Mouse model construction method for severe endometrial injury ineffective in MSCs treatment

Abstract

The invention belongs to the technical field of medical model construction, and particularly relates to a construction method of a severe endometrial injury mouse model ineffective in MSCs treatment. The method specifically comprises the following steps: S1, selecting a female mouse to adapt to the environment for 2-3 days; s2, after the mouse adapts to the environment, anesthetizing the mouse on the 0th day, performing laparotomy to physically scrape the inner uterine wall of the mouse, and then performing injury induction and suturing on the inner uterine wall by using an LPS (Lipopolysaccharide) solution; s3, repeating the step S2 on the third day, and performing laparotomy, curettage, LPS induction and suture on the mouse; s4, preliminarily evaluating the severe endometrial injury condition of the mouse on the fifth day; s5, injecting MSCs into the uterus of the mouse with the severe endometrial injury to treat the endometrial injury; and S6, on the eighth day, collecting a uterus sample to evaluate the repair capability of the MSCs, and screening to obtain the severe endometrial injury mouse model ineffective in MSCs treatment. Through a mode of carrying out physical curettage and lipopolysaccharide induction on the uterus through twice laparotomy, severe IUA patients ineffective in clinical MSCs treatment are better simulated.

Description

Technical Field

[0001] The invention belongs to the technical field of medical model construction, and particularly relates to a method for constructing a mouse model of severe endometrial damage that is ineffective in MSCs treatment. Background Art

[0002] The human menstrual cycle is divided into four stages: the proliferation phase, the secretion phase, the menstrual phase, and the regeneration phase. Maintaining a normal endometrial cycle is the prerequisite for ensuring female physiological and reproductive functions. However, due to the frequent use of cesarean sections and abortions, the resulting problems such as poor endometrial healing, endometrial regeneration disorders, and endometrial receptivity defects have seriously affected the fertility of women of childbearing age. Many researchers believe that curettage and intrauterine surgery are important causes of abnormal endometrial repair, vascular disorder, sparse glands, and thinning of the endometrium, which in turn cause embryo abnormalities, recurrent pregnancy loss, infertility, and a series of gynecological diseases, seriously affecting women's lives and health. At present, the residual amount of endometrium in patients with moderate and severe endometrial damage is very small after large-scale damage, and the existing clinical drug treatment is not effective. Mesenchymal stem cells (MSCs), as a cell drug with powerful immunomodulatory function and tissue repair ability, have a good effect in repairing the endometrium and can prevent the recurrence of endometritis and uterine adhesions, but their treatment efficiency is still insufficient to meet the needs of most patients. There is an urgent need to explore new treatment methods in clinical practice to solve the problem of low cure rate in patients with severe injuries.

[0003] At present, the existing method for establishing the endometrial injury model is in the form of physical scraping plus lipopolysaccharide dual induction, but this modeling method is not well adapted to the physiological process of repeated curettage-induced infertility in clinical practice, cannot characterize the severity of endometrial damage in patients with severe endometrial damage, and cannot simulate severe IUA patients for whom MSCs treatment is ineffective in clinical practice. Therefore, the present invention improves the original modeling method, is more adapted to the repeated curettage process, establishes severe endometrial damage to better simulate severe IUA patients for whom MSCs treatment is ineffective in clinical practice, and provides greater space for further optimization of existing treatment methods, which is of great clinical value and exploratory significance. Summary of the invention

[0004] In view of the shortcomings of the prior art, the purpose of the present invention can be achieved through the following technical solutions:

[0005] A method for constructing a mouse model of severe endometrial damage that is ineffective in MSCs treatment, the method comprising the following steps:

[0006] S1. Select female mice and let them adapt to the environment for 2-3 days;

[0007] S2. After the mice were adapted to the environment, they were anesthetized on day 0, and the uterine lining of the mice was physically scraped by laparotomy. After scraping, LPS solution was used to induce damage to the uterine lining, and then sutured;

[0008] S3. On the third day, the steps of S2 were repeated, and the mice were re-opened, scraped, induced with LPS, and sutured;

[0009] S4, on the fifth day, the mice were preliminarily evaluated for severe endometrial damage;

[0010] S5. Inject MSCs into the uterus of mice with severe endometrial damage to treat endometrial damage;

[0011] S6. On the eighth day, uterine samples were collected to evaluate the repair capacity of MSCs, and a mouse model of severe endometrial damage that was ineffective with MSCs treatment was screened.

[0012] Furthermore, the mice in step S1 are 6-8 week old sexually mature C57BL / 6J female mice, and the breeding conditions are: temperature of 21-23° C., relative humidity of 50-60%, 12h cycle circadian rhythm, and acclimatization to the environment in an SPF breeding room for 2-3 days.

[0013] Furthermore, the specific operation of physical scraping in step S2 is as follows: remove the hair between the 4th and 5th pairs of nipples on the abdomen of the mouse, and then make a longitudinal skin incision about 1.0 cm long on the left side between the 4th and 5th pairs of nipples on the abdomen; use a sterile artery clamp to clamp the end of the uterine horn near the fallopian tube, use a 1mL syringe needle to puncture a small hole in the uterine horn near the vaginal end, and then use a pre-blunted 1mL syringe needle to scrape the inner wall of the uterus, and scrape 10 times respectively until the mouse uterus is obviously congested and the inner wall feels rough.

[0014] Furthermore, the LPS injection concentration is 1 μg / μL, and the injection volume is 25 μL.

[0015] Furthermore, after LPS injection in step S2, the uterine horn near the fallopian tube and the uterus are fixed with vascular clamps to prevent liquid leakage for 5 minutes.

[0016] Furthermore, the detection indicators in step S4 include uterine length, HE staining to detect the endometrial area and the number of glands, and breeding to form embryos to detect the number of embryos, total embryo weight, and the length of the uterus containing the embryos.

[0017] Furthermore, the MSCs were administered intrauterinely. After injection, the uterine horns near the fallopian tube and the uterus were fixed with vascular clamps to prevent fluid leakage for 5 minutes. MSCs were cell suspensions with a density of 1×10 7 / mL, and the injection volume was 25μL.

[0018] Furthermore, the indicators for evaluating the repair capacity of MSCs include uterine length, HE staining to detect the endometrial area, the number of glands, and the resident amount of MSCs.

[0019] A mouse model of severe endometrial damage that is ineffective in MSCs treatment is constructed by the above-mentioned method for constructing a mouse model of severe endometrial damage that is ineffective in MSCs treatment.

[0020] Beneficial effects of the present invention: The present invention performs physical scraping and lipopolysaccharide induction on the uterus twice through laparotomy, which is closer to the pathogenesis of patients with severe endometrial damage caused by repeated curettage in clinical practice, is more consistent with the severity of damage in patients with severe endometrial damage, and better simulates severe IUA patients who are ineffective in clinical MSCs treatment, and has broad application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 A is a representative graph of uterine length measurement after paired modeling of three groups of mice. The blank control group (Sham), endometrial injury group (EI), and severe endometrial injury group (sEI) were paired in pairs. B is a statistical graph of uterine length measurement.

[0023] Figure 2 HE staining images of paired mice in three groups: Sham, EI, and sEI.

[0024] Figure 3 The statistical graphs of endometrial area and gland number of the three groups of Sham, EI, and sEI were obtained through HE staining.

[0025] Figure 4 After the modeling of Sham, EI, and sEI mice was completed, the mice were co-caged with male mice on the 6th day to conceive embryos. Embryonic samples were collected on the 27th to 35th day, and the representative figure of the measurement of uterine length was shown.

[0026] Figure 5 Statistical charts of total embryo weight, embryo length, and embryo number of mice in the Sham, EI, and sEI groups.

[0027] Figure 6 Representative graphs and statistical graphs of uterine length measurements of two groups of mice. Group 4 was paired with EI and EI+MSCs, and Group 5 was paired with sEI and sEI+MSCs.

[0028] Figure 7 HE staining images of two paired groups of mice: EI, EI+MSCs, sEI, and sEI+MSCs.

[0029] Figure 8 The statistical graphs of endometrial area and gland number in the two groups of EI, EI+MSCs, sEI, and sEI+MSCs were obtained through HE staining.

[0030] Fig. 9 Schematic diagram of the modeling and drug administration process for mice with severe endometrial damage. DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Example 1

[0033] Mouse uterus pairing and grouping and environmental adaptation rearing

[0034] (1) The left and right sides of the mouse uterus were divided into different groups. First, three groups were set up, with 6 mice in each group. In group 1, the left uterus was a blank control (Sham) and the right side was endometrial injury (EI). In group 2, the left uterus was a blank control (Sham) and the right side was severe endometrial injury (sEI). In group 3, the left uterus was endometrial injury (EI) and the right side was severe endometrial injury (sEI). The mice were paired to eliminate the differences between individual mice.

[0035] Two groups were set up for efficacy verification, with 6 mice in each group. In group 4, the left uterus was endometrial injury (EI), and the right side was endometrial injury (EI) plus MSCs treatment group. In group 5, the left uterus was severe endometrial injury (sEI), and the right side was severe endometrial injury (sEI) plus MSCs treatment group. Both groups were paired to eliminate individual differences in mice.

[0036] (2) Thirty 6-8 week old sexually mature female C57BL / 6JNifdc SPF mice (about 20 g) (purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd., production license number SCXK (Zhejiang) 2024-0001) were selected and kept under room temperature (22 ± 1 °C), 50-60% relative humidity, and a 12-h cycle circadian rhythm. They were kept in an SPF breeding room for 3 days to acclimate to the environment.

[0037] 2. Modeling steps of severe endometrial injury (sEI) group

[0038] (1) Severe endometrial injury model was established for mice in groups 2, 3, and 5. First, the mice were anesthetized by pouring a small amount of isoflurane into a 50 ml EP tube filled with cotton balls to perform gas anesthesia on the mice. The mice were placed flat on a removable dissecting cardboard, with their limbs naturally spread out and fixed with tape. The 50 ml EP tube containing isoflurane was pointed at the head of the mouse for continuous inhalation anesthesia.

[0039] (2) Remove the hair between the 4th and 5th nipples on the abdomen of the mouse, disinfect the surgical area with iodine, make a skin incision about 1 cm long on the left side between the 4th and 5th nipples, use sterile surgical elbow forceps to find the pre-determined unilateral uterus of the sEI group, clamp the uterine horn near the fallopian tube with a sterile artery clamp, use a 1mL syringe needle to pierce a small hole in the uterine horn near the vaginal end, and then use a pre-blunted 1mL syringe needle to scrape the inner wall of the uterus. Hold the syringe in the right hand, and use the force of scraping to avoid puncturing the uterus. The cone of the syringe needle faces up, and scrape upward, left, and downward 10 times respectively. The scraping area should be as large as possible to complete the mechanical damage to the entire inner wall of the uterus, until the uterus is observed to be obviously red and congested, and the inner wall feels rough.

[0040] (3) Prepare LPS solution in advance, using sterile PBS to prepare a final working solution concentration of 1 μg / μL. Use an insulin syringe to insert it parallel to the uterine cavity and slowly inject 25 μL of LPS solution into the uterine cavity until the cavity is obviously filled. Then, clamp the small opening at the lower end with an artery clamp to keep the liquid in the uterine cavity for 5 minutes.

[0041] (4) The mouse abdominal skin was sutured in two layers, the inner layer and the outer layer, with about 4-5 stitches in each layer. The wound was then disinfected with iodine and the mouse was placed on a 37°C heating pad and waited for the mouse to wake up.

[0042] (5) The first laparotomy for modeling was considered as day 0. The laparotomy was performed on the third day at the right side between the fourth and fifth pairs of nipples. All steps 1 to 4 were repeated except for the different laparotomy locations to cause severe damage to the uterine lining of the mice.

[0043] (6) On the 5th day, mice in groups 2 and 3 were killed by cervical dislocation. The abdominal cavity of the mice was cut open, and the adipose tissue and other connective tissues adhering to the uterus were carefully peeled off. Part of the mouse's ovary was retained as the end point of the uterus, and part of the cervix was retained as the other end point of the uterus. The left and right uteri were not swapped when they were removed. The uterus was allowed to lie flat on the filter paper naturally. The measurements were taken with a ruler and recorded by photographing.

[0044] 3. Modeling steps of endometrial injury (EI) group

[0045] (1) Endometrial injury model was established in mice of groups 1, 3, and 4. First, the mice were anesthetized by pouring a small amount of isoflurane into a 50 ml EP tube filled with cotton balls to perform gas anesthesia on the mice. The mice were placed flat on a removable dissecting cardboard, with their limbs naturally spread out and fixed with tape. The 50 ml EP tube containing isoflurane was pointed at the head of the mice for continuous inhalation anesthesia.

[0046] (2) Remove the hair between the 4th and 5th nipples on the mouse abdomen, disinfect the surgical area with iodine, make a skin incision about 1 cm long between the 4th and 5th nipples, use sterile surgical elbow forceps to find the pre-determined unilateral uterus of the EI group, clamp the uterine horn near the fallopian tube with a sterile artery clamp, use a 1mL syringe needle to pierce a small hole in the uterine horn near the vaginal end, and then use a pre-blunted 1mL syringe needle to scrape the inner wall of the uterus. Hold the syringe in the right hand, and the force of scraping should not puncture the uterus. The cone of the syringe needle faces upward, and scrape upward, left, and downward 10 times respectively. The scraping area is as large as possible to complete the mechanical damage to the entire inner wall of the uterus, until the uterus is observed to be obviously red and congested, and the inner wall feels rough. The EI side uterus model of group 3 mice was completed when the abdominal cavity was opened for the first time for sEI modeling.

[0047] (3) Prepare LPS solution in advance, using sterile PBS to prepare a final working solution concentration of 1 μg / μL. Use an insulin syringe to insert it parallel to the uterine cavity and slowly inject 25 μL of LPS solution into the uterine cavity until the cavity is obviously filled. Then, clamp the small opening at the lower end with an artery clamp to keep the liquid in the uterine cavity for 5 minutes.

[0048] (4) The mouse abdominal skin was sutured in two layers, the inner layer and the outer layer, with about 4-5 stitches in each layer. The wound was then disinfected with iodine and the mouse was placed on a 37°C heating pad and waited for the mouse to wake up.

[0049] (5) On the 5th day, mice in groups 1 and 3 were killed by cervical dislocation. The abdominal cavity of the mice was cut open, and the adipose tissue and other connective tissues adhering to the uterus were carefully peeled off. Part of the mouse's ovary was retained as the end point of the uterus, and part of the cervix was retained as the other end point of the uterus. The left and right uteri were not swapped when they were removed. The uterus was allowed to lie flat on the filter paper naturally. The measurements were taken with a ruler and recorded by photographing.

[0050] 4. Blank control group (Sham) treatment

[0051] The uteri of mice in group 1 and group 2 were set as blank controls, and no treatment was done on the blank control uteri of group 1 and group 2 during modeling. The blank control group and experimental group were set up in the same mice to eliminate the differences caused by surgery on different groups.

[0052] 5. Drug administration steps for the efficacy experimental group

[0053] (1) The uterus of the mice in groups 4 and 5 was injected with MSCs. First, the mice were anesthetized by pouring a small amount of isoflurane into a 50 ml EP tube filled with cotton balls. The mice were placed flat on a removable dissecting cardboard, with their limbs naturally spread out and fixed with tape. The 50 ml EP tube containing isoflurane was pointed at the head of the mouse for continuous inhalation anesthesia.

[0054] (2) Remove the hair between the 4th and 5th pairs of nipples on the mouse abdomen, disinfect the surgical area with iodine, make a skin incision about 1 cm long on the right side between the 4th and 5th pairs of nipples, use sterile surgical elbow forceps to find the unilateral uterus of the pre-determined dosing group, and use a sterile artery clamp to clamp the uterine horn near the fallopian tube end.

[0055] (3) Prepare MSCs cell suspension in advance, digest the pre-cultured MSCs into single cells, and resuspend them in PBS to a density of 1×10 7 Use an insulin syringe to insert the cell suspension of cells / mL into the uterine cavity in parallel, and slowly inject 25μL of cell suspension into the uterine cavity to fill the cavity significantly. Then clamp the small opening at the lower end with an artery clamp to maintain the liquid in the uterine cavity for 5 minutes.

[0056] (4) The mouse abdominal skin was sutured in two layers, the inner layer and the outer layer, with about 4-5 stitches in each layer. The wound was then disinfected with iodine and the mouse was placed on a 37°C heating pad and waited for the mouse to wake up.

[0057] (5) On the 8th day, mice in groups 4 and 5 were killed by cervical dislocation. The abdominal cavity of the mice was cut open, and the adipose tissue and other connective tissues adhering to the uterus were carefully peeled off. Part of the mouse's ovary was retained as the end point of the uterus, and part of the cervix was retained as the other end point of the uterus. The left and right uteri were not swapped when they were removed. The uterus was allowed to lie flat on the filter paper naturally. The measurements were taken with a ruler and recorded by photographing.

[0058] 6. Evaluation of the establishment of a severe injury model

[0059] (1) Uterine length measurement

[0060] The mice were killed by cervical dislocation on the 5th day, and the fat and connective tissue adhering to the uterus were gently peeled off. The ovaries and cervix at both ends of the uterus were retained and then transferred to the filter paper. The left and right uteri were not flipped or exchanged, so that the uterus was placed naturally in a "V" shape. A ruler was placed under the uterus and photographed. The distance between the uterus of each mouse and the camera was kept fixed for subsequent analysis using Image J.

[0061] Image J analysis of uterine length steps: open the image to be analyzed, select the straight line tool, draw a straight line of unit length on the ruler, select Analyze-Set Scale, enter the length of the straight line in Known distance, enter the unit in Unit of length, check the Global checkbox, click OK, use the connection between the uterine cavity and the ovary as the starting point and the connection with the contralateral uterus as the end point, draw the length with a straight line segment, and click Measure to get the length of the uterus in the Result window. Use GraphPad for data analysis and pairwise comparison, and the statistical method is t test.

[0062] like Figure 1 As shown, A is the measurement pictures of the uterus of three groups of mice, the left uterus of group 1 is blank control (Sham), and the right side is endometrial injury (EI), the left uterus of group 2 is blank control (Sham), and the right side is severe endometrial injury (sEI), the left uterus of group 3 is endometrial injury (EI), and the right side is severe endometrial injury (sEI), B is the bar graph after statistical analysis, the results show that compared with the control group, the length of the uterus in the EI group decreased significantly, and the sEI group was significantly lower than the control group and the EI group, which can prove that the sEI group caused more severe endometrial damage, resulting in shortening of the mouse uterus.

[0063] (2) HE staining to observe the endometrial area and gland number

[0064] The left and right sides of the separated mouse uterus were cut open from the middle connection, and the front 1 / 2 of the uterus close to the ovarian end was taken, keeping the sampling position of the left and right uteruses consistent. The uteruses were fixed with 4% paraformaldehyde for 24 hours, and after paraffin embedding and sectioning, HE staining was performed to observe the endometrial area and the number of glands.

[0065] Paraffin embedding steps: ① After the fixed tissue is taken out, put it into the dehydration frame and rinse it with running tap water for 4-5 hours, then stop fixing and rinsing the fixative; ② Dehydrate the tissue step by step with alcohol from low to high concentrations, first dehydrate with 70% ethanol for 12 hours, dehydrate with 80% ethanol for 1 hour, and dehydrate with 95% ethanol twice, with durations of 1 hour and 45 minutes respectively; finally dehydrate with 100% ethanol twice for 30 minutes each time. ③ Make the tissue transparent and soak the tissue in xylene for 15 minutes twice. ④ Put the tissue into the melted paraffin for 1.5 hours to allow the paraffin to penetrate the tissue. ⑤ Place the dehydration frame in the wax liquid tray, with the cross-section of the uterus facing down, and place it on the cooling table to wait for the paraffin to solidify.

[0066] HE staining steps: ① Fix the wax block on the slicer, adjust the slice thickness to 10 microns, place the sliced ​​slices on a 45℃ bleaching and drying machine, and then stick them flat on a clean slide. ② Wait for the slices to dry and put them in an oven (60-65℃) for 4 hours to bake the slices so that the slices stick firmly. ③ Dewax the slices with xylene, put them in xylene for 15 minutes, and then put them in xylene again for 10 minutes. Use ethanol with decreasing concentrations to hydrate the slices, put the slices in anhydrous ethanol for 5 minutes, then 95% ethanol for 5 minutes, 80% ethanol for 5 minutes, and finally put them in distilled water. ④ Stain with hematoxylin for 5 minutes, wash with water for 10 minutes, 1% hydrochloric acid ethanol for 30 seconds, wash with water for 30 seconds, eosin stain for 1-3 minutes, and wash with distilled water for 30 seconds. 80% ethanol for 30 seconds, 95% ethanol for 1 minute twice, anhydrous ethanol for 3 minutes twice, xylene for 3 minutes twice, and finally seal the slices with neutral resin.

[0067] Image J was used to analyze the endometrial area. Open the image and use the polygon tool to circle the total area within the basal layer, excluding the myometrium. Click Measure, and the Area display will calculate the total area. Click type-8bit to convert the image format. Click process-find edges. Click Image-adjust-threshold to select the cavity part. Click the magic wand tool to select the cavity part. Measure to get the area of ​​the cavity part. Subtract the total area from the cavity area to get the endometrial area. Image J was used to analyze the number of endometrial glands. The multi-point tool was selected to count the glands in the uterine cavity. GraphPad was used for data analysis and pairwise comparisons were performed. The statistical method was t-test.

[0068] like Figure 2 The HE-stained sections were taken under an upright fluorescence microscope at 10 times magnification. The pictures show that the mouse uterine cavity is composed of the serosa, myometrium, and endometrium from the outside to the inside. The endometrium in the Sham group is thick and composed of neatly arranged single-layer columnar epithelial cells and interstitial cells, while the endometrial epithelial cells in the EI and sEI groups are disordered and the number of glands is significantly reduced. Figure 3 In order to obtain the statistical results of endometrial area and gland number of three different groups based on the HE staining results, the above statistical results show that the endometrial area and gland number of the endometrial injury group EI are significantly lower than those of the Sham group, while the endometrial area and gland number of the severe injury group sEI are significantly lower than those of the EI group, which indicates that the functional morphology of the sEI endometrium is more severely damaged than that of the EI group, thereby simulating the physiological state of the endometrium in patients with severe injury.

[0069] (3) Mouse uterine embryo receptivity test

[0070] Embryo receptivity is an endpoint indicator that characterizes endometrial function. The degree of endometrial damage is determined by the ability of the endometrium to accept embryo implantation. Another 18 mice were selected and modeled according to the above grouping and modeling steps. On the fifth day, three groups of Sham, EI and sEI severe endometrial damage model mice were obtained. On the sixth day, the female modeling mice were mated with 6-8 week old sexually mature male C57BL / 6JNifdc SPF mice at a ratio of 2:1 and raised normally in the experimental animal center. On about the 21st to 42nd day, the abdomen of the female modeling mice was obviously bulging, indicating that the female mice were pregnant. After the pregnant mice were killed by cervical dislocation, the abdominal cavity was cut open, and the embryo was clearly visible inside the uterus. Carefully peel off the fat tissue around the uterus, retain part of the ovary and cervix, and naturally spread the uterus containing the mouse embryo on the filter paper and place it in a "V" shape. Place a ruler at the lower edge of the uterus for photography and recording. Use scissors to remove the ovaries and cervix, separate the embryos on the left and right sides, weigh the embryos on the left and right sides, and record the number of embryos.

[0071] like Figure 4 The following are representative images of embryo acceptance in three groups of paired mice. The number and quality of embryo implantation reflect the health and integrity of endometrial function. In the control group, the number of embryos in the sham side uterus can reach 4-6, and the total length of the uterus and the total weight of the embryos are significantly better than those in the EI group and the sEI group. The endometrium is damaged after modeling, and the ability to accept embryo implantation is reduced. The number of embryos in the sEI group is reduced to 0-3, and the length of the uterus and the total weight of the embryos are significantly reduced. Figure 5 As shown in the figure, a bar graph was made based on the total weight of embryos, uterine length, and number of embryos, and statistical analysis was performed using a t-test. The above results indicate that the endometrium of mice in the sEI group was severely damaged, and even lost the ability to nurture embryos, and the quality of embryos was significantly reduced, which is consistent with the clinical situation of patients with severe endometrial damage and infertility due to multiple curettage or abortion, indicating that the mouse model of severe endometrial damage was successfully established.

[0072] (4) MSCs efficacy verification

[0073] like Figure 6 The pictures and statistical graphs of the uterine length measurements of mice in groups 4 and 5 are shown. The left uterus of group 4 was endometrial injury (EI), and the right uterus was the EI plus MSCs treatment group. The left uterus of group 5 was severe endometrial injury (sEI), and the right uterus was the sEI plus MSCs treatment group. The results show that the phenomenon of shortening of uterine length was restored after mesenchymal stem cell treatment of endometrial injury (EI), but it did not repair the shortening of uterine length caused by severe endometrial injury (sEI), which indicates that MSCs cannot repair the shortening of uterine length caused by severe endometrial injury.

[0074] Figure 7 , Figure 8 HE staining sections and statistical results of endometrial area and gland number of mice in group 4 and group 5 showed that the endometrial area and gland number increased significantly after MSCs treatment of endometrial injury (EI), indicating that endometrial function was significantly improved and repaired. However, it did not repair the endometrial thinning and gland reduction caused by severe endometrial injury (sEI). The above results indicate that a mouse model of severe endometrial injury that simulates clinical MSCs treatment is ineffective has been successfully constructed.

[0075] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0076] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A method for constructing a mouse model of severe endometrial damage that is ineffective in MSCs treatment, characterized in that: The construction method comprises the following steps: S1. Select female mice and let them adapt to the environment for 2-3 days; S2. After the mice were adapted to the environment, they were anesthetized on day 0, and the uterine lining of the mice was physically scraped by laparotomy. After scraping, LPS solution was used to induce damage to the uterine lining, and then sutured; S3. On the third day, the steps of S2 were repeated, and the mice were re-opened, scraped, induced with LPS, and sutured; S4, on the fifth day, the mice were preliminarily evaluated for severe endometrial damage; S5. Inject MSCs into the uterus of mice with severe endometrial damage to treat endometrial damage; S6. On the eighth day, uterine samples were collected to evaluate the repair capacity of MSCs, and a mouse model of severe endometrial damage that was ineffective with MSCs treatment was screened.

2. The method for constructing a mouse model of severe endometrial damage that is ineffective in MSCs treatment according to claim 1, characterized in that: The mice in step S1 are 6-8 week old sexually mature C57BL / 6J female mice, and the breeding conditions are: temperature of 21-23° C., relative humidity of 50-60%, 12h cycle circadian rhythm, and acclimatization to the environment in an SPF breeding room for 2-3 days.

3. The method for constructing a mouse model of severe endometrial damage that is ineffective in MSCs treatment according to claim 1, characterized in that: The specific operation of physical scraping in step S2 is as follows: remove the hair between the 4th and 5th pairs of nipples on the abdomen of the mouse, and then make a longitudinal skin incision about 1.0 cm long on the left side between the 4th and 5th pairs of nipples on the abdomen; use a sterile artery clamp to clamp the end of the uterine horn near the fallopian tube, use a 1mL syringe needle to pierce a small hole in the uterine horn near the vaginal end, and then use a pre-blunted 1mL syringe needle to scrape the inner wall of the uterus, and scrape 10 times respectively until the mouse uterus is obviously congested and the inner wall feels rough.

4. The method for constructing a mouse model of severe endometrial damage that is ineffective in MSCs treatment according to claim 1, characterized in that: The LPS injection concentration was 1 μg / μL, and the injection volume was 25 μL.

5. The method for constructing a mouse model of severe endometrial damage that is ineffective in MSCs treatment according to claim 1, characterized in that: In step S2, after LPS injection, the uterine horn near the fallopian tube and the uterus are fixed with vascular clamps to prevent fluid leakage for 5 minutes.

6. The method for constructing a mouse model of severe endometrial damage that is ineffective in MSCs treatment according to claim 1, characterized in that: The detection indicators in step S4 include uterine length, HE staining to detect the endometrial area and the number of glands, and the number of embryos, total embryo weight, and length of the uterus containing embryos formed by mating.

7. The method for constructing a mouse model of severe endometrial damage that is ineffective in MSCs treatment according to claim 1, characterized in that: MSCs were administered intrauterinely. After injection, the uterine horns near the fallopian tube and the uterus were fixed with vascular clamps to prevent fluid leakage for 5 minutes. MSCs were cell suspensions with a density of 1×10 7 / mL, and the injection volume was 25μL.

8. The method for constructing a mouse model of severe endometrial damage that is ineffective in MSCs treatment according to claim 1, characterized in that: The indicators for evaluating the repair ability of MSCs include uterine length, HE staining to detect the endometrial area, the number of glands, and the resident amount of MSCs.

9. A mouse model of severe endometrial damage that is ineffective in MSCs treatment is constructed by the method for constructing a mouse model of severe endometrial damage that is ineffective in MSCs treatment according to any one of claims 1 to 9.

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