Construction method of novel ovarian endometriosis mouse model

Abstract

The invention discloses a construction method of a novel ovarian endometriosis mouse model. Longitudinally cutting the uterus tissue of the donor mouse into small pieces; then putting the small uterus blocks into a collagenase solution for incubation; then removing collagenase from the small uterus blocks; attaching the small uterus block to the notch of the female mouse of which the envelope on the ovary surface is cut, so that the small uterus block covers the exposed ovary surface, pushing back into the abdominal cavity as soon as possible, nailing the skin and muscles by using a suturing nail, disinfecting and putting back into the mouse cage; after 24 hours of fasting, water can be freely drunk; and conventionally feeding after 24 hours. The method does not need preoperative / postoperative estradiol management, has the advantages of simplicity in operation, time saving, good effect, high molding success rate (about 90%) and the like, and is worthy of further popularization. The constructed ovarian endometriosis mouse model can evaluate the ovarian function condition and the influence of infertility, and overcomes the defect that the existing endometriosis mouse model lacks special ovarian types.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a method for constructing a novel ovarian endometriosis mouse model. Background Art

[0002] Ovarian endometriosis (OEMS) is a gynecological disease caused by the ectopic location of active endometrial tissue in the ovaries. It is also known as ovarian chocolate cysts, or simply chocolate cysts. Clinical manifestations of OEMS include dysmenorrhea, chronic pelvic pain, dyspareunia, and pelvic adhesions. Its incidence is increasing, with 50% of OEMS cases affecting both ovaries. Approximately 40%-50% of patients experience infertility, accounting for 35% of infertile women and a leading cause of infertility in women of reproductive age. OEMS disrupts the reproductive process in a comprehensive manner, including decreased endometrial receptivity, abnormal follicular development, an inflammatory microenvironment in the pelvis that is detrimental to the normal function of sperm, eggs, embryos, and fallopian tubes, epigenetic alterations, postoperative effects on ovarian function, and pelvic adhesions. Current treatment options include medication and surgery. However, the unclear pathogenesis of endometriosis presents limitations in its treatment. Therefore, in order to gain a deeper understanding of endometriosis, establishing and identifying relevant models is the key to studying and treating this disease.

[0003] Current models of endometriosis include immortalized cells, primary cell models, and animal models. The advantage of immortalized cell lines is that they can be passaged multiple times, facilitating mechanistic studies. However, their transformed phenotypes fail to maintain the three-dimensional (3D) structure and physiological properties of the original tissue. Primary cell models, on the other hand, offer the advantage of better preserving the properties of the original tissue. However, the limited number of glandular epithelial and stromal cells isolated from both eutopic and ectopic endometrium makes stable passage difficult, leading to significant inter-individual variability in results. Currently, there are reports of mouse models of endometriosis that mimic human disease, including peritoneal lesions and deep endometriosis. Most previous reports of endometriosis mouse models were based on Sampson's theory and induced by syngeneic transplantation of uterine tissue. Although these models exhibit cystic lesions in the peritoneum of the ectopic endometrium, ovarian endometriosis is rarely induced. Therefore, establishing a suitable and reliable animal model of ovarian endometriosis is crucial for exploring the pathophysiological mechanisms and new treatments for OEMS. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides a method for constructing an ovarian endometriosis mouse model. Through ovarian function and fertility capacity tests, the research results show that the ovarian endometriosis mouse model was successfully constructed and is consistent with the relevant phenotypes of clinical patients. It provides an ideal model for exploring the pathogenesis of ovarian endometriosis and is expected to promote research in this field.

[0005] To achieve the above object, the present invention provides a method for constructing an ovarian endometriosis mouse model, which is characterized by comprising the following steps:

[0006] S1. Obtaining the donor mouse endometrium: 9±2-week-old BALB / c female donor mice were sacrificed and dissected to obtain endometrial tissue. The whole piece was dissected and the fibrous adipose tissue was removed in phosphate-buffered saline. The uterine tissue was longitudinally cut into small pieces of 2±0.5 mm × 2±0.5 mm. The uterine pieces were then placed in a 1±0.2 mg / ml collagenase solution and incubated at 37±2°C for 30±5 min. The uterine pieces were then centrifuged, the supernatant removed, and resuspended in PBS. The supernatant was removed by a second centrifugation to remove the collagenase. The pieces were then placed in sterile PBS and awaited transplantation.

[0007] S2. Surgical transplantation in recipient mice: Pull out the ovaries of 9-week-old BALB / c female mice after the adaptation period, scratch the cyst on the surface of the ovary, only create a gap, expose part of the surface, and do not peel it off completely. Then attach the uterine pieces obtained in step S1 to the gap so that the uterine pieces cover the exposed surface of the ovary. Place 4-6 uterine pieces obtained in step S1 on each ovary, and then push them back into the abdominal cavity as quickly as possible. Nail the skin and muscles with sutures, and put them back into the mouse cage after disinfection; fasten them for 24 hours, but allow free drinking water; and feed them normally after 24 hours.

[0008] Furthermore, in the step S1, the conditions for the first centrifugation and the second centrifugation are both 8000×g for 3-8 minutes; more preferably, the conditions for the first centrifugation and the second centrifugation are both 8000×g for 5 minutes.

[0009] Furthermore, in step S1, the volume ratio of the added uterine pieces to the collagenase solution is 1:3.

[0010] Furthermore, in step S2, the step of pulling out the ovaries of 9-week-old BALB / c female mice after the acclimation period is to use isoflurane to induce and maintain general anesthesia in the 9-week-old BALB / c female mice after the acclimation period, and to make a 5-7 mm incision on the skin and muscle layer on both sides of the back of the mouse to find the ovaries; after finding the ovaries, use tweezers to clamp the fat pad connected to the ovaries and pull out the ovaries as a whole.

[0011] Furthermore, in step S2, the dosage of isoflurane during induction is 3%; and the dosage of isoflurane during maintenance is 2.5%.

[0012] The present invention provides a novel ovarian endometriosis mouse model that can assess ovarian function and the impact of infertility. This overcomes the lack of a dedicated ovarian endometriosis model in existing mouse models. This method, which requires no pre- or post-operative estradiol administration, offers advantages such as simplicity, time-saving, excellent results, and a high modeling success rate (approximately 90%), making it worthy of further promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a diagram of the establishment and analysis of ovarian endometriosis in mice.

[0014] Figure 2 This is the establishment and analysis of ovarian endometriosis in mice: HE staining and histological confirmation of endometriosis.

[0015] Figure 3 The expression of follicle-stimulating hormone receptor (FSHR) is significantly decreased in ovarian endometriosis.

[0016] Figure 4 The results of MASSON triple staining showed that the fibrosis area in the OE group was significantly higher than that in the C and S groups.

[0017] Figure 5 The expression of fibrosis marker molecule α-SMA is significantly increased in ovarian endometriosis.

[0018] Figure 6 This figure shows the results of TUNEL apoptosis staining showing increased apoptosis signals in ovarian endometriosis.

[0019] Figure 7 This figure shows that the expression of the proliferation-related molecule Ki-67 is low in ovarian endometriosis.

[0020] Figure 8 This is a graph showing the results of a fertility test. DETAILED DESCRIPTION

[0021] Below in detail embodiments of the present invention, the example of described embodiment is shown in the accompanying drawings, wherein identical or similar reference numerals represent identical or similar elements or elements with identical or similar functions throughout. The embodiment described below by reference to the accompanying drawings is exemplary, is intended to be used for explaining the present invention, and is not to be construed as limiting the present invention. In the embodiment, those not indicating specific techniques or conditions are carried out according to the technology or conditions described in the document in this area or according to the product specification. Reagents used or instruments not indicating manufacturers are conventional products that can be obtained commercially.

[0022] Example 1: Establishment and analysis of a mouse ovarian endometriosis model

[0023] 1. Establishment of an ovarian endometriosis model in mice

[0024] A total of 82 eight-week-old Balb / c mice (76 females and 6 males, 6 of which were used for fertility testing) were used. Prior to the experiment, they were acclimated for seven days and maintained at 23-25°C with a 12-hour dark / light cycle, standard food, and specific pathogen-free water. Their cages were changed weekly. They were divided into three groups: a control group (n=19), a sham-operated group (n=16), and a modeling group (n=27). Fourteen mice were used as donors.

[0025] Endometrial tissue was obtained by sacrificing donors and dissecting them en bloc. Fibrofatty tissue was removed in phosphate-buffered saline (PBS). The uterine tissue was cut longitudinally into small pieces of approximately 2 mm × 2 mm. A complete uterus can be cut into 15-20 pieces. The minced uterine tissue was digested and incubated with 1% collagenase at 37°C for 30 minutes. The volume ratio of collagenase to mouse uterine tissue pieces was approximately 3:1. The tissue was then centrifuged at 8000 × g for 5 minutes to remove the supernatant and resuspended in 100 μl of PBS. The supernatant was then removed by washing with an additional centrifugation (8000 × g, 5 minutes) to remove the collagenase. The tissue was then placed in sterile PBS and used immediately for modeling.

[0026] Modeling group (OEMs group, ovarian endometriosis): General anesthesia was induced and maintained with isoflurane (3% induction; 2.5% maintenance). A 5-7 mm incision was made bilaterally in the dorsal skin and muscle layers to locate the ovaries. Once the ovaries were located, forceps were used to grasp the attached fat pad and pull the entire ovary out to secure it for further manipulation. Ophthalmic forceps were used to puncture the ovarian capsule, creating a partial incision and exposing the ovarian surface without complete excision. Pre-treated uterine tissue was then attached to the incision, allowing a small uterine fragment to cover the exposed ovarian surface. Approximately 4-6 fragments of the processed uterine fragments from the donors were placed on each ovary and rapidly advanced back into the abdominal cavity. The skin and muscle were stapled. After disinfection with PVP-I, the mice were returned to their cages and fasted for 24 hours, with free access to water. After 24 hours, normal feeding was resumed. Samples were collected 4 and 6 weeks after modeling, and histological examination confirmed the establishment of a successful mouse ovarian endometriosis model.

[0027] Sham operation group (S group, sham): only the same ovarian exposure and suturing operations as the modeling group were performed, without attaching uterine fragments.

[0028] Control group (Group C, control): No treatment was performed and the mice were raised under the same conditions as the sham operation group and the model group.

[0029] Verification of successful model:

[0030] The prepared mouse ovarian endometriosis model mice were killed and dissected in situ. The results are shown in Figure 1 . Figure 1 There are three groups of mice. The upper part shows the in situ observation after the three groups of mice were dissected 4 weeks later to observe the formation of endometriosis lesions in the ovaries; the lower part shows the uterus and ovaries of any 6 mice in the control group, the uterus and ovaries of any 3 mice in the sham operation group, and the uterus and ovaries of any 7 mice in the modeling group were removed for comparative observation. It can be seen that single or multiple cystic lesions can be seen in the ovaries of the ovarian endometriosis modeling group (OEMs group), while no changes were observed in the ovaries of the blank control group (Group C) and the sham operation group (Group S). There are obvious adhesions between the cysts and adjacent organs such as the intestine, pancreas and abdominal wall. From Figure 1 It can also be seen that the photos of the lesion formation in the ovaries of multiple mice in the modeling group can reflect that the modeling success rate is high (about 90%) and the effect is good.

[0031] The ovaries and lesions were paraffin-embedded, sectioned, and HE-stained. Figure 2 Histological examination confirmed endometriosis, indicating that the mouse ovarian endometriosis model was successfully established.

[0032] 2. Analysis of the Mouse Ovarian Endometriosis Model

[0033] (1) The development of follicles is mainly controlled by pituitary follicle-stimulating hormone (FSH). FSH must rely on the expression of the corresponding receptor FSHR to exert its effect, so the expression regulation of FSHR is very important for follicle development.

[0034] The mouse ovarian endometriosis model group prepared in this example was compared with the blank control group without surgery. The ovaries of the two groups of mice were paraffin-embedded, sectioned, and then immunohistochemically stained for FSHR molecules. Figure 3 Among them, control is the control group (C group), sham is the sham operation group (S group), Ovarianendometriosis is the ovarian endometriosis model group (OEMs group), Figure 3 It can be seen that the expression of follicle-stimulating hormone receptor (FSHR) was significantly decreased in ovarian endometriosis compared with the control group.

[0035] (2) Ovarian endometriosis (OEMs) is characterized by adhesions and scar formation, leading to clinical manifestations such as pain and infertility. Fibrosis, accompanied by follicular loss, can be observed in the ovaries of patients with OEMs, indicating that the disease leads to altered ovarian function. Fibrosis was assessed using Masson's tristaining, and α-SMA, a marker of fibrosis, was also detected by histochemical analysis.

[0036] α-SMA is an indicator for detecting the transition of cells from epithelial to mesenchymal. As a marker protein of myofibroblasts, α-SMA is widely expressed in the ovaries. It is expressed not only in the theca cells of preantral follicles and antral follicles, but also in the periphery of atretic follicles. α-SMA is mainly expressed in glandular epithelial cells and some interstitial cytoplasm. The ovaries of the two groups of mice were paraffin-embedded, sectioned, and then immunohistochemically stained for α-SMA molecules. The results are shown in Figure 2. Figure 4 and Figure 5 Among them, control is the control group, sham is the sham operation group, and Ovarian endometriosis is the ovarian endometriosis model group. Figure 4-Figure 5 The results showed that the degree of fibrosis was increased in ovarian endometriosis and the expression of fibrosis marker molecule α-SMA was significantly increased in ovarian endometriosis.

[0037] (3) The TUNEL (Terminal deoxynucleotidyl transferase dUTP nick endlabeling) staining experiment uses the fact that during the process of apoptosis, cells activate some DNA endonucleases, which cut the genomic DNA between nucleosomes. When the genomic DNA breaks, the exposed 3'-OH can be catalyzed by terminal deoxynucleotidyl transferase (TdT) and fluorescein or biotin-labeled dUTP can be added. This allows the use of fluorescence microscopy or chemical color development methods to determine whether the cells have undergone apoptosis. At the same time, by measuring the intensity of the fluorescent signal at the ends of the labeled DNA breaks, the degree of cell apoptosis can be quantitatively assessed. Normal cells or necrotic cells rarely have DNA breaks, so there is no 3'-OH exposure and therefore they cannot be stained. The ovaries of the two groups of mice were paraffin-embedded and sectioned, and the paraffin sections were fluorescently stained using the TUNEL staining kit. The results are shown in the figure. Figure 6 Among them, control is the control group, sham is the sham operation group, and Ovarian endometriosis is the ovarian endometriosis model group. From top to bottom are the TUNEL, DAPI and MERGE results of the two. Figure 6 The results showed that the apoptosis level in the OE group was higher than that in the control group and the sham operation group.

[0038] (4) Ki-67 protein is a cell marker for proliferation and one of the important markers of immunohistochemistry. It is closely related to cell proliferation. In interphase, Ki-67 antigen can only be detected in the cell nucleus, while in mitosis, most of the protein is relocated to the chromosome surface. Ki-67 protein is present in all active phases of the cell cycle (G1, S, G2 and mitosis), but is absent in resting (G0 phase) cells. The cellular content of Ki-67 protein increases significantly during the cell progression in the S phase of the cell cycle. Therefore, the expression level of Ki-67 can indicate whether cell proliferation is active. When the Ki-67 level is high, it indicates that cell division and proliferation activities are very active. The ovaries of the two groups of mice were paraffin-embedded, sectioned and then immunohistochemically stained for Ki-67 molecules. The results are shown in Figure 2. Figure 7 Among them, control is the control group, sham is the sham operation group, and Ovarian endometriosis is the ovarian endometriosis model group. Figure 7 Histochemical results showed that the Ki-67 expression in the ovarian endometriosis model group was lower than that in the other groups.

[0039] (5) Mouse fertility experiment:

[0040] The untreated, normally reared control group and the ovarian endometriosis model establishment group consisted of six female mice, each weighing approximately 20-22g. The mice were co-housed at 6:00 PM Beijing time, with a female:male ratio of 3:1. Each group was divided into two cages, with a female:male ratio of 3:1 per cage, for a total of four cages. A plug was tested at 9:00 AM the following day of co-housing. Pregnancy was recorded as a result of the presence of a plug, and the mice were placed in separate cages until birth, to count the number of live offspring.

[0041] To investigate whether ovarian endometriosis affects mouse fertility, the differences in the number of live-born pups obtained in each group were counted after mating.

[0042] In the blank group (n=6) and OEMs model group (n=6), a total of 52 and 22 pups were obtained from the mothers, respectively. The mean number (and range) of live-born offspring in the blank group and OEMs model group were 8.7 (7-10) and 3.7 (1-5), respectively. Compared with the blank group, the mean number of live-born offspring in the OEMs model group was significantly lower, as shown in the results. Figure 8 (See Table 1, blank group vs. OEMs model group, P < 0.05). This indicates that the ovarian endometriosis model group mice have low fertility.

[0043] Table 1 Statistics of the number of live-born offspring in the blank group and the OEMs modeling group

[0044]

[0045] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and purpose of the present invention.

Claims

1. A method for constructing an ovarian endometriosis mouse model, characterized in that: The following steps are included: S1. Obtaining the endometrium of donor mice: 9±2-week-old donor BALB / c female mice were killed and dissected to obtain endometrial tissue, and the whole piece was dissected and the fibrous adipose tissue was removed in phosphate-buffered saline; the uterine tissue was cut longitudinally into small pieces of 2±0.5mm×2±0.5mm; the small pieces of uterus were then placed in 1±0.2mg / ml collagenase solution and incubated at 37±2℃ for 30±5min; the small pieces of uterus were then centrifuged for the first time, the supernatant was removed and resuspended in PBS, and the supernatant was removed by a second centrifugation and washing to remove the collagenase; and then placed in sterile PBS to wait for transplantation; S2. Surgical transplantation in recipient mice: Pull out the ovaries of 9-week-old BALB / c female mice after the adaptation period, cut the cystic membrane on the surface of the ovaries, only create a gap to expose part of the surface, and do not peel it off completely. Then attach the small pieces of uterus obtained in step S1 to the gap so that the small pieces of uterus cover the exposed surface of the ovaries. Place 4-6 small pieces of uterus obtained in step S1 on each ovary, push them back into the abdominal cavity as quickly as possible, staple the skin and muscles with sutures, and put them back into the mouse cage after disinfection; fasten for 24 hours, but allow free drinking water; feed normally after 24 hours.

2. The construction method according to claim 1, characterized in that: In the step S1, the conditions for the first centrifugation and the second centrifugation are both 8000×g for 3-8 minutes; more preferably, the conditions for the first centrifugation and the second centrifugation are both 8000×g for 5 minutes.

3. The construction method according to claim 1, characterized in that: In step S1, the volume ratio of the uterine pieces to the collagenase solution is 1:

3.

4. The construction method according to claim 1, characterized in that: In the step S2, the step of pulling out the ovaries of 9-week-old BALB / c female mice after the adaptation period is to use isoflurane to induce and maintain general anesthesia for 9-week-old BALB / c female mice after the adaptation period, and make a 5-7 mm incision on the skin and muscle layer on both sides of the back of the mouse to find the ovaries; after finding the ovaries, use forceps to clamp the fat pad connected to the ovaries and pull out the ovaries as a whole.

5. The construction method according to claim 1, characterized in that: In the step S2, the dosage of isoflurane during induction is 3%; and the dosage of isoflurane during maintenance is 2.5%.

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