Construction method of endometriosis animal model

Abstract

The invention provides a construction method used for researching endometriosis. The method comprises the following steps: transplanting human ovary-derived ectopic endometrial tissues into an immunodeficient animal body, and additionally adding estrogen for local stimulation, so as to construct a subcutaneous endometriosis xenotransplantation model. The method has the advantages that: 1, tissues are derived from a human body, and an enzyme system and a drug target are both derived from the human body; 2, the transplanted ectopic endometrial tissue has the characteristics of structural disorder and mixing of glands and interstitial substances, and is highly close to the disease state of a human body; 3, subcutaneous transplantation operation is simple, observation is direct, and intervention is convenient; 4, local estrogen stimulation simulates a high-estrogen environment of ectopic intima, so that the survival rate and the survival time of the graft are improved. Test results show that the graft locally injected with estradiol is high in survival rate after being transplanted for 30 days, the structure is kept compact, the living cell form is complete, and the graft is closer to the original tissue. The model is closer to the pathological characteristics of human endometriosis, and a more ideal platform is provided for pathogenesis research and drug development.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for constructing an animal model of endometriosis. Background Art

[0002] Endometriosis (EMs) is a common gynecological disease, characterized by the presence of endometrial tissue with growth function outside the uterine cavity. This disease mainly affects women of reproductive age, with an incidence rate of 6%-10%, and may occur in postmenopausal women with relatively high estrogen levels, especially in patients receiving estrogen replacement therapy. The main symptoms of endometriosis include dysmenorrhea, dyspareunia, abnormal menstruation, infertility, etc., seriously affecting the quality of life of patients. Currently, there is still a lack of effective radical drugs in clinical practice, and the demand for drug research and development for endometriosis remains strong.

[0003] Recent studies have shown that endometriosis is an estrogen-dependent disease. The local estrogen synthesis in the eutopic endometrium and ectopic lesions of patients increases, thus promoting the growth of endometrial tissue. Ectopic endometrial tissue can synthesize estrogen by itself, and due to long-term inflammatory stimulation, its structural characteristics are significantly different from those of the eutopic endometrium, showing structural disorder, obvious fibrosis, rich stroma, etc. These pathological characteristics pose challenges to the research on the pathogenesis of endometriosis and the development of therapeutic drugs.

[0004] To deeply study the pathogenesis of endometriosis and develop effective treatment methods, the establishment of an animal model is crucial. An ideal animal model should be able to simulate the natural course of human diseases, including pathological characteristics, hormonal regulation, and immune responses, etc. However, the existing endometriosis lacks a good test model, and its animal models have certain limitations. For example, although the primate endometriosis animal model is highly similar to humans in terms of physiology and anatomical structure, its high maintenance cost and complex operation limit its wide application in drug development. Rodent animal models (such as mice and rats) are widely used due to their low cost, fast reproduction, consistent genetic background, etc., but their pathological characteristics are somewhat different from human diseases, and the observation is relatively difficult.

[0005] In rodent models, autotransplantation models and allotransplantation models are two common construction methods. The autotransplantation model simulates the occurrence process of diseases by transplanting the animal's own endometrial tissue to parts such as the abdominal cavity. However, its pathological characteristics and molecular characteristics are significantly different from those of human diseases, and the observation is relatively difficult. The allotransplantation model involves transplanting endometrial tissue in situ in the human uterine cavity into the peritoneum or abdominal cavity of immunodeficient nude mice or SCID (severe combined immunodeficiency) mice. Although the transplantation based on human endometrial tissue already has certain advantages compared with the autotransplantation model, mice do not have a menstrual cycle and do not naturally develop endometriosis. This means that there are significant differences in the physiological background between mice and humans, and the pathophysiological process of human endometriosis cannot be fully simulated. The occurrence of human endometriosis is closely related to the menstrual cycle, while mice lack this periodic change, resulting in the inability to accurately simulate some key pathological mechanisms. The transplanted endometrial tissue in situ in the human uterine cavity does not have the characteristics of structural disorder of endometriotic tissue and cannot simulate features such as obvious structural disorder, significant fibrosis, and rich stroma.

[0006] Moreover, traditional peritoneal or abdominal cavity transplantation methods have many disadvantages in the study of endometriosis, including difficult observation, complex operation, low graft survival rate, incomplete simulation of pathological characteristics, and poor reproducibility of results. In immunodeficient nude mice, the survival rate of most grafts is relatively low, usually 33% - 66% within 3 weeks, and the glandular cells will gradually become flattened, and the glandular lumen will dilate and form vesicles, and its structure is quite different from that of the endometrium in clinical true endometriosis. Summary of the Invention

[0007] The present invention provides a method for constructing an endometriosis animal model, aiming to solve the technical problem that the animal model constructed by the existing method for constructing an endometriosis animal model has a large difference in pathological characteristics from human pathological characteristics.

[0008] To solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0009] In the first aspect, the present invention provides a method for constructing an endometriosis animal model, including the following steps:

[0010] S1. Obtain ectopic endometrial tissue from the ovaries of a patient with endometriosis, wash and trim it, remove blood clots and fascia, and cut it into grafts with a size of 3 - 5 mm 3 ;

[0011] S2. Immerse the graft in matrix glue to evenly wrap the graft with matrix glue, and then put the graft wrapped with matrix glue into a trocar.

[0012] S3. Anesthetize immunodeficient animals and complete the transplantation by subcutaneous injection of the graft using a trocar;

[0013] S4. Starting from the second day after transplantation, estradiol is injected around the subcutaneous inoculum at a dose of 0.45 - 0.55 μg per 18 - 22 g body weight once a week for a total of 2 times to complete the construction of the endometriosis animal model.

[0014] Preferably, in step S3, the immunodeficient animals are nude mice or severe combined immunodeficient mice.

[0015] Based on the above protocol, when human tissue is transplanted into normal mice, the immune system of immunocompetent animals will recognize the transplanted tissue as "foreign" and initiate an immune response, resulting in the rejection and destruction of the transplanted tissue. Immunodeficient animals such as nude mice cannot normally generate mature T cells. The lack of T cells means that nude mice cannot produce a strong cellular immune rejection response to the transplanted xenogeneic tissue. Moreover, nude mice are hairless all over their bodies, which is convenient for observing the growth of the transplanted tissue, especially in the case of subcutaneous transplantation. Due to gene mutations, SCID mice lack T cells and B cells. T cells and B cells are the main cells responsible for cellular immunity and humoral immunity in the immune system. The lack of these two types of cells makes it almost impossible for SCID mice to produce an immune rejection response. In addition, the natural killer (NK) cell activity of SCID mice is relatively low. NK cells usually play a role in the early immune response, and their low activity further reduces the immune attack on the transplanted tissue.

[0016] Preferably, in step S3, the immunodeficient animals are 4 - 6 - week - old Nude nude mice, and the Nude nude mice need to be adapted in a SPF environment for at least 1 week.

[0017] Based on the above protocol, 4 - 6 - week - old Nude nude mice are in their juvenile stage, with relatively stable physiological states, and their body weights are usually between 18 - 22 grams. Moreover, the immune systems of mice at this age have not yet fully developed, and the characteristics of immunodeficiency are more obvious, enabling them to better accept xenogeneic transplanted tissues. The SPF environment refers to an environment free of specific pathogens. Placing the mice in a SPF environment in advance for 1 week can reduce the stress response of the mice and effectively reduce the risk of the mice being infected by pathogens. Pathogen infection may affect the immune status and health of the mice, thereby interfering with the test results.

[0018] Preferably, in step S3, the transplantation is completed by subcutaneous injection of the graft into the axillary region using a trocar.

[0019] Based on the above - mentioned scheme, the subcutaneous tissue in the axillary region is rich in blood supply, which can provide sufficient nutrition and oxygen for the transplanted ectopic endometrial tissue, facilitating the survival and growth of the graft. Moreover, the subcutaneous tissue in the axillary region is relatively thin, and it is easy to observe the growth of the graft in this area, which is convenient for recording the characteristics of the graft such as size, color, texture, etc., and also convenient for subsequent pathological examinations.

[0020] In a preferred embodiment, in step S4, the estrogen is estradiol.

[0021] Based on the above - mentioned scheme, estradiol can promote the proliferation and growth of endometrial cells and plays an important role in the pathophysiological process of endometriosis. Estradiol can simulate the high - estrogen environment in the body and support the growth and survival of the graft.

[0022] In a preferred embodiment, the concentration of estradiol is 0.5 μg / 20 g body weight.

[0023] In a second aspect, the present invention provides a method for screening and evaluating drugs for treating endometriosis, which screens and evaluates drugs for treating endometriosis based on the endometriosis animal model constructed by the construction method described in the first aspect.

[0024] The beneficial effects of the present invention are as follows:

[0025] The present invention provides a method for constructing an endometriosis animal model. The ectopic endometrial tissue derived from the patient's ovary is transplanted into an immunodeficient animal, and local stimulation is carried out by additionally adding estrogen to establish a subcutaneous endometriosis xenograft model. Compared with the prior art, the present invention has the following remarkable advantages:

[0026] 1. Simulate the high - estrogen environment and extend the survival time of the model. Since mice are non - menstrual animals, there are significant differences in hormone levels between their bodies and those of human patients with endometriosis. Traditional transplantation methods often overlook this problem, resulting in differences between the studied pathological characteristics and actual human pathological characteristics. To accurately simulate human pathological characteristics, the present invention innovatively adopts the method of locally adding estrogen. By locally stimulating hormones around the graft, a local high - concentration hormone environment is successfully created, thus more precisely simulating the high - estrogen conditions in which ectopic endometrial tissue is located clinically. This method not only improves the survival rate and growth effect of the graft in the animal body, but also significantly increases the success rate of the animal model and extends the survival time of the graft in the body, providing a more generous treatment intervention time window for related research. Therefore, the animal model constructed by the construction method provided by the present invention shows significant advantages in studying the pathogenesis of endometriosis and drug development, and is closer to the clinical real situation.

[0027] 2. Different from the traditional method of choosing intraperitoneal transplantation, the present invention selects subcutaneous tissue in the armpit or other parts for transplantation, which is simple to operate and easy to implement. Compared with the traditional intraperitoneal transplantation, subcutaneous transplantation reduces the surgical difficulty and trauma to animals, improves the repeatability and success rate of the experiment. Moreover, subcutaneous transplantation enables the direct observation of the growth of the graft, facilitating the recording of characteristics such as the size, color, and texture of the graft. This provides more intuitive data for the experiment and is convenient for subsequent pathological examination and analysis. The graft for subcutaneous transplantation is easy to perform intervention operations, such as drug injection and tissue sampling. Especially in the armpit area, the local blood supply is rich, which is beneficial to the survival and growth of the graft, further improving the stability and reliability of the model.

[0028] 3. High survival rate and typical pathological characteristics: It has been proven by experiments that the grafts locally injected with E2 still have a relatively high survival rate 30 days after transplantation and have the characteristics of typical ectopic endometrium, such as unclear endometrial structure, obvious stroma, disordered structure, and the mixture of glands, endometrial stroma, and endometrial epithelium that are difficult to distinguish. These characteristics are closer to the original tissue, indicating that the model of the present invention can more accurately simulate the pathological characteristics of human endometriosis.

[0029] 4. Consistent enzyme system and drug targets: The ectopic endometrial tissue used in the present invention is directly derived from the patient's ovary, and its enzyme system and possible drug targets are all human-derived. Compared with the traditional animal models, it can more accurately simulate the pathological characteristics of human diseases.

[0030] Through estrogen stimulation and subcutaneous tissue injection, the present invention has achieved the construction of an animal model with high survival rate and typical pathological characteristics, providing a more ideal experimental platform for the research on the pathogenesis of endometriosis and drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can also be obtained based on these drawings without creative efforts.

[0032] Figure 1 It is a diagram of ectopic endometrial tissue in the experiment of the present invention.

[0033] Figure 2 It is a schematic diagram of the graft after treatment in the experiment of the present invention.

[0034] Figure 3 It is a diagram of the state of a nude mouse transplanted with ectopic endometrium with or without estrogen used locally.

[0035] Figure 4 It is a schematic diagram for exploring the effect of estrogen on graft survival in the present invention.

[0036] Figure 5 It is a comparison diagram of the graft structure of the experimental group and the control group with the original tissue in the present invention. Specific Embodiments

[0037] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative labor belong to the protection scope of the present invention.

[0038] Embodiment 1:

[0039] This embodiment provides a method for constructing an endometriosis animal model, including the following steps:

[0040] S1. Obtain ectopic endometrial tissue from the ovaries of animals with endometriosis, wash, trim it, remove blood clots and fascia, and cut it into grafts of 3 - 5 mm 3 in size;

[0041] S2. Immerse the grafts in Matrigel so that the grafts are evenly coated with Matrigel, and then put the grafts evenly coated with Matrigel into a trocar;

[0042] S3. Anesthetize immunodeficient animals and use a trocar to perform subcutaneous injection of the grafts to complete the transplantation;

[0043] S4. Starting from the second day after transplantation, at a dose of 0.45 - 0.55 μg per 18 - 22 g body weight, perform puncture injection of estrogen around the subcutaneous inoculum once a week for a total of 2 times to complete the construction of the endometriosis animal model.

[0044] Specifically, the estrogen is estradiol, and the concentration of the estradiol is 0.5 μg per 20 g body weight.

[0045] Among them, in step S3, in order to prevent the animal's immune system from recognizing the transplanted tissue as "foreign matter" and initiating an immune response, resulting in rejection and destruction of the transplanted tissue, the immunodeficient animals are nude mice or severe combined immunodeficient mice.

[0046] Preferably, the immunodeficient animals are 4 - 6 - week - old Nude nude mice, and the Nude nude mice need to be adapted in an SPF environment for at least 1 week.

[0047] In step S3, since the subcutaneous tissue in the armpit area is relatively thin, the graft is likely to be observable after growing in this area. A trocar is used to perform subcutaneous injection of the graft into the armpit area to complete the transplantation.

[0048] Example 2:

[0049] This example provides a method for screening and evaluating drugs for treating endometriosis. An endometriosis animal model constructed based on the construction method described in Example 1 is used to screen and evaluate drugs for treating endometriosis.

[0050] The present invention will be further explained and illustrated below with reference to experiments:

[0051] I. Reagents and consumables:

[0052] 1. Nude mice: A total of 10 Balb / C Nude mice.

[0053] 2. Grafts: Post-surgical medical waste of human origin for transplantation: ovarian chocolate cysts (ectopic endometrium of ovarian origin).

[0054] 3. Sterile instruments: including scalpel, surgical forceps, surgical scissors, trocar, etc., for performing subcutaneous transplantation surgery.

[0055] 4. Disinfectant: such as alcohol, for disinfecting the skin at the injection site.

[0056] 5. Anesthetic: such as sodium pentobarbital, for anesthetizing nude mice.

[0057] 6. Matrix gel: such as Matrigel, for wrapping tissue blocks, which helps in the fixation and growth of tissues under the skin of nude mice.

[0058] 7. Estrogen: human E2 estrogen.

[0059] 8. Other reagents and consumables: such as normal saline, syringes, etc.

[0060] II. Test methods:

[0061] 1. Selection of nude mice: Select Nude mice at 4 - 6 weeks of age, and they need to be acclimated in an SPF environment for 1 week in advance.

[0062] 2. Tissue preparation: Refer to Figure 1 - Figure 2 , wash the ovarian chocolate cysts obtained after surgery, trim them with sterile surgical instruments in a sterile environment, remove tissues such as blood clots and fascia, and cut them into small pieces of 3 - 5 mm for injection in the experiment. At the same time, retain a part of the original tissue for paraffin embedding for pathological examination, which is used for subsequent structural comparison with the graft.

[0063] 3. Anesthetize nude mice: Use anesthetics such as sodium pentobarbital to anesthetize nude mice at appropriate concentrations and doses.

[0064] 4. Fix the nude mice: Fix the anesthetized nude mice on the test bench.

[0065] 5. Disinfect the skin: Use disinfectants such as alcohol to disinfect the skin at the injection site.

[0066] 6. Inject the graft: Refer to Figure 3 , soak the graft in Matrigel so that the graft is evenly wrapped with Matrigel, then put it into a trocar, and use the trocar to perform subcutaneous injection of the graft at the disinfected site. After injection, gently withdraw the trocar to avoid pulling out the graft.

[0067] 7. Estrogen support: Starting from the second day after transplantation, inject estradiol around the subcutaneous inoculum at a dose of 0.5 μg / 20 g body weight once a week for a total of 2 times.

[0068] 8. Observe the growth of the graft: Record the growth of the graft, such as size, color, texture, etc.

[0069] 9. Sample collection and pathological examination: Sacrifice the animals at appropriate time points, take out the grafts for paraffin embedding, and perform pathological examination to observe the similarities and differences between the structure of the graft and the original tissue.

[0070] Experimental group setting:

[0071]

[0072] III. Experimental results

[0073] Refer to Figure 4 , where Figure 4 shows the effect of local use or non-use of estrogen support on the survival of grafts in nude mice transplanted with ectopic endometrium. From the results, it can be seen that in the control group with only subcutaneous transplantation of ectopic endometrium in nude mice, there is a certain construction rate, that is, a small number of grafts can survive under the skin of nude mice 30 days after transplantation.

[0074] Refer to Figure 5 , Figure 5 -A is the original tissue structure at 0 day of injection, Figure 5 -B is the graft structure of the control group at 30 days of injection in the control group. From the results, it can be seen that compared with the original tissue, the surviving grafts have the characteristics of typical ectopic endometrium, including unclear endometrial structure, obvious stroma, disordered structure, and confusion of glands, endometrial stroma, and endometrial epithelium that are difficult to distinguish. However, due to the low survival rate of the tissue, its structure is loose and the proportion of living cells is significantly reduced.

[0075] Refer to Figure 5 -C,Figure 5 - A is the original tissue structure at day 0 of injection. Figure 5 - C is the graft structure of the experimental group at day 30. In contrast, in the experimental group of nude mice locally injected with estradiol (E2), the grafts still had a relatively high survival rate 30 days after transplantation. These grafts also had typical pathological features of ectopic endometrium, but their structures remained compact, the morphology of living cells was well-preserved, and the proportion of living cells was significantly higher, being closer to the original tissue. This indicates that the additional estrogen stimulation significantly improved the survival rate of the grafts and the maintenance of typical pathological features.

[0076] IV. Conclusion

[0077] In this experiment, an animal model of endometriosis was successfully constructed by transplanting ectopic endometrium subcutaneously in nude mice and locally injecting estradiol (E2). The results showed that although the control group that only transplanted ectopic endometrium had a certain establishment rate, the survival rate of the grafts was low, the structure was loose, and the proportion of living cells was low; while the grafts in the experimental group had a high survival rate, a compact structure, intact morphology of living cells and a high proportion, being closer to the original tissue. This indicates that this model has the following advantages: First, the additional estrogen stimulation simulates a high E2 environment, which helps the survival of the grafts, improves the success rate of the model, extends the survival time of the model, and increases the treatment intervention time window; Second, the transplanted ectopic endometrial tissue has typical pathological features; Third, the subcutaneous transplantation operation is simple, the observation is direct, and the intervention is convenient; Fourth, human-derived tissues are used, and the enzyme system and drug targets are human-derived.

[0078] In summary, this model is closer to the pathological features of human endometriosis and provides a more ideal experimental platform for related research and drug development.

[0079] The present invention is not limited to the above optional embodiments. Any person can obtain other various forms of products under the inspiration of the present invention. However, no matter what changes are made in its shape or structure, as long as the technical solutions fall within the scope defined by the claims of the present invention, they are all within the protection scope of the present invention.

Claims

1. A method for constructing an animal model of endometriosis, characterized in that It includes the following steps: S1. Obtain ectopic endometrial tissue from the ovaries of patients with endometriosis, wash and trim it, remove blood clots and fascia, and cut it into grafts of 3-5 mm 3 ; S2. Immerse the graft in Matrigel, evenly coat the graft with Matrigel, and then put the graft evenly coated with Matrigel into a trocar; S3. Anesthetize the immunodeficient animal, and use the trocar to perform subcutaneous injection of the graft to complete the transplantation; S4. Starting from the second day after transplantation, at a dose of 0.45 - 0.55 μg per 18 - 22 g body weight, perform punctate injection of estrogen around the subcutaneous inoculum once a week for a total of 2 times to complete the construction of the endometriosis animal model.

2. The method for constructing an animal model of endometriosis according to claim 1, characterized in that, In step S3, the immunodeficient animal is a nude mouse or a severe combined immunodeficient mouse.

3. The construction method of an endometriosis animal model according to claim 1, characterized in that, In step S3, the immunodeficient animal is a 4 - 6 - week - old Nude mouse, and the Nude mouse needs to be adapted in an SPF environment for at least 1 week.

4. The construction method of an endometriosis animal model according to claim 1, characterized in that, In step S3, use the trocar to perform subcutaneous injection of the graft at the axillary site to complete the transplantation.

5. The construction method of an endometriosis animal model according to claim 1, wherein, In step S4, the estrogen is estradiol.

6. The construction method of an endometriosis animal model according to claim 1, characterized in that, It is characterized in that the concentration of the estradiol is 0.5 μg per 20 g body weight.

7. A method for screening and evaluating drugs for treating endometriosis, characterized in that, Screen and evaluate the drugs for treating endometriosis using the endometriosis animal model constructed based on the construction method according to any one of claims 1 to 6.

Images