolides, aminoglycosides, tetracyclines, quinolones and the like are required to be avoided from being used together with the probiotic preparation.
The invention provides an optimized adipose tissue mesenchymal stem cell separation scheme, which can ensure that a harvested cell population shows higher proliferation rate and cell activity, overcomes the defects of the existing method for obtaining mesenchymal stem cells, and provides a practical, simple and efficient method for separating mesenchymal stem cells from adipose tissues and culturing the mesenchymal stem cells.
The mesenchymal stem cell primary cells are adipose-derived mesenchymal stem cell primary cells, and the mesenchymal stem cell primary cells are obtained by adopting the following technical scheme:
Adipose tissue was extracted and minced: extracting fat particles, removing visible micro blood vessels and muscle tissues, washing with an equal volume of tissue washing liquid for 3 times, and finely cutting into paste with surgical scissors and a surgical knife to enable the paste to be smaller than 1mm 3; treated with 2 volumes of 0.075% type I collagenase for 1 hour at 37℃and gently stirred to release the cells. Collagenase activity was neutralized with an equal volume of DPBS and the suspension was centrifuged at 1000x g for 10 min at 20 ℃. The pellet was resuspended in DPBS and debris removed by passing through a 100 μm cell filter. The filtered cells were centrifuged at 1000x g for 10 min, resuspended in low-sugar DMEM containing 15% fetal bovine or human serum, and inoculated into T175 flasks at a density of 5X 10 3 cells/cm 2 and incubated at 37℃in a 5% CO 2 incubator. The primary liquid exchange is carried out on the next day, then the culture medium is changed every 3 days, and the primary cells of the adipose-derived mesenchymal stem cells can be obtained after the culture for 6 days. When the cells reached 80% confluence, incubation with trypsin-EDTA for 3 minutes will separate, wash, and then re-inoculate the flask at the same density. And finally, replacing a serum-free culture medium, collecting the mesenchymal stem cells of the 3 rd to 5 th generation, and freezing for later use.
The mesenchymal stem cells of the present invention are derived from human or other mammals. Adipose tissue mesenchymal stem cells can be obtained from autologous fat aspirates, are less invasive, have a large number of obtained stem cells, have similar functions to bone marrow-derived stem cells, and can be easily grown under standard tissue culture conditions. Therefore, the mesenchymal stem cells in the present invention are preferably derived from adipose tissue.
The invention also provides an extraction and cell preparation scheme of the dermal tissue-derived mesenchymal stem cells, which has the unique advantages of convenient material taking, wide sources, hematopoietic recovery promotion and the like, and the dermal mesenchymal stem cells have wide potential clinical application prospects, can be used for repairing skin and nerve injury, can be applied to skin aging resistance and skin tissue engineering, and are more beneficial to white body transplantation, so that the dermal mesenchymal stem cells have great potential to become important seed cells for stem cell treatment. The general operational flow of separation, extraction and cultivation comprises the following steps:
The back skin of SPF-class BALB/c female mice from a new generation for 2-3 days was placed in an operation tray, washed 3 times with tissue washing liquid to remove impurities and blood stasis, the subcutaneous adipose tissue was removed, washed 3 times with DPBS, then treated with 2 volumes of 0.075% type I collagenase for 1 hour in a37℃water bath, and gently shaken to release the cells. After the dermis and epidermis are seen to separate, the epidermis is removed with forceps, the dermis tissue is sheared into 1mm 3 small pieces in another petri dish with scissors, 0.075% type I collagenase is added in 3-4 times the tissue volume, and the pieces are treated in a37 ℃ water bath for 1 hour. When the digestion was seen under a microscope to produce a large number of cells, collagenase activity was neutralized with an equal volume of low-sugar DMEM, and then the suspension was centrifuged at 1000x g for 10 minutes at 20 ℃. The pellet was resuspended in DPBS and filtered through a 100 μm cell filter to remove debris. The filtered cells were centrifuged at 1000x g for 10min, resuspended in low-sugar DMEM containing 15% fetal calf serum, and inoculated into T175 flasks at a density of 5X10 3 cells/cm 2 and incubated in a 5% CO2 incubator at 37 ℃. The first liquid change is carried out in the next day, then the culture medium is changed every 3 days, and the autologous adipose-derived mesenchymal stem cells primary cells can be obtained after 6 days of culture. When the cells reached 80% confluence, the medium was discarded, washed 2 times with DPBS, digested 3 minutes with 0.05% trypsin-EDTA, stopped by adding DMEM, centrifuged to collect the cells, and then re-inoculated to the flask at the same density. And finally, replacing a serum-free culture medium, collecting the mesenchymal stem cells of the 3 rd to 5 th generation, and freezing for later use. For example, the cells after collection were frozen, added with a frozen stock solution (DMEM: inactivated fetal bovine serum: dmso=5:4:1), and stored in liquid nitrogen.
The purity and quality of the obtained dermal tissue mesenchymal stem cells in the present invention are slightly inferior to those of adipose-derived mesenchymal stem cells, and the present invention prefers adipose-derived mesenchymal stem cells in consideration of convenience of obtaining and material cost.
In the scheme of the invention, the adipose-derived mesenchymal stem cell transplantation method is to locally and targeted inject the ovary, the final concentration of mesenchymal stem cell transplantation is 5×10 6/ml, and the mesenchymal stem cell slow-release liquid is physiological saline containing 0.05% sodium hyaluronate and 2% human serum albumin.
The tissue cleanser in the invention is DPBS and contains 1% of diabody (streptomycin and gentamicin).
Preferably, the adipose-derived mesenchymal stem cell primary cell culture medium is low-sugar DMEM containing 15% fetal bovine or human serum.
Preferably, the digestive juice in the adipose mesenchymal stem cell primary cell isolation and extraction protocol is 1x DPBS containing 0.075% collagenase (type I), and the digestive juice in the adipose mesenchymal stem cell primary cell culture protocol is 1x DPBS of 0.05% trypsin.
As a preferred embodiment of the use according to the invention, the mesenchymal stem cells are passaged in a stem cell medium for 3 to 5 passages.
The mesenchymal stem cell preparation of the invention, wherein the cell purity of each generation of the obtained adipose-derived mesenchymal stem cells is more than 90%. Besides using 0.05% sodium hyaluronate physiological saline as a slow release solution to prepare stem cell preparations, the invention also provides other schemes including physiological saline, physiological saline containing 0.05% sodium hyaluronate and physiological saline containing 2% human serum albumin. Each slow release solution scheme can ensure that the CD73 expression of the obtained primary cells is more than 80 percent, the CD45 is not expressed, the content of the mesenchymal stem cells in the obtained primary cells reaches more than 70 percent, and the concentration of the obtained stem cells is very high. When the slow release solution is physiological saline containing 0.05% of sodium hyaluronate and 2% of human serum albumin, the content of mesenchymal stem cells in primary cells is more than 90%, and is usually in the range of 93-96%.
The invention uses 0.4% trypan blue staining method to count the number of living cells before and after freezing.
The invention relates to an intermediate mesenchymal stem cell pollution detection method, which is used for detecting whether cells are polluted by fungi and bacteria or not by collecting cell culture solution, and mainly comprises the steps of detecting whether the cells are infected by one or more selected from the following steps: mycoplasma, hepatitis B virus, hepatitis C virus, HIV, cytomegalovirus, EB virus, syphilis, etc.
The invention provides an evaluation of the combination of mesenchymal stem cell transplantation and a bifidobacterium triple viable bacteria preparation in the treatment effect of premature ovarian failure, which comprises different combination modes (7 days, 14 days and 21 days of continuous supplementation) and treatment effects of the mesenchymal stem cell and the bifidobacterium triple viable bacteria preparation.
Preferably, the invention considers that the bifidobacterium triple viable bacteria preparation is continuously supplemented for more than 14 days, so that the treatment effect of the mesenchymal stem cells on premature ovarian failure is greatly promoted.
In addition, the invention also provides the evaluation of the combination effect of mesenchymal stem cell transplantation and lactobacillus preparation in the treatment of premature ovarian failure, comprising different combination modes (7 days, 14 days and 21 days of continuous supplementation) and treatment effects of the mesenchymal stem cells and the lactobacillus preparation. Although the mesenchymal stem cell transplantation treatment effect can be well promoted, the combined scheme of the mesenchymal stem cell transplantation and the bifidobacterium triple viable bacteria preparation is not the same.
The terms "comprising," "including," "containing," "having," or other variations thereof herein are intended to cover a non-closed inclusion, without distinguishing between them. The term "comprising" means that other steps and ingredients may be added that do not affect the end result. The term "comprising" also includes the terms "consisting of …" and "consisting essentially of …". The compositions and methods/processes of the present invention comprise, consist of, and consist essentially of the essential elements and limitations described herein, as well as additional or optional ingredients, components, steps, or limitations of any of the embodiments described herein.
All numbers or expressions referring to amounts of components, process conditions, etc. used in the specification and claims are to be understood as modified in all instances by "about". All ranges directed to the same component or property are inclusive of the endpoints, which endpoints are independently combinable. Because these ranges are continuous, they include every value between the minimum and maximum values. It should also be understood that any numerical range recited herein is intended to include all sub-ranges within that range.
In order to make the objects, technical solutions and advantages of the present application more apparent, the technical solutions of the present application will be more fully described below with reference to the embodiments. The described embodiments are some, not all, embodiments of the application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available.
Example 1 intestinal probiotic preparation for promoting mesenchymal Stem cells to treat premature ovarian failure
The probiotic preparation comprises a bifidobacterium triple viable bacteria capsule (national drug standard code S10950032, shanghai drug Yi pharmaceutical factory Co., ltd.) or a composite lactobacillus enteric capsule (national drug standard code H19980184, jiangsu Meitong pharmaceutical Co., ltd.).
In the invention, the concentration of the bifidobacterium triple viable bacteria or the compound lactobacillus in the preparation is preferably 1g/ml, the physiological saline is 0.9 percent sodium chloride, the concentration of the sodium hyaluronate in the preparation is 0.05 percent, and the concentration of the human serum albumin in the preparation is 2 percent. The pH value of the preparation is 6.5-7.0, and the pH value of the preparation is 6.8, and the preparation is stored in a refrigerator at 4 ℃ in a dark place.
The preparation is prepared according to the following method: weighing 100mg of sodium hyaluronate and 4g of human serum albumin, adding into 200ml of sterile medical physiological saline (containing 0.9% of sodium chloride) to obtain a slow release solution of the preparation, sterilizing a matrix, and filtering and sterilizing by adopting a 0.22 mu m filter head; removing the viable bacillus bifidus capsule or the composite lactobacillus enteric capsule, respectively, taking out the content, suspending in the sustained release solution (ensuring the temperature to be lower than 25 ℃), and packaging into a disposable prefilled syringe. All the above preparation processes need to be carried out under aseptic conditions.
The preferred slow-release liquid in the invention is physiological saline containing 0.05% of sodium hyaluronate and 2% of human serum albumin, and as a control, the invention also provides a slow-release liquid group comprising physiological saline, physiological saline containing 0.05% of sodium hyaluronate and physiological saline containing 2% of human serum albumin, and the slow-release liquid group is prepared into a bifidobacterium triple viable bacteria and composite lactobacillus preparation.
Example 2, effect of bifidobacterium triple viable bacteria and compound lactic acid bacteria preparation on improving symptoms of premature ovarian failure in mice construction of premature ovarian failure mouse model:
(1) SPF-class 6-week-old female BALB/c mice with normal estrus cycle were randomly selected, 70 total, and 2mg/kg cisplatin was intraperitoneally injected. All were injected 1 time per day for 14 days. Vaginal abscission cytology was performed at fixed points daily to determine if the estrus cycle of mice was disturbed. Hormone levels of Follicle Stimulating Hormone (FSH), estradiol (E2), and Luteinizing Hormone (LH) were measured in the serum of each group of mice using ELISA kit.
(2) After cisplatin is injected continuously for 14 days, model groups are randomly divided into 7 groups, 10 mice in each group are divided into a control group, physiological saline is supplemented by a lavage mode, bifidobacteria triple viable bacteria of a physiological saline preparation containing 0.05% of sodium hyaluronate is filled into 2 groups, bifidobacteria triple viable bacteria of a physiological saline preparation containing 3 groups of 2% of human serum albumin is filled into the stomach, bifidobacteria triple viable bacteria of a physiological saline preparation containing 0.05% of sodium hyaluronate and 2% of human serum albumin is filled into 4 groups, composite lactic acid bacteria of a physiological saline preparation containing 0.05% of sodium hyaluronate is filled into 5 groups, composite lactic acid bacteria of a physiological saline preparation containing 2% of human serum albumin is filled into 6 groups, and composite lactic acid bacteria of a physiological saline preparation containing 0.05% of sodium hyaluronate and 2% of human serum albumin is filled into 7 groups. The dosage is 0.5mg/10g body weight. Each group was administered continuously for 21 days. Mice were sacrificed 21 days later to evaluate 9 inter-group estrus cycles and ovarian tissue pathology. Wherein, the estrus cycle of the mice is shown in the table 1:
Table 1 shows that the preparation of the bifidobacterium triple viable bacteria or the compound lactobacillus preparation by taking the physiological saline of 0.05 percent of sodium hyaluronate and 2 percent of human serum albumin as the slow release liquid improves the cycle disturbance condition of mice with premature ovarian failure, compared with the bifidobacterium triple viable bacteria preparation by taking the physiological saline of 0.05 percent of sodium hyaluronate and 2 percent of human serum albumin as the slow release liquid, the effect of the bifidobacterium triple viable bacteria preparation is better than that of the compound lactobacillus preparation. In addition, as shown in the HE staining result of FIG. 1, the bifidobacterium triple viable bacteria preparation group (FIG. 1-G) with 0.05% sodium hyaluronate and 2% human serum albumin as physiological saline as slow release liquid has the best effect, and the repairing degree of the granular cells is better than that of other groups, so that the follicle tissue morphology tends to be normal. As shown in figure 1-H, the recovery of the ovarian secondary follicle of the compound lactobacillus preparation group is obvious, but the damage of the granular cells is not recovered, and the effect is slightly weaker than that of the bifidobacterium triple viable bacteria preparation group taking 0.05% sodium hyaluronate and 2% human serum albumin physiological saline as slow release liquid. Therefore, the following examples of the present invention mainly include bifidobacterium triple viable bacteria preparations in which physiological saline containing 0.05% sodium hyaluronate and 2% human serum albumin is used as a sustained release solution.
FIGS. 1-A and 1-B are respectively a group of physiological saline supplemented by a gastric lavage method, FIG. 1-C is a bifidobacterium triple viable bacteria of a physiological saline preparation containing 0.05% sodium hyaluronate by gastric lavage, FIG. 1-E is a bifidobacterium triple viable bacteria of a physiological saline of 2% human serum albumin by gastric lavage, FIG. 1-G is a bifidobacterium triple viable bacteria of a physiological saline preparation containing 0.05% sodium hyaluronate and 2% human serum albumin by gastric lavage, FIG. 1-D is a composite lactic acid bacteria of a physiological saline preparation containing 0.05% sodium hyaluronate by gastric lavage, FIG. 1-F is a composite lactic acid bacteria of a physiological saline preparation containing 0.05% sodium hyaluronate and 2% human serum albumin by gastric lavage, and FIG. 1-H is a composite lactic acid bacteria of a physiological saline preparation containing 0.05% sodium hyaluronate and 2% human serum albumin by gastric lavage.
All groups of mice were anesthetized with isoflurane, serum was collected from heart and assayed for levels of E2, FSH and LH by ELISA. As a result, as shown in FIG. 2, the treatment with either the bifidobacterium triple viable bacteria or the composite lactic acid bacteria, using the physiological saline containing 0.05% of sodium hyaluronate and 2% of human serum albumin as a slow-release solution, can promote the level of E2 in mice with premature ovarian failure and reduce the expression of LH and FSH. In addition, the effect of the bifidobacterium triple viable bacteria preparation is slightly better than that of the composite lactobacillus preparation.
Example 3 evaluation of the Effect of bifidobacteria triple viable bacteria preparation on improving the symptoms of premature ovarian failure in mice
Construction of an ovarian failure mouse model:
(1) Randomly dividing SPF female mice with age of 6-7 weeks into a control group and a model group, wherein 6 normal mice are subjected to intraperitoneal injection of physiological saline, and the volume of the normal mice is 20ml/kg; model group 18, 2mg/kg of cisplatin was intraperitoneally injected. All were injected 1 time per day for 14 days.
(2) After continuous 14 days of cisplatin injection, the model groups were randomly divided into 4 groups of 6 mice each, wherein 1 group was a control group, and physiological saline containing 0.05% sodium hyaluronate and 2% human serum albumin was supplemented by gastric lavage. The other 3 groups of bifidobacterium triple viable bacteria preparations are supplemented by the stomach filling mode, and the dosage is 0.5g/10g of body weight. Each group was administered continuously for 7 days, 14 days and 21 days, respectively.
The mice of each group were monitored daily for behaviours and body weights during the experiment, and serum was collected after 7 days, 14 days and 21 days of continuous administration of the bifidobacterium triple viable bacteria preparation, and hormone levels of FSH, E2, LH and the like in the serum of each group were detected using an ELISA kit. The results are shown in figure 3, and the FSH and LH of the mice serum of the cisplatin-induced model group are obviously increased, and the E2 is obviously reduced, so that the mice serum meets the biochemical index of the mice model of premature ovarian failure. Continuous administration of the bifidobacteria triple viable bacteria preparation can reduce the level of FSH and E2, promote E2 release, and exhibit statistical differences after 14 consecutive days of administration. Furthermore, the effect of 21 days of continuous administration was significantly better than the effect of 7 and 14 days of continuous administration.
Example 3 extraction and isolation of adipose tissue-derived mesenchymal Stem cells
(1) Taking 2 SPF-grade BALB/c female mice of 6-7 weeks old, sucking isoflurane into the mice for anesthesia, killing the mice by adopting a cervical dislocation method, and completely soaking the mice in 75% alcohol for 20min to achieve sterilization treatment;
(2) The mice were fixed on a fixed mount in a sterile console, the bilateral groin was opened, rubbed 3 times in turns with iodophor and alcoholic cotton ball, dissected the abdomen with surgical scissors, and then inguinal adipose tissue was separated with tissue forceps and tissue scissors, fascia and blood vessels around the adipose tissue were removed, washed 3 times with DPBS (containing 1% streptomycin and gentamicin, purchased from Gibco company), finely minced to a paste of less than 1mm 3 using surgical scissors and a surgical knife, and placed in a sterile 50ml centrifuge tube.
(3) The cells were released by treatment with 2 volumes of 0.075% collagenase type i (purchased from Sigma) for 1 hour in a 37 ℃ water bath and gently shaking.
(4) Collagenase activity was neutralized with an equal volume of low-sugar DMEM, and the suspension was centrifuged at 1000x g for 10 minutes at 20 ℃. The pellet was resuspended in DPBS and filtered through a 100 μm cell filter to remove debris.
(5) The filtered cells were centrifuged at 1000x g for 10 min, resuspended in low-sugar DMEM containing 15% fetal calf serum, and inoculated into T175 flasks at a density of 5X 10 3 cells/cm 2 and incubated in a 5% CO2 incubator at 37 ℃. The first liquid change is carried out in the next day, then the culture medium is changed every 3 days, and the autologous adipose-derived mesenchymal stem cells primary cells can be obtained after 6 days of culture.
(6) When the cells reached 80% confluence, the medium was discarded, washed 2 times with DPBS, digested 3 minutes with 0.05% trypsin-EDTA, stopped by adding DMEM, centrifuged to collect the cells, and then re-inoculated to the flask at the same density. And finally, replacing a serum-free culture medium, collecting the mesenchymal stem cells of the 3 rd to 5 th generation, and freezing for later use.
(7) When frozen, the cells collected can be subjected to the procedure described in (6) above, and frozen stock solution (DMEM: inactivated fetal bovine serum: dmso=5:4:1) is added thereto and stored in liquid nitrogen.
(8) If the recovery step is carried out, the frozen cells are taken out from liquid nitrogen, quickly thawed in a water bath kettle at 37 ℃, centrifugally collected after DMEM is added, resuspended in low-sugar DMEM containing 15% inactivated fetal bovine serum, inoculated in a T175 culture flask and cultured in a 5% CO2 incubator at 37 ℃.
(9) The 3 rd generation adipose-derived mesenchymal stem cells (figure 4) with good growth state are taken, inoculated into 24-well plates at the density of 1X 10 4/well after pancreatin digestion, 3-well cell digestion counts are taken every day, each well is counted for 3 times, the average value is calculated, the continuous measurement is carried out for 7 days, and the growth curve of the adipose-derived mesenchymal stem cells is drawn. As shown in fig. 5, after a incubation period of one or two days after the adipose-derived mesenchymal stem cells were inoculated, the cells began to enter a logarithmic growth phase on day 3, proliferate by rapid cell division, gradually slowed down after day 5, reached a plateau phase, and the cell number reached a peak and did not substantially continue to increase.
Example 4 Induction of differentiation of adipose tissue-derived mesenchymal Stem cells
(1) The fat-forming differentiation method is as follows: the induction medium components were low-sugar DMEM, 10% dbs, 1 μm dexamethasone, 0.5mm 3-isobutyl-1-methylxanthine, 10 μm bovine insulin, 60 μm indomethacin and 1% glutamine. The logarithmic phase cells with good growth state of 3 rd generation are inoculated into a 6-hole plate at the density of 1.5X10 5/mL, the culture medium is discarded after the culture is carried out for 48 hours in a 5% CO2 incubator at 37 ℃, the culture medium is added into the culture medium for induction after 3 times of washing by dPBS, and then the liquid is changed every 2 days. Photographing is observed every day until the cells become round and lipid droplets are formed. Then carrying out oil red O staining identification, discarding the induction culture medium, washing 3 times with dPBS, and fixing methanol for 2min at room temperature; rinsing with 70% ethanol for 3 times, dyeing with 2% oil red O for 15min, and rinsing with 70% ethanol for 3 times; and (5) observing under a microscope, photographing and counting.
(2) The osteogenic differentiation method is as follows: the induction medium components were low-sugar DMEM, 10% dbs, 1 μm dexamethasone, 50 μm ascorbic acid, 10mM sodium beta-glycerophosphate and 1% glutamine. Cells of the 3 rd generation in the log phase with good growth state were inoculated in 6-well plates at a density of 1.5X10 5/mL. After 48h of growth, the medium was discarded, washed 3 times with pbs and induced by addition of induction medium, after which the medium was changed every 2 days. Photographing is carried out every day until calcium nodules are formed on the cell surface, and induction is completed. Identification by alizarin red staining, discarding the induction medium, and washing 3 times with dPBS; fixing with 95% ethanol at room temperature for 10min, rinsing with deionized water for 3 times, dyeing with alizarin red for 30min, and rinsing with deionized water for 3 times. And (5) observing under a microscope, photographing and counting.
The results are shown in FIG. 6, and the mesenchymal stem cells isolated by the method of the present invention have the capacity of adipogenic, osteogenic and chondrogenic differentiation, indicating that the mesenchymal stem cells obtained by the method of the present invention have stem cell characteristics.
FIG. 6-A is uninduced adipose mesenchymal stem cells (. Times.100); FIG. 6-B shows the appearance of black round small particles (x 100) in cells after osteoinduction by alkaline phosphatase calcium cobalt staining; FIG. 6-C is alizarin red staining showing mineralized calcium nodule formation in cells after osteoinduction (×100); FIG. 6-D is an oil red O stain showing the appearance of more red circular lipid droplets (x 100) in cells after lipid induction.
Example 5 flow cytometry identification of adipose mesenchymal Stem cell phenotype
(1) The 3 rd generation adipose mesenchymal stem cells were taken to prepare a cell suspension, the morphological observation result of the cells is shown in fig. 4, 500x g is centrifuged for 5min, the supernatant is discarded, and the cells are washed twice with 1x PBS.
(2) After discarding the supernatant, 800. Mu.l of FACS buffer (1 XPBS with 2% FBS, 0.5% paraformaldehyde and 0.1% NaN 3) was added, gently mixed and split into 7 EP tubes, 100. Mu.l of cell suspension number 1-6 per tube.
(3) Tube 1 was added with an equal amount of isotype control antibody, i.e., negative control. Tube No. 2-6 was added with 0.5. Mu.l FITC anti-mouse CD90, CD105, CD34, CD45, CD73 (all purchased from BD company) respectively, and the mixture was blown and mixed well and incubated at room temperature for 30min in the absence of light.
(Wherein CD105, CD73 and CD90 are typical mesenchymal stem cell surface marker characteristics and CD45 and CD34 are lineage specific marker characteristics)
(4) Add 500. Mu.l of FACS buffer to each tube, gently blow wash cells, centrifuge for 5min at 500x g, discard supernatant; after repeating the washing process 3 times, 200 μl of FACS buffer was added to each centrifuge tube, and the mixture was blown and mixed uniformly, and the mixture was sequentially subjected to machine inspection using a flow cytometer model BD FACSCalibur.
The test results are shown in FIG. 7, wherein the positive rates of CD73 and CD90 are greater than 95% and the positive rates of CD34 and CD45 are less than 2%, indicating that the cell suspension is adipose tissue mesenchymal stem cells.
The embodiment of the invention also provides a scheme for separating, extracting and identifying the dermal mesenchymal stem cells. The dermal mesenchymal stem cells have the unique advantages of convenient material taking, wide sources, hematopoietic recovery promotion and the like, have wide potential clinical application prospects, can be used for repairing skin and nerve injury, can be applied to resisting skin aging and skin tissue engineering, and is more beneficial to white body transplantation, so that the dermal mesenchymal stem cells have great potential to become important seed cells for stem cell treatment.
Example 5 isolation and extraction and culture of dermal mesenchymal Stem cells
(1) 2 SPF-grade BALB/c female mice (female mice are adopted in the invention) which are newly born for 2-3 days are taken, and the female mice are soaked in 75% alcohol for 5 minutes after the new mice are killed by a cervical dislocation method.
(2) The mice were immobilized on their limbs, the back skin was cut and placed in DPBS with l% diabody added for washing.
(3) Subcutaneous adipose tissue was removed, washed 3 times with DPBS, treated with 2 volumes of 0.075% type I collagenase for 1 hour in a 37℃water bath, and gently shaken to release cells.
(4) After the dermis and epidermis are seen to separate, the epidermis is removed with forceps, the dermis tissue is sheared into 1mm 3 small pieces in another petri dish with scissors, 0.075% type I collagenase is added in 3-4 times the tissue volume, and the pieces are treated in a 37 ℃ water bath for 1 hour.
(5) When the digestion was seen under a microscope to produce a large number of cells, collagenase activity was neutralized with an equal volume of low-sugar DMEM, and then the suspension was centrifuged at 1000x g for 10 minutes at 20 ℃. The pellet was resuspended in DPBS and filtered through a 100 μm cell filter to remove debris.
(6) The filtered cells were centrifuged at 1000x g for 10 min, resuspended in low-sugar DMEM containing 15% fetal calf serum, and inoculated into T175 flasks at a density of 5X 10 3 cells/cm 2 and incubated in a 5% CO2 incubator at 37 ℃. The first liquid change is carried out in the next day, then the culture medium is changed every 3 days, and the autologous adipose-derived mesenchymal stem cells primary cells can be obtained after 6 days of culture.
(7) When the cells reached 80% confluence, the medium was discarded, washed 2 times with DPBS, digested 3 minutes with 0.05% trypsin-EDTA, stopped by adding DMEM, centrifuged to collect the cells, and then re-inoculated to the flask at the same density. And finally, replacing a serum-free culture medium, collecting the mesenchymal stem cells of the 3 rd to 5 th generation, and freezing for later use.
(8) When frozen, the cells collected can be subjected to the procedure described in (7) above, and frozen stock solution (DMEM: inactivated fetal bovine serum: dmso=5:4:1) can be added thereto and stored in liquid nitrogen.
Example 6 induced differentiation of dermal mesenchymal Stem cells
The method for adipogenic and osteogenic differentiation of dermal mesenchymal stem cells is the same as "induced differentiation of adipose tissue mesenchymal stem cells of example 4", and the results are shown in fig. 8, and the dermal mesenchymal stem cells isolated by the method of the present invention have the capacity of adipogenic, osteogenic and chondrogenic differentiation, indicating that the dermal mesenchymal stem cells obtained by the method of the present invention have stem cell characteristics.
FIG. 8-A is uninduced dermal mesenchymal stem cells (. Times.100); FIG. 8-B is a graph showing the appearance of black round small particles (100) in cells after osteoinduction by alkaline phosphatase calcium cobalt staining; FIG. 8-C is alizarin red staining showing mineralized calcium nodule formation in cells after osteoinduction (×100); FIG. 8-D is an oil red O stain showing the appearance of more red circular lipid droplets (x 100) in cells after lipid induction.
Example 7 flow cytometry identification of dermal mesenchymal Stem cell phenotype
The flow cytometry identification method of the phenotype of the dermal mesenchymal stem cells is consistent with the adipose mesenchymal stem cell scheme. The results of the test are shown in FIG. 9, wherein the positive rates of CD73 and CD90 are greater than 85% and the positive rates of CD34 and CD45 are less than 2%, which indicates that the cell suspension is dermal mesenchymal stem cells, but the purity and quality are not as good as those of adipose mesenchymal stem cells. Therefore, the embodiment 8 of the invention adopts a triple viable bacteria preparation scheme of adipose tissue-derived stem cells combined with bifidobacteria and evaluates the treatment effect of the combined scheme on premature ovarian failure.
Example 8 evaluation of the efficacy of the adipose-derived mesenchymal Stem cell-Bifidobacterium triple viable bacteria preparation on treatment of premature ovarian failure
(1) The cisplatin method is used for constructing an premature ovarian failure mouse model, and the method is the same as in the part of the example 1;
(2) Dividing cisplatin model mice into model groups (5) and treatment groups (20) by adopting a random grouping mode; the treatment groups are 4 subgroups, namely a bifidobacterium triple viable bacteria preparation group with continuous 7 days of intragastric administration, a bifidobacterium triple viable bacteria preparation group with continuous 14 days of intragastric administration, a bifidobacterium triple viable bacteria preparation group with continuous 21 days of intragastric administration and a non-administration group (but for adipose tissue mesenchymal stem cell transplantation treatment, a control group is called), and each subgroup comprises 5 mice. The following groups are referred to as control group, 7 days, 14 days, and 21 days, respectively. It should be noted that, the following day (i.e. the beginning of day 15) after the end of day 14 of cisplatin molding, the mice of each treatment group were continuously dosed with bifidobacterium intragastric triple viable bacteria for 7 days, 14 days and 21 days, respectively; the model group was supplemented with physiological saline containing 0.05% sodium hyaluronate and 2% human serum albumin by gastric lavage for 21 days.
(3) After 7 days, 14 days and 21 days of continuous supplementation of the bifidobacterium triple viable bacteria preparation, transplantation treatment of adipose-derived mesenchymal stem cells was started, and in the scheme of the invention, the adipose-derived mesenchymal stem cells were used for local targeted injection into ovaries, the final concentration of the mesenchymal stem cells was 5×10 6/ml, and the mesenchymal stem cells were resuspended in physiological saline containing 0.05% sodium hyaluronate and 2% human serum albumin and injected at a dose of 5×10 6 cells/kg. Each treatment group was injected 1 time every 3 days for a total of 3 times. On day 2 after the last 1 injection, all groups of mice were anesthetized with isoflurane, serum was collected by heart blood collection, and levels of E2, FSH and LH in serum were measured by ELISA. The results are shown in FIG. 10, and after supplementing the bifidobacterium triple viable bacteria preparation, the adipose tissue mesenchymal stem cell transplantation treatment is performed, so that the E2 level of the premature ovarian failure mice can be promoted, and the expression of LH and FSH can be reduced. In addition, the bifidobacterium triple viable bacteria preparation can promote the therapeutic effect of stem cell transplantation, and the effect is shown that the serum E2 level can be continuously increased in a group of 14 days (the group of 14 days is compared with a control group).
(4) The ovaries of the mice were collected, weighed wet, paraffin sections were prepared, stained for conventional HE, and morphological observations of ovarian tissue and follicular counts were observed. As shown in fig. 11, the combined treatment of adipose mesenchymal stem cells and bifidobacterium triple viable bacteria preparation can significantly increase the follicular number of mice with premature ovarian failure, especially in the 14-day and 21-day groups. Therefore, the supplementation of the bifidobacterium triple viable bacteria preparation can promote the effectiveness of the adipose mesenchymal stem cell transplantation in treating premature ovarian failure.
In conclusion, the therapeutic effectiveness of the mesenchymal stem cell and bifidobacterium triple viable bacteria preparation in premature ovarian failure is considered. The scheme has little ethical dispute and low immunogenicity, and is an ideal treatment means. The stem cells are directly injected into the ovary parts in a targeting way, so that the effective repair of the ovary functions can be ensured, the function ovulation can be promoted, and various symptoms of climacteric period can be relieved; compared with the existing intravenous infusion intervention way, the adverse reactions of intravenous thrombolysis, such as cell embolism, immune reaction and the like, and abdominal and local wounds caused by intervention treatment are avoided, and side effects and damages caused by treatment of patients can be effectively prevented.
The present embodiment is only for explanation of the present invention and is not to be construed as limiting the present invention, and modifications to the present embodiment, which may not creatively contribute to the present invention as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present invention.
Claims (10)
1. The application of the probiotic combined with the mesenchymal stem cells in preparing the ovarian anti-aging medicament is characterized in that the concentration of the bifidobacterium triple viable bacteria capsule or the composite lactobacillus enteric capsule in the probiotic preparation is 1g/ml; the final concentration of the mesenchymal stem cells in the cell slow-release liquid is 5 multiplied by 10 6/ml.
2. The use according to claim 1, wherein the pH of the probiotic preparation is in the range of 6.5 to 7.0.
3. The use according to claim 2, wherein the preparation method of the probiotic preparation comprises the following steps: under the aseptic condition, weighing 100mg of sodium hyaluronate and 4g of human serum albumin, adding into 200ml of aseptic medical physiological saline to obtain a preparation slow-release liquid, sterilizing a matrix, and filtering and sterilizing by adopting a 0.22 mu m filter head; removing the bifidobacterium triple viable bacteria capsule or the composite lactobacillus enteric capsule respectively, taking out the content, suspending in the preparation slow release liquid, and subpackaging into a disposable prefilled syringe.
4. The use according to any one of claims 1 to 3, wherein the probiotic preparation is administered orally at a dose of 0.5g/10g body weight for a duration of more than 14 days.
5. The use according to claim 1, wherein the mesenchymal stem cells are adipose tissue mesenchymal stem cells or dermal mesenchymal stem cells.
6. The use according to claim 5, wherein the mesenchymal stem cells are cultured in a medium of low-sugar DMEM containing 15% fetal bovine serum at a seed density of 5 x 10 3/cm 2, and the ratio of DMEM, inactivated fetal bovine serum and DMSO in the frozen stock solution is 5:4:1.
7. The use according to claim 6, wherein the mesenchymal stem cells are isolated and cultured using DPBS as tissue washing liquid, 1% of diabody (streptomycin and gentamicin), 1 xDPBS% of collagenase (type I) as tissue digestion liquid, and 1 xDPBS% of trypsin as cell digestion liquid of 0.05%.
8. The use according to claim 7, wherein the mesenchymal stem cells are transferred to passage 4 in stem cell medium, FACSbuffer is 1xPBS containing 2% fbs, 0.5% paraformaldehyde and 0.1% nan 3.
9. The use according to claim 5, wherein the adipose-derived differentiation-inducing medium components of the mesenchymal stem cells are low-sugar DMEM, 10% dbs, 1 μΜ dexamethasone, 0.5mm 3-isobutyl-1-methylxanthine, 10 μΜ bovine insulin, 60 μΜ indomethacin and 1% glutamine; or the osteogenic differentiation induction medium is composed of low sugar DMEM, 10% dbs, 1 μm dexamethasone, 50 μm ascorbic acid, 10mM sodium beta-glycerophosphate and 1% glutamine.
10. The use according to claim 5, wherein the adipose tissue mesenchymal stem cells are derived from the groin and/or abdomen of an animal; the dermal tissue is derived from the skin of the back of an animal.
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