CN113041258A

CN113041258A - Biological composition for repairing intrauterine adhesion and preparation method thereof

Info

Publication number: CN113041258A
Application number: CN202110331876.4A
Authority: CN
Inventors: 赵云霞; 林树
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-03-29
Filing date: 2021-03-29
Publication date: 2021-06-29

Abstract

A biological composition for repairing intrauterine adhesions comprising ShakeGel^TM3D and autologous ADSCs identified by flow, adipogenic induced differentiation, osteogenic induced differentiation, chondrogenic induced differentiation, in a volume of 1:1, and mixing uniformly. The preparation method of the biological composition comprises the following steps: extracting autologous ADSCs from the sample, identifying the ADSCs by flow, adipogenic induced differentiation, osteogenic induced differentiation and chondrogenic induced differentiation, and mixing with Shakegel^TM3D combined culture. The invention Shakegel^TMThe 3D can create a feasible three-dimensional growth environment for autologous ADSCs, Shakegel^TMCo-transplantation of 3D with autologous ADSCs compositions promotes damaged endometrium by increasing BMP7-Smad5 signalingThe recovery of the tissue thickens the endometrium, increases the number of glands and reduces the fibrosis, thereby recovering the fertility of the mouse with the conglutinated uterine cavity.

Description

Biological composition for repairing intrauterine adhesion and preparation method thereof

Technical Field

The invention relates to the technical field of automobile biology, in particular to a biological composition for repairing intrauterine adhesion and a preparation method thereof.

Background

Intrauterine adhesion (IUA) is the partial or total occlusion of the uterine cavity and/or cervix due to damage to the underlying layer of the endometrial lining, which is also the most common cause of uterine infertility. There are many hypotheses about the pathogenesis of IUA, including fibrosis proliferation theory, neuroreflex theory, abnormal stem cell differentiation, uterine cavity microenvironment change and fibrosis, abnormal signal pathway regulation, promotion of inflammatory response, etc. Since the pathological features of IUA are related to endometrial fibrosis, the theory of fibrotic hyperplasia is the most widely studied hypothesis at present. Intrauterine adhesions are a serious complication of uterine surgery and can lead to thousands of health problems in the female reproductive system, such as abnormal menstruation, recurrent miscarriage, periodic abdominal pain, infertility and pregnancy related complications. Thus, reestablishing a normal uterine cavity and restoring uterine function are the goal of IUA treatment.

The traditional treatment method of intrauterine adhesion is the application of various auxiliary treatments after the adhesion is removed under a hysteroscope. However, these anti-adhesion strategies have several drawbacks and deficiencies, such as recurrence of secondary surgery, limited isolated area, induction of intrauterine inflammatory response, difficulty in endometrial regeneration, etc., which compel continued research into intrauterine adhesions and the exploration of new treatment regimens. Therefore, the biological composition for repairing the intrauterine adhesion and the preparation method thereof are provided.

Disclosure of Invention

The invention provides a biological composition for repairing intrauterine adhesion and a preparation method thereof, and provides a good treatment medicament for intrauterine adhesion patients.

The invention adopts the following technical scheme:

a biological composition for repairing intrauterine adhesions comprising ShakeGel^TM3D gel liquid and autologous adipose-derived mesenchymal stem cells identified by flow, adipogenic induced differentiation, osteogenic induced differentiation and chondrogenic induced differentiation are mixed according to the volume of 1:1, and mixing uniformly.

Further, the autologous adipose-derived mesenchymal stem cells are extracted from white adipose tissues of the groin of the animal.

The invention also provides a preparation method of the biological composition for repairing intrauterine adhesion, which comprises the following steps:

(1) extracting and culturing autologous fat of miceMesenchymal stem cells; (2) carrying out osteogenic induction, adipogenic induction differentiation and chondrogenic induction differentiation treatment on the adipose-derived mesenchymal stem cells in the step (1); (3) taking the culture solution of the adipose-derived mesenchymal stem cells treated in the step (2) for flow cytometry analysis; (4) adding GFP chronic virus culture medium mixed liquor into the adipose tissue-derived mesenchymal stem cells in a good state for continuous culture; (5) shakegel^TMAnd (3) mixing the 3D gel solution with the autologous adipose-derived stem cells obtained by the culture in the step (4) according to the volume ratio of 1:1, centrifuging and mixing uniformly to prepare the biological composition.

Further, the extraction and culture process of the mouse autologous adipose-derived mesenchymal stem cells in the step (1) is as follows: 1) stripping and collecting 3-4 weeks old female mouse inguinal adipose tissues, and putting the tissues into a centrifuge tube filled with KRP buffer preheated to 37 ℃ in advance; 2) adipose tissue was transferred from KRP buffer to cell culture dishes, washed twice with pre-warmed PBS, and concentrated in 4:1, putting a new KRP buffer, and cutting adipose tissues into small pieces; 3) sucking the upper fat suspended matter, adding into a new sterile test tube, and adding collagenase II with the equal volume concentration of 0.75 mg/ml; 4) sealing the sterile test tube, placing the sterile test tube into a water bath at 37 ℃, slowly shaking for 45 minutes to digest the fat suspended matter until a separated cell layer is suspended at the uppermost layer of the liquid and separated from other precipitated tissue fragments, adding preheated PBS (phosphate buffer solution), neutralizing collagenase II, and stopping digestion; 5) centrifugally separating the adipose-derived stem cell sediment, and sucking and removing the upper mixed solution; 6) adding erythrocyte lysate for resuspending the precipitate, lysing for 10min, centrifuging, discarding supernatant, and leaving adipose-derived stem cell precipitate; 7) the adipose stem cell culture medium resuspended cell pellets were plated in cell culture dishes.

Further, the process of inducing the adipose tissue-derived mesenchymal stem cells into bone in the step (2) is as follows: 1) placing the primary extracted adipose-derived mesenchymal stem cells at 37 deg.C and 5% CO₂When the cell fusion degree reaches 80-90%, digesting with 0.25% Trypsin-0.04% EDTA; 2) inoculating the digested mouse adipose-derived stromal stem cells into a six-hole plate which is coated with 0.1% gelatin in advance, and adding a complete culture medium into each hole; 3) then, the cells were incubated at 37 ℃ with 5% CO₂The culture is carried out in the incubator (2),when the cell fusion degree reaches 60-70%, sucking away the complete culture medium in the hole, and adding 2mL of C57BL/6 mouse adipose-derived stromal stem cell osteogenic induced differentiation complete culture medium into a six-hole plate; 4) replacing a fresh C57BL/6 mouse adipose-derived mesenchymal stem cell osteogenic induced differentiation complete culture medium every 3 days; 5) after 2-4 weeks of induction, alizarin red staining was performed, and the effect of bone formation staining was observed under a microscope.

Further, the adipogenic induction differentiation process of the adipose-derived mesenchymal stem cells in the step (2) is as follows: 1) placing mouse adipose-derived mesenchymal stem cells at 37 deg.C and 5% CO₂When the cell fusion degree reaches 80-90%, digesting with 0.25% Trypsin-0.04% EDTA; 2) inoculating the digested mouse adipose-derived stromal stem cells into a six-hole plate which is coated with 0.1% gelatin in advance, and adding a complete culture medium into each hole; 3) then, the cells were incubated at 37 ℃ with 5% CO₂Culturing in the incubator, changing the culture solution every three days until the cell fusion degree reaches 100% or the cell fusion degree is over-fused, sucking the complete culture medium of the mesenchymal stem cells, and adding the fat-forming induced differentiation culture medium A solution of the mouse adipose mesenchymal stem cells into a six-hole plate; 4) after 3 days of induction, sucking away the solution A in a six-hole plate, and adding 2mL of solution B of a C57BL/6 mouse adipose-derived mesenchymal stem cell adipogenic induction differentiation culture medium; 5) after 24h, sucking away the solution B, replacing the solution A for induction, after the solution A and the solution B alternately act for 3-5 times, continuing to maintain and culture for 4-7 days by using the solution B until lipid droplets become large enough and round, and replacing with fresh solution B every 2-3 days during the period of maintaining and culturing by using the solution B; 6) and after the lipogenic induced differentiation is finished, alizarin red is adopted for dyeing, and the osteogenic dyeing effect is observed under a microscope.

Further, the chondrogenic induction differentiation process of the adipose-derived mesenchymal stem cells of the step (2) is as follows: 1) mixing 3-4X 10⁵Transferring the individual cells into a centrifuge tube, centrifuging for 4min, 2) sucking supernatant, adding 0.5 mL of premix, resuspending the precipitate obtained by the centrifugation in the previous step to clean the adipose-derived mesenchymal stem cells of the mouse, and centrifuging for 5min at 150 g at room temperature; 3) repeating the step 2), and cleaning the cells again; 4) resuspending the precipitate obtained in the last step by using 0.5 mL of complete culture medium for chondrogenic induced differentiation of mouse adipose-derived mesenchymal stem cells; 5) centrifuging at room temperature for 5min, and unscrewing the centrifugal tube cover to facilitate gas exchangePlacing the mixture in an incubator at 37 ℃ and 5% CO2 for culture; 6) when the cell clusters are gathered, the bottom of the centrifugal tube is flicked to separate the cartilage balls from the tube bottom and suspend the cartilage balls in the liquid; 7) replacing fresh chondrogenic induction differentiation complete culture medium every 2-3 days by calculation from the beginning of inoculation; 8) after the liquid is changed, flicking the bottom of the centrifuge tube to separate the cartilage ball from the tube bottom and suspend the cartilage ball in the liquid, slightly unscrewing the centrifuge tube cover, and putting the centrifuge tube cover into an incubator at 37 ℃ and 5% CO2 for continuous induction culture; 9) after 21-28 days of continuous induction, the chondrocytes were formalin-fixed and paraffin-embedded, and finally stained with alistin blue, and the staining effect of alistin blue was observed under a microscope.

Further, the specific process of step (3) is as follows: 1) sucking a supernatant culture solution of the adipose-derived mesenchymal stem cells, and adding preheated PBS for washing; 2) adding 1ml trypsin, shaking to make the cells totally infiltrate, and digesting for 2min in a 5% CO2 cell culture box at 37 deg.C; 3) adding an adipose-derived mesenchymal stem cell culture medium to terminate the reaction, fully transferring the cells to a centrifuge tube, and centrifuging and precipitating the cells; 4) removing supernatant, adding 1ml adipose-derived mesenchymal stem cell culture medium, and counting after resuspending cells; 5) take 5 x10⁵Adding the adipose-derived mesenchymal stem cells into an EP tube, and centrifuging and precipitating the cells; 6) the supernatant was aspirated off, then 200. mu.l of Facs buffer was added for washing, and centrifugation was performed; 7) the supernatant was aspirated off, 50. mu.l of cell surface antibody staining solution was added, and the mixture was iced for 30 min; 8) Adding 200 μ l Facs buffer for washing, centrifuging, removing supernatant, and repeating twice; 9) 200 μ l of Facs buffer resuspended cells, protected from light and ready for loading for assay.

The specific process of the step (4) is as follows: 1) well-conditioned adipose-derived mesenchymal stem cells were seeded in a 24-well plate at 6 × 10⁴cells/well density is plated, 500ul of culture medium is added into each well, and the fusion rate is ensured to reach 30% -40% when the next day is infected with virus; 2) the next day, cells grew to 30% -40%. The original medium was aspirated off according to: cell number × MOI value/virus stock titer × 103= virus infection amount (UI) to dilute the virus to 2ml while adding 5 μ g/ml polybren, and continuing the culture after adding the virus culture medium mixture; 3) after 8 h of viral infection, the medium was replaced with fresh medium, followed by every other intervalChanging the liquid once in 48 hours; 4) cells were observed under a fluorescent microscope 72h after infection with the virus to determine the efficiency of cell infection.

Further, the specific process of the step (5) is as follows: 1) uniformly mixing the gel liquid and the cell suspension in a centrifugal tube according to the volume ratio of 1:1, uniformly blowing and beating for a plurality of times, slightly and rapidly shaking on a vortex instrument for 1s to fully mix the gel liquid and the cell suspension, and repeatedly shaking for 1-2 times; 2) wetting each hole of the cell culture plate by a proper amount of PBS, discarding the wetted hole, sucking the fully and uniformly mixed cell gel mixture into the hole of the plate by using a pipette gun, and slightly shaking to uniformly spread the gel; 3) Incubating the cell plate in a constant temperature incubator at 37 ℃ for 5-10min to allow the cell gel mixture to form a gel; 4) after the gel incubation is finished, slightly adding a culture solution with a proper volume along the hole wall, and continuously placing the mixture in an incubator for incubation so as to ensure that the cells are fully attached to the gel; 5) after 24h of incubation, about 1/2 of the culture broth was gently aspirated off with a pipette and an equal amount of fresh culture broth was added.

As can be seen from the above description of the present invention, compared with the prior art, the present invention has the following advantages:

the autologous adipose-derived mesenchymal stem cells (ADSCs) of the invention express CD105(99.1%), CD29(99.6%), CD73(98.9%), CD34 (0.46%), CD45 (3.26%), and the cells can induce adipogenesis, osteogenesis and chondrogenesis, Shakegel^TMThe 3D gel liquid can maintain the functions of the autologous ADSCs and create a feasible three-dimensional growth environment for the autologous adipose-derived mesenchymal stem cells. Shakegel^TMThe combined transplantation of 3D and autologous ADSCs composition promotes the recovery of damaged endometrial tissue by increasing BMP7-Smad5 signaling, thickening the endometrium, increasing the number of glands, reducing fibrosis, and thus restoring fertility to mice with uterine cavity adhesions.

Detailed Description

The following describes specific embodiments of the present invention. Numerous details are set forth below in order to provide a thorough understanding of the present invention, but it will be apparent to those skilled in the art that the present invention may be practiced without these details. Well-known components, methods and processes are not described in detail below.

Organism for repairing intrauterine adhesionComposition comprising ShakeGel^TM3D gel liquid and autologous adipose-derived mesenchymal stem cells identified by flow, adipogenic induced differentiation, osteogenic induced differentiation and chondrogenic induced differentiation are mixed according to the volume of 1:1, and mixing uniformly.

The preparation method of the biological composition for repairing intrauterine adhesion comprises the following steps:

(1) extracting and culturing autologous adipose-derived mesenchymal stem cells of the mice; (2) carrying out osteogenic induction, adipogenic induction differentiation and chondrogenic induction differentiation treatment on the adipose-derived mesenchymal stem cells in the step (1); (3) taking the culture solution of the adipose-derived mesenchymal stem cells treated in the step (2) for flow cytometry analysis; (4) adding GFP chronic virus culture medium mixed liquor into the adipose tissue-derived mesenchymal stem cells in a good state for continuous culture; (5) shakegel^TMAnd (3) mixing the 3D gel solution with the autologous adipose-derived stem cells obtained by the culture in the step (4) according to the volume ratio of 1:1, centrifuging and mixing uniformly to prepare the biological composition.

1. Extraction and culture of mouse autologous ADSCs

1) The inguinal adipose tissues of 3-4 week-old female C57BL/6 mice were dissected and collected, taking care that the dissected adipose tissues were to exclude animal hair, blood vessels and connective tissues. The separated adipose tissues were placed in a 15 ml centrifuge tube with KRP buffer pre-heated to 37 ℃. 2) Adipose tissue was transferred from the KRP buffer to a 10 cm cell culture dish and washed twice with pre-warmed PBS to further remove visible vascular and connective tissue. 3) Putting the washed adipose tissues into a new 10ml KRP buffer, wherein the volume ratio of the fat to the KRP buffer is 4:1, and cutting the adipose tissues into small pieces by using a clean scissors, so that the adipose tissues can be fully contacted with and digested by digestive juice. 4) The upper fat suspension was aspirated by 1ml rubber tip, a new 15 ml sterile tube was added, and an equal volume of collagenase II was added to the newly obtained suspension to a final concentration of 0.75 mg/ml. 5) The test tube cover is screwed down, the sealing film is sealed and then put into a water bath with the temperature of 37 ℃ to slowly shake for 45 minutes, and the digestion condition is observed every 10 minutes. Digestion until the separated cell layer is suspended in the uppermost layer of liquid separated from other sedimented tissue debris. 6) The reaction was stopped by adding pre-warmed 1 x PBS to a total volume of 14 ml. Lightly, theThe tube was inverted, the collagenase was neutralized sufficiently, and digestion was terminated. 7) Centrifuging for 5min, separating adipose-derived stem cell precipitate, and removing upper layer mixed solution. 8) The pellet was resuspended in 10ml of erythrocyte lysate and lysed for 10 min. 9) Centrifuge for 5min, and discard the supernatant by aspiration, leaving a pellet of adipose stem cells. 10) Adipose-derived stem cell culture medium resuspend the cell pellet at 6X 10⁴/cm²The ratio of (A) to (B) was spread on a 10 cm cell culture dish.

2.1 osteogenic Induction of adipose mesenchymal Stem cells

(1) Preparation of complete culture medium for osteogenesis induced differentiation

1) Before use, the serum was thawed overnight at 2-8 ℃ until the serum was completely dissolved, and the vial was gently shaken to ensure that the serum was well mixed. 2) The ascorbic acid, sodium beta-glycerophosphate, bisantibody and glutamine were dissolved at room temperature about 30min before preparation, and the reagent tubes were turned upside down gently to ensure uniform mixing of the reagents. 3) Dexamethasone was dissolved at room temperature 10min before use. 4) The open outer wall of each bottle/tube in the kit was wiped with 70% ethanol and left at room temperature for several seconds to evaporate the ethanol. 5) The above bottles/tubes were opened aseptically in a clean bench. 6) Ascorbic acid, beta-sodium glycerophosphate, fetal calf serum special for the C57BL/6 mouse adipose-derived stromal stem cells, diabody and glutamine are all added into a C57BL/6 mouse adipose-derived stromal stem cell osteogenic induced differentiation basal medium. 7) The bottles/tubes were washed aseptically with a small amount of basal medium, and all components were added as intact to the basal medium as possible. 8) Finally, dexamethasone is added into the basal medium, 0.5 mL of the basal medium is absorbed to wash the reagent tube, and the mixture is added into the basal medium together. 9) Operation step 8) is repeated. 10) And (3) lightly shaking the prepared complete culture medium to ensure that the culture medium can be used after being uniformly mixed.

(2) Gelatin coating on the surface of the culture vessel.

1) Adding appropriate amount of 0.1% gelatin into the culture vessel to cover the whole culture vessel. 2) The liquid was shaken up to cover the entire bottom surface of the culture vessel. 3) The culture vessel, which was plated with 0.1% gelatin, was placed on a clean bench for at least 30 min. 4) And removing the gelatin after 30min, and airing the culture vessel to inoculate the cells.

(3) Osteogenic induced differentiation procedure

1) Placing the primary extracted adipose-derived mesenchymal stem cells at 37 ℃ and 5% CO₂Cultured in an incubator. 2) When the cell fusion degree reaches 80-90%, digestion is carried out by using 0.25% Trypsin-0.04% EDTA. 3) Digesting the C57BL/6 mouse adipose-derived mesenchymal stem cells according to the ratio of 2X 10⁴cells/cm²The cell density of (a) was seeded in six-well plates previously coated with 0.1% gelatin, and 2mL of complete medium was added per well. 4) The cells were incubated at 37 ℃ with 5% CO₂The incubator of (2) for cultivation. 5) When the cell fusion degree reaches 60% -70%, carefully sucking away the complete culture medium in the hole, and adding 2mL of C57BL/6 mouse adipose-derived stromal cell osteogenic induced differentiation complete culture medium into a six-hole plate. 6) The complete medium for osteogenic induction and differentiation of the fresh C57BL/6 mouse adipose-derived mesenchymal stem cells is replaced every 3 days (preheated to 37 ℃ before use). 7) After 2-4 weeks of induction, cells were stained with alizarin red, depending on morphological changes and growth.

(4) Alizarin red staining assay

1) After the osteogenic induction differentiation was completed, the medium for complete osteogenic induction differentiation in the six-well plate was aspirated and washed 1 to 2 times with 1 × PBS. 2mL of 4% neutral formaldehyde solution was added to each well and fixed for 30 min. 2) The neutral formaldehyde solution was aspirated and washed 2 times with 1 × PBS. Adding 1mL alizarin red staining solution into each hole for staining for 3-5 min. 3) The alizarin red stain was aspirated and washed 2-3 times with 1 × PBS. 4) The plates were placed under a microscope to observe the effect of osteogenic staining.

2.2 adipogenic induced differentiation of adipose-derived mesenchymal Stem cells

(1) Preparation of adipogenic differentiation induction culture medium A liquid

1) Before use, please place the serum in an environment of 2-8 ℃ to thaw overnight until the serum is completely dissolved, and shake the reagent bottle gently to ensure that the serum is mixed uniformly. 2) Dexamethasone, insulin, IBMX, rosiglitazone, diabesin and glutamine were dissolved at room temperature around 30min before preparation, and the reagent tubes were turned upside down gently to ensure uniform mixing of the reagents. 3) The open outer wall of each bottle/tube in the kit was wiped with 70% ethanol and left at room temperature for several seconds to evaporate the ethanol. 4) In the superclean bench, special fetal calf serum, double antibody and glutamine for the C57BL/6 mouse adipose-derived mesenchymal stem cells are all added into a liquid A basal medium of a C57BL/6 mouse adipose-derived mesenchymal stem cell adipogenic induced differentiation medium. 5) And (4) aseptically sucking a small amount of the A liquid basic culture medium to wash each bottle/tube, and completely adding all components into the A liquid basic culture medium as far as possible. 6) Dexamethasone, insulin, IBMX and rosiglitazone were all added to liquid A basal medium. 7) And (4) aseptically sucking a small amount of the A liquid basic culture medium to wash each bottle/tube, and completely adding all components into the A liquid basic culture medium as far as possible. 8) Operation step 7) is repeated. 9) And (3) lightly shaking the prepared complete culture medium to ensure that the culture medium can be used after being uniformly mixed.

(2) Preparation of adipogenic differentiation induction culture medium B liquid

1) Before use, the serum was thawed overnight at 2-8 ℃ until the serum was completely dissolved, and the vial was gently shaken to ensure that the serum was well mixed. 2) The insulin, the diabase and the glutamine are dissolved at room temperature about 30min before preparation, and the reagent tube is slightly turned upside down to ensure that the reagents are mixed evenly. 3) The open outer wall of each bottle/tube in the kit was wiped with 70% ethanol and left at room temperature for several seconds to evaporate the ethanol. 4) In the superclean bench, special fetal calf serum, double antibody and glutamine for the adipose-derived mesenchymal stem cells of the C57BL/6 mice are all added into a B liquid basal medium of a mesenchymal stem cell adipogenic differentiation culture medium. 5) And (4) aseptically sucking a small amount of the B liquid basic culture medium to wash each bottle/tube, and adding all the components into the B liquid basic culture medium completely as far as possible. 6) Insulin was added to liquid B basal medium. 7) And (4) aseptically sucking a small amount of the liquid B basal medium to wash the reagent tube, and completely adding all the components into the liquid B basal medium as far as possible. 8) Operation step 7) is repeated. 9) And (3) lightly shaking the prepared complete culture medium to ensure that the culture medium can be used after being uniformly mixed.

(3) Adipogenic induced differentiation procedure

1) Placing C57BL/6 mouse adipose-derived mesenchymal stem cells at 37 deg.C with 5% CO₂Cultured in an incubator. 2) When the cell fusion degree reaches 80% -90%, digesting with 0.25% Trypsin-0.04% EDTA. 3) The digested mesenchymal stem cells are arranged according to 2 x10⁴ cells/cm²The cell density of (2) was seeded in six-well plates, 2 per wellmL complete medium. 4) The cells were incubated at 37 ℃ with 5% CO₂The incubator of (2) for cultivation. 5) Fluid was changed every third day until cell confluence reached 100% or over-confluency. 6) Carefully sucking the complete culture medium of the mesenchymal stem cells, and adding a C57BL/6 mouse adipose mesenchymal stem cell adipogenic induction differentiation culture medium A liquid into a six-well plate. 7) After 3 days of induction, the solution A in the six-well plate is sucked away, and 2mL of solution B of the culture medium for inducing differentiation by adipogenic induction of the C57BL/6 mouse adipose-derived mesenchymal stem cells is added. 8) After 24h, the solution B was aspirated and replaced with solution A for induction. 9) After the alternating action of solution A and solution B for 3-5 times (12-20 days), the culture is continued for 4-7 days with solution B until the lipid droplets become large enough and round. During the maintenance culture period of the solution B, fresh solution B is required to be replaced every 2-3 days.

(4) Oil Red O staining analysis

1) After the lipogenesis induction differentiation is finished, the adipose-derived mesenchymal stem cell lipogenesis induction differentiation culture medium in the six-hole plate is sucked away, and the medium is washed 1-2 times by 1 XPBS. 2mL of 4% neutral formaldehyde solution was added to each well and fixed for 30 min. 2) The neutral formaldehyde solution was aspirated and washed 2 times with 1 × PBS. Adding 1mL of oil red O dye working solution into each hole for dyeing for 30min (working solution preparation method: oil red O stock solution: distilled water =3:2, mixing uniformly and filtering with neutral filter paper). 3) The oil red O stain was aspirated and washed 2-3 times with 1 XPBS. 4) The plates were placed under a microscope to observe the effect of adipogenic staining.

2.3 chondrogenic induced differentiation of adipose-derived mesenchymal Stem cells

(1) Preparation of complete culture medium for chondrogenic induced differentiation

1) The open outer wall of each bottle/tube in the kit (dexamethasone, ascorbic acid, ITS additive, sodium pyruvate, proline, C57BL/6 mouse adipose-derived mesenchymal stem cell chondrogenesis induction differentiation basal medium) is wiped by 70% ethanol, and the ethanol is volatilized after the kit is placed at room temperature for a plurality of seconds. 2) The dexamethasone, the ascorbic acid, the ITS additive, the sodium pyruvate and the proline are all added into a basic culture medium for inducing and differentiating the cartilage of the C57BL/6 mouse adipose-derived mesenchymal stem cells in an ultra-clean workbench to prepare a premixed solution of a complete culture medium for inducing and differentiating the cartilage. 3) And (3) aseptically sucking a small amount of chondrogenesis induced differentiation basic culture medium to wash each bottle/tube, completely adding all components into the basic culture medium as far as possible, and possibly influencing the product performance by a small amount of residues. 4) And (4) lightly shaking the prepared premixed liquid, and uniformly mixing the premixed liquid and the premixed liquid for use.

(2) Dilution of TGF-beta 3

1) And subpackaging a small amount of TGF-beta 3 into a low temperature resistant sample storage tube, and storing at the temperature of-20 ℃ or lower and finishing using within 6 months. 2) According to the proportion (10 mu L of TGF-beta 3 is added into 1mL of the premixed solution), the TGF-beta 3 with the required dosage for the experiment is absorbed and added into the premixed solution with the corresponding volume to prepare a complete culture medium for the cartilage induced differentiation, and the reagent tubes are slightly turned upside down to ensure that the reagents are uniformly mixed.

(3) Chondrogenic induced differentiation procedure

1) Cells after routine digestion need to be counted before chondrogenic differentiation experiments are performed. 2) Mixing 3-4X 10⁵The cells were transferred to a 15 mL centrifuge tube and centrifuged at 250g for 4 min. 3) The supernatant was aspirated off, 0.5 mL of the premix was added, and the pellet from the previous centrifugation step was resuspended to wash C57BL/6 mouse adipose-derived mesenchymal stem cells and centrifuged at 150 g for 5min at room temperature. 4) Repeat step 3), again washing the cells. 5) And (3) resuspending the precipitate obtained in the previous step in 0.5 mL of complete chondrogenesis induction differentiation culture medium of C57BL/6 mouse adipose-derived mesenchymal stem cells. 6) Centrifuge at 150 g for 5min at room temperature. 7) The centrifuge tube cap was unscrewed to facilitate gas exchange and placed at 37 ℃ with 5% CO₂Cultured in an incubator. 8) When the cell mass is gathered (generally 24h or 48 h later, actually according to the cell growth condition), the bottom of the centrifugal tube is flicked to separate the cartilage ball from the tube bottom and suspend the cartilage ball in the liquid. 9) Cells were replaced with fresh chondrogenic differentiation complete medium every 2-3 d, approximately 0.5 mL chondrogenic differentiation complete medium per tube, calculated from the start of inoculation. 10) After the liquid is changed, the bottom of the centrifuge tube is flicked to separate the cartilage ball from the tube bottom and suspend the cartilage ball in the liquid. Slightly unscrewing the centrifugal tube cover, placing at 37 deg.C and 5% CO₂The induction culture is continued in the incubator. 11) Typically, after 21-28 days of continuous induction, the chondrocytes can be formalin-fixed and paraffin-embedded and finally stained with Alisinum blue.

(4) Alisinblue staining analysis

1) The cartilage ball was embedded in paraffin and sectioned. 2) And (3) dyeing: a) dewaxing and dewatering; b) dyeing with Alisin blue dye liquor for 30 min; c) washing with tap water for 2 min; d) distilled water was washed 1 time. 3) The staining effect of Alisin blue was observed under a microscope, and the staining portion of Alisin blue showed endogenic acid mucopolysaccharides in cartilage tissue.

3. Flow cytometry analysis

1) And (3) absorbing and removing the supernatant culture solution of the adipose tissue-derived mesenchymal stem cells, adding preheated 2ml of 1X PBS, and washing. 2) Adding 1ml of trypsin, shaking all around to infiltrate all cells, and culturing at 37 deg.C with 5% CO₂And digesting in a cell culture box for 2 min. 3) The reaction was terminated by addition of adipose mesenchymal stem cell culture medium and the cells were transferred well to a 15 ml centrifuge tube. 4) Centrifuging for 5min, and precipitating cells. 5) And (4) removing the supernatant, adding 1ml of adipose mesenchymal stem cell culture medium, and counting after resuspending the cells. 6) Take 5 x10⁵Adipose-derived mesenchymal stem cells were added to a 1.5 ml EP tube. The cells were pelleted by centrifugation at 300 g for 5min. 7) The supernatant was aspirated and washed with 200. mu.l of Facs buffer, 300 g, 5min centrifugation. 8) The supernatant was discarded, and 50. mu.l of cell surface antibody staining solution was added thereto and the mixture was frozen for 30 min. 9) Add 200. mu.l Facs buffer for washing, centrifuge for 5min, aspirate the supernatant and repeat twice. 10) 200 μ l of Facs buffer resuspended cells, protected from light and ready for loading for assay.

4. GFP lentivirus infected adipose mesenchymal stem cells

1) Well-conditioned adipose-derived mesenchymal stem cells were seeded in a 24-well plate at 6 × 10⁴cells/well density plated with 500ul of medium per well. Ensuring that the fusion rate reaches 30-40% when the virus is infected the next day. 2) The next day, cells grew to 30% -40%, the original medium was aspirated off according to: cell number MOI value/Virus stock titer 10³= viral infection amount (UI) dilution virus to 2ml, at the same time add 5 u g/ml polybren, add virus culture medium mixture and then continue the culture. 3) After 8 h of virus infection, the medium was replaced with fresh medium, followed by changing the medium every 48 hours. 4) Cells were observed under a fluorescent microscope 72h after infection with the virus to determine the efficiency of cell infection. These cells were then injected into the uterus for localization experiments.

5. Preparation of ShakeGelTM3D binding mouse autologous adipose-derived stem cells

1) Mixing the gel solution and the cell suspension in a centrifugal tube according to a volume ratio of 1:1 (strictly according to the sequence of adding the gel first and then adding the cell suspension), uniformly blowing and beating for a plurality of times, slightly and rapidly shaking on a vortex instrument (vortex) for 1s to fully mix, and repeatedly shaking for 1-2 times. 2) Wetting each hole of the cell culture plate by a proper amount of PBS, discarding the wetted hole, sucking the fully and uniformly mixed cell gel mixture into the hole of the plate by using a pipette gun, and slightly shaking to uniformly spread the gel. In addition, the prepared cell gel mixture can also be directly dripped on the surface of any sterile cell culture vessel to prepare the micro-tissue. 3) The cell plate is placed in a constant temperature incubator at 37 ℃ and incubated for 5-10min, so that the cell gel mixture forms gel. 4) After the gel incubation is finished, a proper volume of culture solution is gently added along the hole wall, and the culture solution is continuously placed in an incubator for incubation so as to ensure that the cells are fully attached to the gel. 5) After 24h of incubation, about 1/2 of the culture broth was gently aspirated off with a pipette and an equal amount of fresh culture broth was added. After that, the liquid can be changed normally, and the follow-up experiment can be carried out.

6. Cell counting kit-8 Cell activity detection

1) The CCK8 method is generally used as a colorimetric detection method for detecting the number of living cells in cell proliferation or cytotoxicity experiments. Mitochondrial dehydrogenases within cells reduced WST-8 to orange-yellow, highly water-soluble formazan in the reagent, the number of formazan being proportional to the number of living cells. 2) 100 μ l CCK8 dye was combined with 900 μ l DMEM F12 medium and mixed well. 3) The primary cell culture medium was aspirated along the side walls, 200. mu.l of CCK8 dye mix was added, and shaken gently back and forth and left and right to help the cells and dye come into full contact, taking care not to generate air bubbles and not to disrupt the cell or cell-gel structure. 4) 37 ℃ and 5% CO₂The cell culture box with saturated humidity is incubated for 4 hours, 100 mul of reaction staining solution is taken to a 96-well plate, and the generation of bubbles in the well is avoided, otherwise, OD reading is influenced, and experiment errors are caused. 5) And measuring the absorbance at 450 nm by using a microplate reader.

The main reagents are configured as follows:

KRP buffer solution: adding CaCl₂(0.0222g)、HEPES(0.5956g)、Na₂HPO₄·12H₂O(0.04296g)、MgSO₄(0.0288g)、NaH₂PO₄·12H₂O (0.0128g), NaCl (1.404g) and KCl (0.08944g) were dissolved in double distilled water, and the solution was filtered through a 0.22 μm filter, and then filtered through a 0.45 μm filter at a ratio of 50ml of the first-step solution to BSA (1.5g) before use.

Preparation of collagenase II: the mixture was mixed according to the proportions of DEME HIGH glucose (10ml), collagen II (0.015g) and BSA (0.1 g).

Adipose-derived stem cell culture medium: DMEM F12+0.5% NaHCO₃+2% P/S+10% FBS。

The above is a specific test case of the mouse model using the biological composition of the present invention.

1. Establishment of IUA model

When adipose-derived mesenchymal stem cells grow to the second generation, namely, when the mouse grows for about 6-7 weeks, 1% pentobarbital sodium is given according to the dose of 80mg/kg for intraperitoneal injection anesthesia, after anesthesia, the mouse is fixed on an operation plate in a supine position, the lower abdomen is prepared, 75% alcohol is sterilized, a sterile drape is laid, the lower abdomen is incised in the middle, the distance between the lower abdomen and the pubic symphysis is about 0.5cm, the incision length is about 1.5cm, and all layers of the skin and the abdominal wall are incised once to enter the abdominal cavity to expose the uterus. 50 mice were divided into 5 groups: in sham group (sham group, n =10), uterus was not treated, but the abdominal cavity was opened, followed by layer-by-layer suturing, and observation was performed until the rats were awake; the PBS model group (PBS group, n =10) is used for repeatedly scratching the 1/3 parts of the middle and lower uterine segments at the two sides by a 24G needle for 20 times, then suturing layer by layer, observing until the mouse revives, opening the abdomen after 7d of modeling, injecting 10ul PBS into each of the two uterine horns of the mouse, closing the abdomen layer by layer, and observing until the mouse revives; the autologous ADSCs treatment group (ADSCs group, n =10) uses a 24G needle to scrape at 1/3 positions of the middle and lower sections of the uterus on both sides repeatedly for 20 times, then sutures layer by layer, observation is carried out until the mouse revives, 7 days after modeling, anesthesia and open abdomen operation is carried out again, and according to the mouse ear tag, each lateral uterus is injected with the third generation autologous ADSCs (5 x 10) with corresponding ear tag numbers along the muscle wall respectively (5 x 10)⁶Suspended in 10ul PBS), closed layer by layer, and observed until the mouse revives; shakegel^TM3D model group (gel group, n =10), bilaterally with 24G needleRepeatedly scratching the lower uterine segment 1/3 for 20 times, suturing layer by layer, observing until the mouse revives, performing anesthesia and open abdomen operation 7 days after molding, and injecting 10ul Shakegel into each of two uterine horns^TM3D, closing layer by layer, and observing and waiting for the mouse to wake up; ADSCs combined Shakegel^TM3D treatment group (ADSCs + Gel group, n =10), repeatedly scratching with 24G needle at 1/3 of bilateral uterus inferior and middle segments for 20 times, suturing layer by layer, observing, waiting for mouse reviving, molding for 7 days, opening abdomen again, and combining third generation ADSCs with Shakegel^TM3D (5*10⁶Suspended in 10ul Shakegel^TMIn 3D) injecting the mouse ear tags into the womb at two sides respectively, closing the abdomen layer by layer, and observing to wait for the mouse to revive. After 7 days of treatment in each group, 5 mice were randomly selected from each group, anesthetized with 80mg/kg of 1% sodium pentobarbital, opened to the abdomen, uterine tissue specimens were collected, one part of the specimens was placed in 4% formaldehyde for paraffin sectioning, the other part of the specimens was placed in liquid nitrogen, and then stored at-80 ℃ for qRT-PCR experiments. In addition, 5 mice were left for the mating experiments per group, by following the procedure as described for C57 male 2: 1, combining the two cages, marking the vaginal suppository appearing in female mice as the first day of conception, 14 days of conception, giving 80mg/kg of 1% sodium pentobarbital, cutting the abdomen, observing and recording the number of the uterine horn fetal mice.

2. Uterine tissue paraffin section HE staining

1) Mouse uterus tissue is fixed, embedded in paraffin and sliced. 2) Dewaxing in xylene (I) was carried out for 5min. 3) Fresh xylene (II) was replaced and dewaxing was again carried out for 5min. 4) And 5min with absolute ethyl alcohol. 5)95% ethanol for 2 min. 6)80% ethanol for 2 min. 7)70% ethanol for 2 min. 8) Distilled water for 2 min. 9) Staining with hematoxylin staining solution for 5min. 10) The differentiation fluid was differentiated for 30 s. 11) Soaking in tap water for 15 min. 12) Placing in eosin dye solution for 1 min. 13)95% ethanol (I) 2 s. 14)95% ethanol (II) 2 s. 15) 100% ethanol (I) 2 s. 16) 100% ethanol (II) for 1 min. 17) Xyllithine carbolic acid (3: 1) for 1 min. 18) Xylene (I) for 1 min. 19) Xylene (II) for 1 min. 20) Air-drying, adding a little neutral gum, sealing with a cover glass, air-drying, and taking a picture under microscope. 21) Image pro plus6.0 was used for analysis using Image analysis software to calculate endometrial thickness and number of uterine glands.

3. Masson trichrome stain

1) The slices were dewaxed conventionally to water. 2) Staining with prepared Weigert hematoxylin staining solution for 5min, 3) differentiating with acidic ethanol differentiation solution for 5s, and washing with water. 4) Returning the Masson bluing solution to blue for 3min, and washing with water. 5) Washing with distilled water for 1 min. 6) And dyeing the fuchsin dyeing solution for 5min. 7) During the operation, according to the weight ratio of distilled water: weak acid solution = 2: preparing weak acid working solution in a proportion of 1, and washing for 1min by using the weak acid working solution. 8) Washing with phosphomolybdic acid solution for 1 min. 9) Washing with prepared weak acid working solution for 1 min. 10) Directly placing into aniline blue staining solution for dyeing for 1 min. 11) Washing with prepared weak acid working solution for 1 min. 12) 95% ethanol dehydrates rapidly. 13) Dehydrated 3 times for 5s with anhydrous ethanol. 14) Xylene was clear 3 times for 5s each. 15) Air-drying, adding a little neutral gum, sealing with a cover glass, air-drying, and taking a picture under microscope. 16) The area of blue staining of the detected endometrial fibrosis was calculated using the Image analysis software Image pro plus6.0 for analysis.

4. Survival of ADSCs in uterine tissue

1) Fresh uterine tissue specimens were placed in 4% paraformaldehyde for 24 h. 2) 10% sucrose for 24 h. 3) 20% sucrose for 12 h. 4) 30% sucrose for 12 h. 5) 30% sucrose for 12 h. 6) 1/230% sucrose and 1/2 OCT 12 h. 7) And (5) OCT 24 h. 8) And (3) placing the uterus into an embedding mold for 24 hours, adding enough OCT, placing into a liquid nitrogen tank, quickly freezing, and placing at-80 ℃ for later use. 9) Slicing: the temperature of the freezing microtome is adjusted to-20 ℃, the embedded tissue block is placed on a sample support, the shape of the tissue block is firstly reshaped by 30um, then the thickness is adjusted to 7um, and a tissue section is cut and marked. 10) Dropping a drop (20-50ul) of the anti-fluorescence attenuation mounting solution on the tissue slice, covering a cover glass, and contacting the slice with the mounting solution to avoid bubbles as much as possible. 11) The sections were then observed by fluorescence microscopy and analyzed using Image analysis software Image pro plus6.0 to calculate the proportion of GFP cells.

5. Real-time fluorescent quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

(1) Extraction of total RNA from uterine tissue

1) Fresh or ultra-low frozen samples were quickly added to 1.5 ml sterile tube, 350ul lysis Buffer RL (50 XTT Solution was confirmed before use) was added to 20mg of tissue and then disrupted using a tissue disruptor, and then repeatedly pipetted until no significant precipitation was observed in the lysate. 2) The lysate was centrifuged at 12000 rpm at 4 ℃ for 5min. 3) Carefully aspirate the supernatant into a new 1.5 ml RNase Free Tube. 4) The gDNA Eraser Spin Column was mounted on a 2ml Collection Tube (supplied in the kit). 5) The supernatant was transferred to gDNA Eraser Spin Column. 6) Centrifuge at 12,000 rpm for 1 min. 7) Discard gDNA Eraser Spin Column (please retain when performing genomic DNA extraction). The filtrate in 2ml of Tube was retained. 8) Adding 70% ethanol (possibly precipitating) with the same volume as the liquid in the step 3) or the step 7), and uniformly mixing the solution by using a pipette gun. 9) Immediately, the mixture (containing the pellet) was transferred into an RNA Spin Column (containing 2ml of Collection Tube) (if the volume of the mixture is greater than 600. mu.l, the mixture was added in portions, and the volume of each addition is not greater than 600. mu.l). 10) Centrifuge at 12000 rpm for 1min, and discard the filtrate. The RNA Spin Column was placed back into the 2ml Collection Tube. 11) Mu.l of Buffer RWA was added to the RNA Spin Column, centrifuged at 12000 rpm for 30s, and the filtrate was discarded. 12) Mu.l of Buffer RWB was added to the RNA Spin Column, centrifuged at 12000 rpm for 30s, and the filtrate was discarded. Buffer RWB was added around the wall of the RNA Spin Column tube to help completely flush out the salt attached to the tube wall. 13) Preparation of DNase I reaction solution: mu.l of 10 XDase I Buffer, 4. mu.l of recombined DNase I (RNase Free, 5U/. mu.l), 41. mu.l of RNase Free dH2O were added to a fresh 1.5 ml of RNase Free Tube and mixed well. 50. mu.l of DNase I reaction solution was added to the center of the RNA Spin Column membrane and allowed to stand at room temperature for 15 minutes. To the center of the RNA Spin Column membrane, 350. mu.l of Buffer RWB was added, and the mixture was centrifuged at 12,000 rpm for 30 seconds, and the filtrate was discarded. 14) Operation step 12) is repeated. 15) The RNA Spin Column was re-mounted on a 2ml Collection Tube and centrifuged at 12000 rpm for 2 min. 16) The RNA Spin Column was placed on a 1.5 ml RNase Free Collection Tube (provided in kit), and 50-200. mu.l of RNase Free dH2O or 0.1% DEPC-treated water was added to the center of the RNA Spin Column membrane, followed by standing at room temperature for 5min. 17) The RNA was eluted by centrifugation at 12,000 rpm for 2 min. 18) If the yield of RNA is to be improved, 50-200 μ l of RNase freedH2O or 0.1% DEPC treated water is added to the center of the RNA Spin Column membrane to elute the RNA; if RNA with high concentration is obtained, the first eluate may be added back to the RNA Spin Column, left to stand at room temperature for 5min, and centrifuged at 12,000 rpm for 2min to elute RNA. 19) And (3) detecting the purity and concentration of the RNA: after RNA is mixed uniformly, 1ul of liquid is dripped into an ultraviolet spectrophotometer for detection, and the absorption curve is observed, so that the ratio of 260/280 to 260/230 is used for judging the purity and the concentration of the RNA.

(2) Reverse transcription

1) Reverse transcription system was configured as shown in the following table

5X FastKing-RT superMix	4ul
		Total RNA	2ug
Enzyme-free water	Make up to 20ul

The total mixture was prepared and then aliquoted into 200ul enzyme-free EP tubes, each 20 ul. After the preparation, the liquid adhered to the wall was centrifuged by a centrifuge.

2) Mixing, placing in PCR instrument, cooling to 42 deg.C for 15min and 95 deg.C for 3min, terminating reaction, and storing cDNA product at-20 deg.C.

(3) Real-time fluorescent quantitative PCR

1) The reaction system was configured as follows:

2xSupeuReal PreMix Plus	10ul
		forward primer (10 uM)	0.6ul
Reverse primer (10 uM)	0.6ul
		cDNA template	1ul
Enzyme-free water	Make up to 20ul

Preparing a total mixed solution on ice, adding 19ul of the uniformly mixed solution into each well, finally adding 1ul of the cDNA template into each well, avoiding the generation of bubbles as much as possible, and centrifuging once if necessary. Each primer was set in3 duplicate wells.

Primer sequence for PCR (Table 1)

2) The specific procedure for the PCR amplification reaction is as follows:

after the reaction is finished, judging the specificity of the primer according to the dissolution curve, taking GAPDH as an internal reference gene, and adopting 2^-△△CTCalculating the relative expression of each group of genes.

6. Immunohistochemistry

1) And (6) baking the slices. 2) Dewaxing and hydrating: xylene I5 min- - -xylene II 5min- - -absolute ethanol 5min- - -95% ethanol 2min- - -80% ethanol 2min- - -75% ethanol 2min- - -distilled water 2 min. 3) Antigen retrieval: a) 10ml of citric acid and 1000m of double distilled water; b) the appropriate antigen retrieval buffer is added to the pressure cooker, which is placed on an electric hot plate and brought to maximum power. At this time, the pressure cooker cover is not required to be closed, and the pressure cooker cover is only required to be buckled. While waiting for the pressure cooker to boil, the slices may be dewaxed and rehydrated. After boiling, the slides were transferred from tap water to a pressure cooker. Care was taken to high temperature the solution (please use tweezers). And timing for 3min after the pressure cooker reaches the maximum pressure. After 3min, the electric heating plate is closed, and the pressure cooker is put into the empty water tank. Opening the pressure release valve and flushing the pressure cooker with cold water, after the pressure is reduced, opening the cooker cover and flushing the interior of the pressure cooker with cold water for 10 min. This allows the slide to be cooled sufficiently for subsequent processing, while allowing the antigenic sites to be re-reduced after exposure to high temperatures. Please take care to handle the hot solution. 4) Transparent 10XTBST and double distilled water ddh₂O is as follows: 9, respectively. Tween 20 is prepared into a solution with the final concentration of 0.2 to 0.5 percent in PBS for 3 times and 3 min. 5) And (5) washing with PBS. 6) Tissues were enclosed using an immunohistochemical pen. Appropriate amount of endogenous peroxidase blocking agent was added dropwise to cover the tissue mass and intervention was carried out at room temperature for 10min to remove the effect of endogenous catalase in the tissue. 7) The PBS immersion washing is carried out for 5min, and repeated for 3 times. 8) Blocking serum stock was diluted 10-fold with PBS. And (3) dropwise adding a proper amount of ready-to-use goat serum confining liquid into each slice, and sealing for 1h at room temperature. 9) Throwing off the ready-to-use goat serum, and respectively dropwise adding the pre-diluted primary anti-rabbit anti-mouse. 10) Rewarming at room temperature for 30min, removing antibody, washing with PBS for 5min, and repeating for 3 times. 11) The reaction enhancing solution is added dropwise, incubated at room temperature for 20min, and washed with PBS buffer for 3min3 times. 12) Adding dropwise the goat anti-mouse/rabbit lgG polymer with the enhanced enzyme, incubating at room temperature for 20min, and washing with PBS buffer solution for 3min3 times. 13) Preparing DAB: sequentially adding 1ml of DAB substrate solution (reagent 2) into a special reagent bottle, concentrating 50ul of DAB solution (reagent 1), uniformly mixing, and storing the prepared DAB color development solution at 2-8 ℃ in a dark place. 14) Adding a proper amount of freshly prepared DAB color development solution, and incubating for 1min at room temperature. The color development time is grasped under the mirror until the positive area develops colorTan, while negative control was not colored. 15) And flushing with flowing water for 5min to stop color development. 16) Counterstaining, washing with tap water, and incubating with hematoxylin staining solution for 20 s. 17) Washing with running water for 10min until the cell nucleus is blue. 18) Soaking in 70% ethanol for 2 min. 19) Soaking in 80% ethanol for 3 min. 20) Soaking in 90% ethanol for 4 min. 21) Soaking in anhydrous alcohol for 4 min. 22) Soaking in fresh anhydrous alcohol for 5min. 23) Soaking in xylene for 5min. 24) And then immersed in fresh xylene for 5min. 25) And (4) dropwise adding a little neutral gum, putting a cover glass for sealing, airing, and observing and photographing under a microscope. 26) Immunoreactivity quantification was performed using Image Pro-Plus 6.0, with 3-5 fields randomly selected per plate, and the Mean Optical Density (MOD) was determined.

7. Statistical analysis

Data analysis a comparison between the two groups was performed using F-test and t-test (equal variance). Differences between groups were analyzed using a one-way ANOVA and Tukey's post-hoc test for three or more groups. Graphics and statistics were obtained using GraphPad Prism8 (GraphPad Software inc., La Jolla, CA, USA). Significant values are assigned as P < 0.05, P <0.01, P < 0.001, P <0.0001, all data expressed as mean ± Standard Deviation (SD).

8. Results

8.1 autologous adipose-derived mesenchymal stem cells in Shakegel^TMCharacterization, differentiation and safety evaluation on 3D.

Observing the morphology of the 0 th generation (P0) to the P3 generation of the adipose-derived mesenchymal stem cells cultured by primary extraction, finding that the appearance of the 3 rd generation autologous adipose-derived mesenchymal stem cells forms typical spindle-formed fibroblast-like cells gradually, and the cells are closely arranged and grow in a vortex shape. Fluorescence-activated cell sorting (FACS) of third generation adipose-derived mesenchymal stem cells showed that autologous ADSCs expressed CD105(99.1%), CD29(99.6%) and CD73(98.9%), with little expression of CD34 and CD 45. Inducing differentiation results of the adipose-derived mesenchymal stem cells, wherein alizarin red is seen to be stained into red mineralized nodules after osteogenic induction for 24 days; after 24 days of chondrogenesis induction, the Alisine blue is dyed into blue inner acidic mucopolysaccharide; after 24 days of adipogenic induction, the formation of fat droplets was visible, and oil red O was stained red.

To further evaluate autologous adipose mesenchymeStem cells in Shakegel^TMSecurity on 3D, CCK-8 results validated 1 x10 ^ s⁶Cell + Shakegel^TM3D cultured cells have OD values higher than 5 x10⁵Cell + Shakegel^TMOD values of 3D cultured cells. On day 3 of co-cultivation, the OD values began to increase gradually. By day 7, 1 x10⁶ cell+ ShakeGel^TMOD value of 3D was over 50%.

8.2 autologous adipose-derived mesenchymal stem cells in Shakegel^TMIUA model effect on 3D

First, GFP lentivirus infected adipose mesenchymal stem cells we did not observe GFP positive cells in uterine tissue sections of sham, PBS and Gel groups. While GFP positive cells were observed in uterine tissue sections of both the ADSCs group (2.175%) and the ADSCs + Gel group (9.215%). And the number of GFP positive cells in the uterus tissue section of the ADSCs + Gel group is obviously more than that of the ADSCs group (P = 0.0101). from the appearance of the gross uterine specimen, the uterus appearance of the PBS group is obviously atrophic and not smooth compared with the uterus of the sham group. Transplanting ADSCs and Shakegel on the seventh day^TM3D, ADSCs binding to Shakegel^TM3D, the uterine appearance of these groups gradually recovered like a normal uterine morphology (Figure 7). Analysis of HE staining results showed significant thinning of endometrial thickness in PBS group (163.8 + -6.128 μm) compared to the thickness in sham group (264.5 + -31.75 μm) (P)<0.0001, n = 7;). ADSCs binding Shakegel^TMThe thickness of the endometrium of the 3D combined transplantation uterus group is obviously thicker than that of the PBS group (ADSCs group 190.1 + -8.389 μm, P is less than 0.05; ADSCs + Gel group 228.8 + -2.254 μm, P is less than 0.0001). The endometrium of the ADSCs + Gel group is thicker than the endometrium of the ADSCs group (p)<0.001, n = 7). Compared with the endometrial tissue of the PBS group, the number of glands of the ADSCs group is obviously increased (9.182 + -2.857 versus 1.545 + -0.9342, respecively, per unit area, P)<0.0001), Gel group (14.55. + -. 1.293 versises 1.545. + -. 0.9342, respecitvely, per unit area, P<0.0001) and ADSCs + Gel group ((19.36 + -2.063 versus 1.545 + -0.9342, respecively, per unit area, P)<0.0001) also increased. The ADSCs + Gel group has a greater number of glands than the ADSCs group (P)<0.0001). To further assess the degree of fibrosis, mass fibrous tissue was present in the PBS group using Massons trichrome staining(56.89 + -0.4425%) compared to the sham group (29.44 + -5.2305%). ADSCs (50.10 + -1.280%, P)<0.05)、ShakeGelTM3D (45.17 ± 1.196%, P<0.0001), ADSCs in combination with ShakeGel 3D (39.55 + -0.4125%, P<0.0001) percent area fibrosis after treatment was reduced compared to the PBS group. Furthermore, the percentage of area fibrosis for the ADSCs + Gel group is significantly lower than for the ADSCs group (P)<0.001). To further confirm the experimental fibrosis results, α -SMA gene and protein expression were combined. The methods all show that the fibrosis degree of the ADSCs + Gel group is obviously smaller than that of the ADSCs group. Through RT-PCR and immunohistochemical experiments, the expression quantity of alpha-SMA of the ADSCs + Gel group and the ADSCs group is found to be remarkably different, and the former is smaller than the latter. The mating experimental data of mice show that only the Sham group and the ADSCs + Gel group are pregnant, and no gestational sac is found in other three groups.

8.3ADSCs binding Shakegel^TMThe 3D can enhance BMP7-Smad5 signals to repair intrauterine adhesion.

As a result of QRT-PCR, BMP7 was expressed slightly higher in ADSCs (0.06124 + -0.006388) than in PBS (0.05062 + -0.01281), but the difference was not statistically significant (P = 0.5302). BMP7 expression was higher in ADSCs + Gel group (0.5575 + -0.06289) than in PBS group and was statistically significant (P < 0.0001). Compared with the ADSCs group, the ADSCs + Gel group expresses BMP7 more than the ADSCs group, and has statistical significance (P < 0.0001). Likewise, smad5 was expressed slightly higher in the ADSCs group (0.7127 ± 0.05734) than in the PBS group (0.6843 ± 0.07691), but the difference was not statistically significant (P = 0.9820). The expression level (0.9066 +/-0.07593) of Smad5 mRNA in the ADSCs + Gel group is obviously higher than that in the PBS group (P < 0.01). The expression of Smad5 was higher for the ADSCs + Gel group than for ADSCs, and was also statistically significant (P = 0.0167).

Also, immunohistochemistry results showed that BMP7 of the ADSCs group had a MOD value (0.02330 ± 0.006284) slightly higher than that of the PBS group (0.002817 ± 0.002115), but the difference was not statistically significant (P = 0.1739). BMP7 has a higher MOD than PBS in ADSCs + Gel group (0.1314 + -0.01210), and is statistically significant (P < 0.0001). The MOD value set of BMP7 was significantly elevated for the ADSCs + Gel group compared to the ADSCs group (P < 0.0001). MOD value (0.1676 + -0.06002) of Smad5 in PBS group is lower than that in ADSCs group (0.2391 + -0.02442, P <0.0001), Gel group (0.2910 + -0.009053, P <0.0001), ADSCs + Gel group (0.3372 + -0.004266, P < 0.0001). MOD values for the ADSCs group smad5 were significantly higher than for the ADSCs group (P <0.0001) and Gel group (P = 0.0087).

The above description is only an embodiment of the present invention, but the design concept of the present invention is not limited thereto, and any insubstantial modifications made by using the design concept should fall within the scope of infringing the present invention.

Claims

1. A biological composition for repairing intrauterine adhesions, characterized by: including ShakeGel^TM3D gel liquid and autologous adipose-derived mesenchymal stem cells identified by flow, adipogenic induced differentiation, osteogenic induced differentiation and chondrogenic induced differentiation are mixed according to the volume of 1:1, and mixing uniformly.

2. The biological composition for repairing intrauterine adhesions according to claim 1, characterized in that: the autologous adipose-derived mesenchymal stem cells are extracted from white adipose tissues of animal groin.

3. A method for preparing a biological composition for repairing intrauterine adhesions according to claim 1, comprising the following steps:

(1) extracting and culturing autologous adipose-derived mesenchymal stem cells of the mice;

(2) carrying out osteogenic induction, adipogenic induction differentiation and chondrogenic induction differentiation treatment on the adipose-derived mesenchymal stem cells in the step (1);

(3) taking the culture solution of the adipose-derived mesenchymal stem cells treated in the step (2) for flow cytometry analysis;

(4) adding GFP chronic virus culture medium mixed liquor into the adipose tissue-derived mesenchymal stem cells in a good state for continuous culture;

(5) shakegel^TMAnd (3) mixing the 3D gel solution with the autologous adipose-derived stem cells obtained by the culture in the step (4) according to the volume ratio of 1:1, centrifuging and mixing uniformly to prepare the biological composition.

4. The method for preparing a biological composition for repairing intrauterine adhesion according to claim 3, wherein the extraction and culture process of the mouse autologous adipose-derived mesenchymal stem cells of the step (1) is as follows:

1) stripping and collecting 3-4 weeks old female mouse inguinal adipose tissues, and putting the tissues into a centrifuge tube filled with KRP buffer preheated to 37 ℃ in advance;

2) adipose tissue was transferred from KRP buffer to cell culture dishes, washed twice with pre-warmed PBS, and concentrated in 4:1, putting a new KRP buffer, and cutting adipose tissues into small pieces;

3) sucking the upper fat suspended matter, adding into a new sterile test tube, and adding collagenase II with the equal volume concentration of 0.75 mg/ml;

4) sealing the sterile test tube, placing the sterile test tube into a water bath at 37 ℃, slowly shaking for 45 minutes to digest the fat suspended matter until a separated cell layer is suspended at the uppermost layer of the liquid and separated from other precipitated tissue fragments, adding preheated PBS (phosphate buffer solution), neutralizing collagenase II, and stopping digestion;

5) centrifugally separating the adipose-derived stem cell sediment, and sucking and removing the upper mixed solution;

6) adding erythrocyte lysate for resuspending the precipitate, lysing for 10min, centrifuging, discarding supernatant, and leaving adipose-derived stem cell precipitate;

7) the adipose stem cell culture medium resuspended cell pellets were plated in cell culture dishes.

5. The method for preparing a biological composition for repairing intrauterine adhesion as claimed in claim 3, wherein the step (2) of osteoinduction of adipose mesenchymal stem cells is as follows:

1) placing the primary extracted adipose-derived mesenchymal stem cells at 37 deg.C and 5% CO₂When the cell fusion degree reaches 80-90%, digesting with 0.25% Trypsin-0.04% EDTA;

2) inoculating the digested mouse adipose-derived stromal stem cells into a six-hole plate which is coated with 0.1% gelatin in advance, and adding a complete culture medium into each hole;

3) then, the cells were incubated at 37 ℃ with 5% CO₂When the cell fusion degree reaches 60When the percentage is 70 percent, sucking away the complete culture medium in the hole, and adding 2mL of C57BL/6 mouse adipose-derived stromal stem cell osteogenesis induced differentiation complete culture medium into a six-hole plate;

4) replacing a fresh C57BL/6 mouse adipose-derived mesenchymal stem cell osteogenic induced differentiation complete culture medium every 3 days;

5) after 2-4 weeks of induction, alizarin red staining was performed, and the effect of bone formation staining was observed under a microscope.

6. The method for preparing a biological composition for repairing intrauterine adhesion according to claim 3, wherein the step (2) the adipogenic-induced differentiation process of the adipose-derived mesenchymal stem cells is as follows:

1) placing mouse adipose-derived mesenchymal stem cells at 37 deg.C and 5% CO₂When the cell fusion degree reaches 80-90%, digesting with 0.25% Trypsin-0.04% EDTA;

3) then, the cells were incubated at 37 ℃ with 5% CO₂Culturing in the incubator, changing the culture solution every three days until the cell fusion degree reaches 100% or the cell fusion degree is over-fused, sucking the complete culture medium of the mesenchymal stem cells, and adding the fat-forming induced differentiation culture medium A solution of the mouse adipose mesenchymal stem cells into a six-hole plate;

4) after 3 days of induction, sucking away the solution A in a six-hole plate, and adding 2mL of solution B of a C57BL/6 mouse adipose-derived mesenchymal stem cell adipogenic induction differentiation culture medium;

5) after 24h, sucking away the solution B, replacing the solution A for induction, after the solution A and the solution B alternately act for 3-5 times, continuing to maintain and culture for 4-7 days by using the solution B until lipid droplets become large enough and round, and replacing with fresh solution B every 2-3 days during the period of maintaining and culturing by using the solution B;

6) and after the lipogenic induced differentiation is finished, alizarin red is adopted for dyeing, and the osteogenic dyeing effect is observed under a microscope.

7. The method for preparing a biological composition for repairing uterine cavity adhesion as set forth in claim 3, wherein the step (2) chondrogenic induced differentiation of adipose-derived mesenchymal stem cells is as follows:

1) mixing 3-4X 10⁵Transferring the cells into a centrifuge tube and centrifuging for 4min,

2) sucking off the supernatant, adding 0.5 mL of premix, resuspending the precipitate obtained by centrifugation in the previous step to clean the adipose-derived mesenchymal stem cells of the mouse, and centrifuging for 5min at room temperature at 150 g;

3) repeating the step 2), and cleaning the cells again;

4) resuspending the precipitate obtained in the last step by using 0.5 mL of complete culture medium for chondrogenic induced differentiation of mouse adipose-derived mesenchymal stem cells;

5) centrifuging at room temperature for 5min, unscrewing the centrifuge tube cover to facilitate gas exchange, and culturing in an incubator at 37 deg.C and 5% CO 2;

6) when the cell clusters are gathered, the bottom of the centrifugal tube is flicked to separate the cartilage balls from the tube bottom and suspend the cartilage balls in the liquid;

7) replacing fresh chondrogenic induction differentiation complete culture medium every 2-3 days by calculation from the beginning of inoculation;

8) after the liquid is changed, flicking the bottom of the centrifuge tube to separate the cartilage ball from the tube bottom and suspend the cartilage ball in the liquid, slightly unscrewing the centrifuge tube cover, and putting the centrifuge tube cover into an incubator at 37 ℃ and 5% CO2 for continuous induction culture;

9) after 21-28 days of continuous induction, the chondrocytes were formalin-fixed and paraffin-embedded, and finally stained with alistin blue, and the staining effect of alistin blue was observed under a microscope.

8. The method for preparing a biological composition for repairing intrauterine adhesion as claimed in claim 3, wherein the specific process of the step (3) is as follows:

1) sucking a supernatant culture solution of the adipose-derived mesenchymal stem cells, and adding preheated PBS for washing;

2) adding 1ml trypsin, shaking to make the cells totally infiltrate, and digesting for 2min in a 5% CO2 cell culture box at 37 deg.C;

3) adding an adipose-derived mesenchymal stem cell culture medium to terminate the reaction, fully transferring the cells to a centrifuge tube, and centrifuging and precipitating the cells;

4) removing supernatant, adding 1ml adipose-derived mesenchymal stem cell culture medium, and counting after resuspending cells;

5) take 5 x10⁵Adding the adipose-derived mesenchymal stem cells into an EP tube, and centrifuging and precipitating the cells;

6) the supernatant was aspirated off, then 200. mu.l of Facs buffer was added for washing, and centrifugation was performed;

7) the supernatant was aspirated off, 50. mu.l of cell surface antibody staining solution was added, and the mixture was iced for 30 min;

8) adding 200 μ l Facs buffer for washing, centrifuging, removing supernatant, and repeating twice;

9) 200 μ l of Facs buffer resuspended cells, protected from light and ready for loading for assay.

9. The method for preparing a biological composition for repairing intrauterine adhesion as claimed in claim 1, wherein the specific process of the step (4) is as follows:

1) inoculating adipose-derived mesenchymal stem cells in a good state into a 24-well plate, paving the plate at the density of 6 × 104 cells/well, and adding 500ul of culture medium into each well to ensure that the fusion rate reaches 30% -40% when the cells are infected with viruses the next day;

2) the next day, cells grew to 30% -40%; the original medium was aspirated off according to: cell number × MOI value/virus stock titer × 103= virus infection amount (UI) to dilute the virus to 2ml while adding 5 μ g/ml polybren, and continuing the culture after adding the virus culture medium mixture;

3) after 8 h of virus infection, the fresh medium was changed, followed by changing the solution every 48 hours;

4) cells were observed under a fluorescent microscope 72h after infection with the virus to determine the efficiency of cell infection.

10. The method for preparing a biological composition for repairing intrauterine adhesion as claimed in claim 1, wherein the specific process of the step (5) is as follows:

1) uniformly mixing the gel liquid and the cell suspension in a centrifugal tube according to the volume ratio of 1:1, uniformly blowing and beating for a plurality of times, slightly and rapidly shaking on a vortex instrument for 1s to fully mix the gel liquid and the cell suspension, and repeatedly shaking for 1-2 times;

2) wetting each hole of the cell culture plate by a proper amount of PBS, discarding the wetted hole, sucking the fully and uniformly mixed cell gel mixture into the hole of the plate by using a pipette gun, and slightly shaking to uniformly spread the gel;

3) incubating the cell plate in a constant temperature incubator at 37 ℃ for 5-10min to allow the cell gel mixture to form a gel;

4) after the gel incubation is finished, slightly adding a culture solution with a proper volume along the hole wall, and continuously placing the mixture in an incubator for incubation so as to ensure that the cells are fully attached to the gel;

5) after 24h of incubation, about 1/2 of the culture broth was gently aspirated off with a pipette and an equal amount of fresh culture broth was added.