Uterus in-situ endometrium and ectopic endometrium organ induced canceration model and establishment method and application thereof

Abstract

The invention discloses a uterus in-situ endometrium and ectopic endometrium organ induced canceration model as well as an establishment method and application thereof. The method comprises the following steps: extracting endometriosis endometrial cells and endometriosis in-situ endometrial cells, and constructing paired endometriosis endometrial organs and endometriosis in-situ endometrial organs; the endometriosis ectopic endometrial organs and the endometriosis in-situ endometrial organs are subjected to passage; the high-concentration estrogen is used for stimulating endometriosis ectopic endometrial organs and endometriosis in-situ endometrial organs; according to the method, endometriosis organs have certain cell nucleus heterogeneity, a monolayer epithelium structure is developed into a proliferative multi-layer epithelium structure, and a cell model which is convenient to construct and observe and can be cultured for a long time is provided for the pathological process of endometriosis into endometrial adenocarcinoma. The method has important scientific significance on the mechanism research of the subsequent endometriosis malignant transformation into the endometrial adenocarcinoma and the research on key molecules at the early stage of the ovarian endometrial adenocarcinoma.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a uterine endometrium in situ and ectopic endometrium organoid induced malignant transformation model and an establishment method and application thereof. Background Art

[0002] Endometriosis (ENDOMETRIOSIS) is a chronic, inflammatory gynecological disease characterized by the appearance, growth, and infiltration of endometrial tissue (glands and stroma) in the lining of the uterine cavity and outside the uterus, with repeated bleeding. Endometriosis is the most common type of gynecological disease. About 10% of women of childbearing age suffer from endometriosis, and the incidence tends to increase gradually. Endometriosis is a precursor lesion of many epithelial ovarian cancers, and is most closely related to endometrial adenocarcinoma and clear cell carcinoma. The malignant transformation of endometriosis into ovarian cancer is associated with a variety of high-risk factors, among which the relationship between the occurrence of endometriosis-related ovarian endometrioid adenocarcinoma and estrogen is more clear. Previous literature reported that in a surgically induced endometriosis rat model treated with high concentrations of estrogen for 8 months, 3 cases (6.0%) showed atypical hyperplasia of ectopic endometrium accompanied by simple hyperplasia of the eutopic endometrium, and 2 cases (4.0%) showed ectopic endometrioid adenocarcinoma accompanied by atypical hyperplasia of the eutopic endometrium, proving that high levels of estrogen can induce the malignant transformation of endometriosis into endometrioid adenocarcinoma, and synchronous abnormal hyperplasia of the eutopic endometrium also occurs.

[0003] The basic research on endometriosis and endometriosis malignant transformation has always been constrained by the lack of mature ectopic endometrial cell line models. The primary culture of ectopic endometrial cells from patients with endometriosis is difficult, and the subculture is even more difficult, which makes it difficult to meet the needs of in vitro experiments. Primary endometrial cells from patients with endometriosis are often used as in vitro cell models. According to current literature reports, primary culture stromal cells are mostly used for subculture and molecular mechanism research. Therefore, in the study of the mechanism of endometriosis malignant transformation, the molecular mechanism of the malignant trend of the corresponding ectopic endometrium is often judged by the changes in the malignant biological behavior of primary endometrial stromal cells in situ, and it is difficult to observe direct morphological evidence of malignant transformation. The in vitro models of endometriosis and endometriosis malignant transformation often use rat autologous endometrial transplantation models and human endometrial nude mouse transplantation models. However, animal models often take a long time to establish, have many uncertain factors, and have high experimental costs. In addition, they are species-specific and cannot fully explain the occurrence mechanism of human endometriosis and endometriosis malignant transformation. Therefore, the existing in vivo and in vitro models of endometriosis have obvious limitations, resulting in a serious lag in the basic research on endometriosis and its malignant transformation. Summary of the invention

[0004] The purpose of the present invention is to provide a model of induced malignant transformation of uterine eutopic endometrium and ectopic endometrium organoids and its establishment method and application, so as to solve the problems that traditional primary endometrial cell models of endometriosis are difficult to culture, difficult to pass, and difficult to meet the needs of in vitro experiments.

[0005] In a first aspect, the present invention provides a method for establishing an endometriosis organoid model, comprising: Extract ectopic endometrial cells and eutopic endometrial cells of endometriosis; Based on the ectopic endometrial cells of endometriosis and the eutopic endometrial cells of endometriosis, paired ectopic endometrial organoids of endometriosis and eutopic endometrial organoids of endometriosis are constructed; Passaging of ectopic endometrium organoids and eutopic endometrium organoids; High concentrations of estrogen were used to stimulate ectopic endometrial organoids and eutopic endometrial organoids.

[0006] Further, extracting endometrial cells of ectopic endometrium and endometrial cells of eutopic endometrium includes: Place the ectopic endometrial lesion tissue and eutopic endometrial tissue freshly collected from endometriosis patients in the operating room into a sterile centrifuge tube containing 10 ml of DMEM / F12 culture medium, transport in an ice box, and ensure processing within 6 hours; The tissues were washed three times with sterile PBS solution, the hard fibrous connective tissue in the ectopic lesions was cut off, and the ectopic endometrial lesion tissue and the eutopic endometrial tissue were cut into pieces with sterile ophthalmic scissors, and crushed until there were no obvious particles. The 10mg / ml collagenase IV + 1mg / ml DNase I digestion solution prepared in advance was added to each of them, and digested at 37 degrees for 40-60 minutes until there were no obvious tissue debris. An equal volume of DMEM / F12 complete medium containing 10% fetal bovine serum was added to each of them. After terminating the digestion, the filtrate was retained after passing through a 100μm cell sieve, and the cell pellet was retained after centrifugation for 5 minutes at 1000rpm. Treat the precipitate with 1ml-2ml of red blood cell lysis buffer. After 2 minutes, centrifuge for 5min at 1000rpm, remove the supernatant and retain the cell precipitate.

[0007] Furthermore, based on the endometriosis ectopic endometrial cells and the endometriosis eutopic endometrial cells, paired endometriosis ectopic endometrial organoids and endometriosis eutopic endometrial organoids are constructed, including: Calculate the number of extracted ectopic endometrial cells and eutopic endometrial cells; remove the supernatant, resuspend the cell pellet with 1 ml of basal culture medium, pipette and evenly distribute 5 ul of the pellet, add it to 45 ul of trypan blue dye, count it with a cell counting plate, and centrifuge again for 5 minutes at 1000 rpm; Select a sterile 24-well plate and drop the two groups of cells into the 24-well plate at a ratio of 1x105 cells mixed with 30ul of matrix gel per well to construct paired endometriosis ectopic endometrial organoids and endometriosis eutopic endometrial organoids; After the gel drop solidifies, add 0.5 ml of endometrial organoid culture medium preheated at 37 degrees to each well and culture in a cell culture incubator containing 5% CO2, changing the medium every 3-4 days.

[0008] Furthermore, the components and concentrations of the endometrial organoid culture medium are as follows: The main ingredients include: DMEM / F12 + glutamine and 4-hydroxyethylpiperazineethanesulfonic acid; Add the following ingredients: Recombinant human R-spine protein 1 500ng / ml; Recombinant human Noggin 100 ng / mL; B27 additive 2%; N-2 additive 1%; GlutaMAX TM 1%; Insulin-transferrin-selenium 1%; Penicillin-streptomycin-amphotericin B mixed solution 1%; Nicotinamide 2mM; A83-01 0.5µM; N-acetyl-L-cysteine ​​2 mM; Recombinant human epidermal growth factor 50ng / ml; β-recombinant human basic fibroblast growth factor 2 ng / ml; recombinant human fibroblast growth factor 10 10 ng / ml; SB202190 10µM; Y-27632 10 µM.

[0009] Furthermore, each time, the endometrial organoid culture medium is prepared according to the required amount according to the components and concentration of the endometrial organoid culture medium, filtered through a 0.22 μm syringe filter and stored, and used within one week.

[0010] Furthermore, the endometriosis ectopic endometrium organoids and the endometriosis eutopic endometrium organoids are passaged, including: Observe for 7-14 days, and when the organoid density reaches 70%-80%, subculture at a subculture ratio of 1:2-1:3. Use P3-P6 organoids for subsequent experiments.

[0011] Further, P3-P6 organoids were used for subsequent experiments, including: After removing the culture medium next to the matrix gel, add basal culture medium pre-cooled at 4°C, use the tip of the gun rinsed with anti-adhesion rinse to break up the gel droplets, blow and beat several times, transfer to the rinsed centrifuge tube, centrifuge at 4°C for 5 minutes, 1500 rpm, and remove the clean liquid layer and gel layer; Add 5 volumes of 37°C preheated organoid digestion solution, pipette thoroughly, and incubate at 37°C for 5 minutes; An equal volume of DMEM / F12 complete medium was added to terminate the digestion, and the mixture was centrifuged at 1500 rpm for 3 min at 4°C. DMEM / F12 basal medium was added and centrifuged again at 1500 rpm for 3 min at 4°C to obtain a cell pellet. Take out the corresponding volume of matrix gel that has been liquefied at low temperature, resuspend the cell pellet on ice according to the subculture ratio, pipette evenly and inoculate it into the cell culture plate. After the gel drop solidifies, add the endometrial organoid culture medium preheated at 37°C.

[0012] Furthermore, high concentrations of estrogen are used to stimulate the ectopic endometrial organoids and the eutopic endometrial organoids, including: Use DMSO to dissolve estrogen to prepare an estrogen stock solution with a concentration of 10mM, and dilute it at a ratio of 1:1000 to prepare endometrial organoid culture medium containing 10μM estradiol. Change the medium every 3-4 days and continue stimulation for 4 weeks; the control group is treated with the same concentration of DMSO.

[0013] In a second aspect, the present invention provides an endometriosis organoid model established by the above-mentioned method for establishing an endometriosis organoid model.

[0014] In a third aspect, the present invention provides an application of an endometriosis organoid model in obtaining a trend of change toward ovarian endometrioid adenocarcinoma.

[0015] Beneficial effects of the present invention: The induction malignant transformation model of eutopic endometrium and ectopic endometrium organoids provided by the present invention, as well as the establishment method and application thereof, provide an endometriosis organoid model with a tendency to malignantly transform into endometrioid adenocarcinoma. The method of continuous stimulation with high concentrations of estrogen can make the endometriosis organoids have a certain nuclear heterogeneity, and develop from a single-layer epithelial structure to a proliferative stratified epithelial structure, providing a cell model that is easy to construct, easy to observe, and can be cultured for a long time for the pathological process of endometriosis transforming into endometrioid adenocarcinoma, which has important scientific significance for the subsequent study of the mechanism of endometriosis transforming into endometrioid adenocarcinoma and the exploration of key molecules in the early stages of ovarian endometrioid adenocarcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to further understand the features and technical contents of the present invention, please refer to the following detailed description and drawings of the present invention. However, the drawings are only provided for reference and illustration and are not intended to limit the present invention. Attached photos

[0017] Figure 1 A flowchart of the method for establishing an endometriosis organoid model of the present invention; Figure 2 These are bright field and HE staining images of endometriosis ectopic endometrial organoids treated with high concentrations of estrogen and DMSO for 4 weeks and their control group; Figure 3 These are the immunohistochemical staining images of ovarian endometrial adenocarcinoma markers ER, PR, and proliferation marker Ki-67 in endometriotic endometrial organoids treated with high concentrations of estrogen and DMSO for 4 weeks and their control group. DETAILED DESCRIPTION

[0018] To further illustrate the technical means and effects of the present invention, the following is a detailed description in conjunction with the preferred embodiments of the present invention and the accompanying drawings.

[0019] See also Figure 1 The embodiment of the present invention provides a uterine endometrium and ectopic endometrium organoid induction malignant transformation model and its establishment method and application, including: Step 1: extracting ectopic endometrial cells and eutopic endometrial cells.

[0020] Specifically, the ectopic endometrial lesion tissue and eutopic endometrial tissue freshly collected from endometriosis patients in the operating room were placed in a sterile centrifuge tube with 10 ml of DMEM / F12 culture medium, transported in an ice box, and processed within 6 hours; The tissues were washed three times with sterile PBS solution, the hard fibrous connective tissue in the ectopic lesions was cut off, and the ectopic endometrial lesion tissue and the eutopic endometrial tissue were cut into pieces with sterile ophthalmic scissors, and crushed until there were no obvious particles. The 10mg / ml collagenase IV + 1mg / ml DNase I digestion solution prepared in advance was added to each of them, and digested at 37 degrees for 40-60 minutes until there were no obvious tissue debris. An equal volume of DMEM / F12 complete medium containing 10% fetal bovine serum was added to each of them. After terminating the digestion, the filtrate was retained after passing through a 100μm cell sieve, and the cell pellet was retained after centrifugation for 5 minutes at 1000rpm. Treat the precipitate with 1ml-2ml of red blood cell lysis buffer. After 2 minutes, centrifuge for 5min at 1000rpm, remove the supernatant and retain the cell precipitate.

[0021] Step 2: Based on the endometriosis ectopic endometrial cells and the endometriosis eutopic endometrial cells, paired endometriosis ectopic endometrial organoids and endometriosis eutopic endometrial organoids are constructed.

[0022] Organoids are a type of 3D in vitro cell model that can restore the characteristics and functions of in vivo tissues. They have the ability to self-renew and differentiate, and can retain the genetic characteristics of the original tissues. They are widely used in drug screening, disease model construction, and disease mechanism research. Endometriosis organoids are often composed of ectopic endometrium from endometriosis patients, and can be paired to construct in situ endometrial organoids. Their characteristic is that the cell components are mainly composed of endometrial epithelial cells, which restore the characteristics of ectopic endometrial glandular cells. Compared with traditional primary endometrial cell models of endometriosis, endometriosis organoids have the advantages of being able to be passaged many times, being able to be cultured in vitro for a long time, being able to be frozen, and being able to reflect the structure and function of in vivo tissues.

[0023] Specifically, the number of extracted ectopic endometrial cells and eutopic endometrial cells was calculated; the supernatant was removed, the cell pellet was resuspended with 1 ml of basal culture medium, 5 ul was taken out and added to 45 ul of trypan blue dye solution, the cell was counted using a cell counting plate, and centrifuged again for 5 minutes at 1000 rpm; Select a sterile 24-well plate and inoculate the two groups of cells at 1x10 5 The ratio of 10 cells mixed with 30ul of matrix gel was dropped into a 24-well plate to construct paired endometriosis ectopic endometrial organoids and endometriosis eutopic endometrial organoids; After the gel drop solidifies, add 0.5 ml of endometrial organoid culture medium preheated at 37 degrees to each well and culture in a cell culture incubator containing 5% CO2, changing the medium every 3-4 days.

[0024] The components and concentrations of the endometrial organoid culture medium are as follows: The main ingredients include: DMEM / F12 + L-glutamine and Hepes (4-hydroxyethylpiperazineethanesulfonic acid); Add the following ingredients: R-spondin 1 (recombinant human R-spondin 1) 500ng / ml; Noggin (recombinant human noggin) 100 ng / mL; B27 additive 2%; N-2 additive 1%; GlutaMAX TM 1%; Insulin-transferrin-selenium 1%; Penicillin-streptomycin-amphotericin B mixed solution 1%; Nicotinamide 2mM; A83-01 0.5µM; N-acetyl-L-cysteine ​​2 mM; EGF (recombinant human epidermal growth factor) 50ng / ml; βFGF (recombinant human basic fibroblast growth factor) 2ng / ml; FGF-10 (recombinant human fibroblast growth factor 10) 10 ng / ml; SB202190 10µM; Y-27632 10µM.

[0025] Each time, the endometrial organoid culture medium was prepared according to the required amount according to the composition and concentration of the endometrial organoid culture medium, filtered through a 0.22 μm syringe filter and stored, and used within one week.

[0026] Step three, passage the ectopic endometrial organoids and eutopic endometrial organoids.

[0027] Observe for 7-14 days, and when the organoid density reaches 70%-80%, subculture at a subculture ratio of 1:2-1:3. Use P3-P6 organoids for subsequent experiments.

[0028] After removing the culture medium next to the matrix gel, add basal culture medium pre-cooled at 4°C, use the tip of the gun rinsed with anti-adhesion rinse to break up the gel droplets, blow and beat several times, transfer to the rinsed centrifuge tube, centrifuge at 4°C for 5 minutes, 1500 rpm, and remove the clean liquid layer and gel layer; Add 5 volumes of 37°C preheated organoid digestion solution, pipette thoroughly, and incubate at 37°C for 5 minutes; An equal volume of DMEM / F12 complete medium was added to terminate the digestion, and the mixture was centrifuged at 1500 rpm for 3 min at 4°C. DMEM / F12 basal medium was added and centrifuged again at 1500 rpm for 3 min at 4°C to obtain a cell pellet. Take out the corresponding volume of matrix gel that has been liquefied at low temperature, resuspend the cell pellet on ice according to the subculture ratio, pipette evenly and inoculate it into the cell culture plate. After the gel drop solidifies, add the endometrial organoid culture medium preheated at 37°C.

[0029] Step 4: Use high concentration of estrogen to stimulate the ectopic endometrial organoids of endometriosis and the eutopic endometrial organoids of endometriosis.

[0030] Use DMSO to dissolve estrogen to prepare an estrogen stock solution with a concentration of 10mM, and dilute it at a ratio of 1:1000 to prepare endometrial organoid culture medium containing 10μM estradiol. Change the medium every 3-4 days and continue stimulation for 4 weeks; the control group is treated with the same concentration of DMSO.

[0031] Based on the characteristics of the organoid model, the present invention proposes for the first time to synchronously culture the ectopic endometrium and eutopic endometrial epithelial cell organoids of endometriosis patients, use estrogen to stimulate the organoids for a long time, and construct an organoid model of ectopic endometrium and eutopic endometrium malignant transformation of endometriosis patients with malignant transformation to endometrioid adenocarcinoma. The establishment of this model can circumvent some of the shortcomings of traditional cell and animal models, better simulate the process of endometriosis lesions in the malignant transformation to endometriosis-related endometrioid adenocarcinoma, and can be applied to the study of the mechanism of endometriosis malignant transformation, which has important scientific significance.

[0032] Identification of endometriosis organoids with malignant transformation tendency of endometriosis-related endometrial adenocarcinoma: The organoids were collected and fixed, embedded in paraffin, and then paraffin sections were made. HE staining and immunohistochemistry staining were performed to observe the morphological changes of organoids and the expression changes of endometrial adenocarcinoma markers and proliferation marker Ki-67 after continuous treatment: It can be seen that the endometriosis ectopic endometrial organoids after high estrogen treatment produced structural wall thickening and enlargement of some cell nuclei. In addition, the expression of ovarian endometrial adenocarcinoma markers ER and PR was increased, and the expression of Ki-67 was increased, which proved that the proliferation of endometriosis organoids was enhanced under high estrogen treatment and they had a trend of changing into ovarian endometrial adenocarcinoma.

[0033] See also Figure 2 From the bright field (50X) and HE staining images (200X) of the endometriosis organoids treated with high concentrations of estrogen and DMSO for 4 weeks and their control group, it can be seen that the endometriosis organoids treated with DMSO present a single layer of columnar epithelium arranged in a glandular vacuolar structure, and the endometriosis organoids stimulated by high concentrations and continuous stimulation locally proliferate to form a complex layer, and the nuclei of some cells are enlarged.

[0034] See also Figure 3 From the immunohistochemical staining images of ovarian endometrial adenocarcinoma markers ER, PR, and proliferation marker Ki-67 in endometriosis organoids treated with high concentrations of estrogen and DMSO for 4 weeks and their control group (200X), it can be seen that the staining of ER, PR, and Ki-67 is enhanced in endometriosis organoids continuously stimulated by estrogen.

[0035] Traditional primary endometriosis ectopic endometrium is difficult to culture in vitro, and it is difficult to meet the experimental requirements of in vitro regulation and induction like endometriosis ectopic endometrial organoids. Although endometriosis eutopic endometrial cells can be cultured in vitro, and can be subjected to gene regulation and in vitro stimulation and induction, and their malignant biological behavior and expression changes of related molecules can be observed, primary endometriosis eutopic endometrial cells cultured in a plane lose their characteristic glandular structure and cannot be cultured for a long time, and it is impossible to intuitively observe the morphological changes brought about by different treatment conditions, and it is also impossible to fully represent the pathophysiological characteristics of endometriosis lesions, and therefore it is impossible to achieve the purpose of the present invention.

[0036] The present invention is that the endometriosis organoid model is an in vitro model that is superior to the traditional primary endometriosis in situ endometrial cells in terms of culture time, tissue structure, etc. The in vitro induction and regulation of the endometriosis organoid model allows us to observe the trend of endometriosis changing to ovarian endometrial adenocarcinoma, providing a strong technical basis for the study of the early initiation mechanism of its malignant transformation.

[0037] Based on the close relationship between estrogen and ovarian endometrial adenocarcinoma, long-term treatment of endometriosis organoids with high concentrations of estrogen is a unique treatment method that can obtain an endometriosis organoid model with a tendency to malignantly transform into ovarian endometrial adenocarcinoma.

[0038] The above-described embodiments of the present invention do not limit the protection scope of the present invention.

Claims

1. A model of uterine endometrium and ectopic endometrium organoids for inducing malignant transformation and its establishment method and application, characterized in that: include: Extract ectopic endometrial cells and eutopic endometrial cells of endometriosis; Based on the ectopic endometrial cells of endometriosis and the eutopic endometrial cells of endometriosis, paired ectopic endometrial organoids of endometriosis and eutopic endometrial organoids of endometriosis are constructed; Passaging of ectopic endometrium organoids and eutopic endometrium organoids; High concentrations of estrogen were used to stimulate ectopic endometrial organoids and eutopic endometrial organoids.

2. The uterine eutopic endometrium and ectopic endometrium organoid induced malignant transformation model and its establishment method and application as claimed in claim 1, characterized in that: Extraction of ectopic endometrial cells and eutopic endometrial cells, including: Place the ectopic endometrial lesion tissue and eutopic endometrial tissue freshly collected from endometriosis patients in the operating room into a sterile centrifuge tube containing 10 ml of DMEM / F12 culture medium, transport in an ice box, and ensure processing within 6 hours; The tissues were washed three times with sterile PBS solution, the hard fibrous connective tissue in the ectopic lesions was cut off, and the ectopic endometrial lesion tissue and the eutopic endometrial tissue were cut into pieces with sterile ophthalmic scissors, and crushed until there were no obvious particles. The 10mg / ml collagenase IV + 1mg / ml DNase I digestion solution prepared in advance was added to each of them, and digested at 37 degrees for 40-60 minutes until there were no obvious tissue debris. An equal volume of DMEM / F12 complete medium containing 10% fetal bovine serum was added to each of them. After terminating the digestion, the filtrate was retained after passing through a 100μm cell sieve, and the cell pellet was retained after centrifugation for 5 minutes at 1000rpm. Treat the precipitate with 1ml-2ml of red blood cell lysis buffer. After 2 minutes, centrifuge for 5min at 1000rpm, remove the supernatant and retain the cell precipitate.

3. The uterine eutopic endometrium and ectopic endometrium organoid induced malignant transformation model and its establishment method and application as claimed in claim 1, characterized in that: Based on endometriosis ectopic endometrial cells and endometriosis eutopic endometrial cells, paired endometriosis ectopic endometrial organoids and endometriosis eutopic endometrial organoids are constructed, including: Calculate the number of extracted ectopic endometrial cells and eutopic endometrial cells; remove the supernatant, resuspend the cell pellet with 1 ml of basal culture medium, pipette and evenly distribute 5 ul of the pellet, add it to 45 ul of trypan blue dye, count it with a cell counting plate, and centrifuge again for 5 minutes at 1000 rpm; Select a sterile 24-well plate and drop the two groups of cells into the 24-well plate at a ratio of 1x105 cells mixed with 30ul of matrix gel per well to construct paired endometriosis ectopic endometrial organoids and endometriosis eutopic endometrial organoids; After the gel drop solidifies, add 0.5 ml of endometrial organoid culture medium preheated at 37 degrees to each well and culture in a cell culture incubator containing 5% CO2, changing the medium every 3-4 days.

4. The uterine eutopic endometrium and ectopic endometrium organoid induced malignant transformation model and its establishment method and application as claimed in claim 3, characterized in that: The components and concentrations of the endometrial organoid culture medium are as follows: The main ingredients include: DMEM / F12 + glutamine and 4-hydroxyethylpiperazineethanesulfonic acid; Add the following ingredients: Recombinant human R-spine protein 1 500ng / ml; Recombinant human Noggin 100 ng / mL; B27 additive 2%; N-2 additive 1%; GlutaMAX TM 1%; Insulin-transferrin-selenium 1%; Penicillin-streptomycin-amphotericin B mixed solution 1%; Nicotinamide 2mM; A83-01 0.5µM; N-acetyl-L-cysteine ​​2 mM; Recombinant human epidermal growth factor 50ng / ml; β-recombinant human basic fibroblast growth factor 2 ng / ml; recombinant human fibroblast growth factor 10 10 ng / ml; SB202190 10µM; Y-27632 10µM.

5. The uterine eutopic endometrium and ectopic endometrium organoid induced malignant transformation model and its establishment method and application as claimed in claim 4, characterized in that: Each time, the endometrial organoid culture medium was prepared according to the required amount according to the composition and concentration of the endometrial organoid culture medium, filtered through a 0.22 μm syringe filter and stored, and used within one week.

6. The uterine eutopic endometrium and ectopic endometrium organoid induced malignant transformation model and its establishment method and application as claimed in claim 1, characterized in that: Passaging of ectopic endometrium organoids and eutopic endometrium organoids, including: Observe for 7-14 days, and when the organoid density reaches 70%-80%, subculture at a subculture ratio of 1:2-1:

3. Use P3-P6 organoids for subsequent experiments.

7. The uterine eutopic endometrium and ectopic endometrium organoid induced malignant transformation model and its establishment method and application as claimed in claim 5, characterized in that: Subsequent experiments using P3-P6 organoids include: After removing the culture medium next to the matrix gel, add basal culture medium pre-cooled at 4°C, use the tip of the gun rinsed with anti-adhesion rinse to break up the gel droplets, blow and beat several times, transfer to the rinsed centrifuge tube, centrifuge at 4°C for 5 minutes, 1500 rpm, and remove the clean liquid layer and gel layer; Add 5 volumes of 37°C preheated organoid digestion solution, pipette thoroughly, and incubate at 37°C for 5 minutes; An equal volume of DMEM / F12 complete medium was added to terminate the digestion, and the mixture was centrifuged at 1500 rpm for 3 min at 4°C. DMEM / F12 basal medium was added and centrifuged again at 1500 rpm for 3 min at 4°C to obtain a cell pellet. Take out the corresponding volume of matrix gel that has been liquefied at low temperature, resuspend the cell pellet on ice according to the subculture ratio, pipette evenly and inoculate it into the cell culture plate. After the gel drop solidifies, add the endometrial organoid culture medium preheated at 37°C.

8. The uterine eutopic endometrium and ectopic endometrium organoid induced malignant transformation model and its establishment method and application as claimed in claim 1, characterized in that: Using high concentrations of estrogen to stimulate endometriosis ectopic endometrial organoids and endometriosis eutopic endometrial organoids, including: Use DMSO to dissolve estrogen to prepare an estrogen stock solution with a concentration of 10mM, and dilute it at a ratio of 1:1000 to prepare endometrial organoid culture medium containing 10μM estradiol. Change the medium every 3-4 days and continue stimulation for 4 weeks; the control group is treated with the same concentration of DMSO.

9. An endometriosis organoid model established by the method for establishing an endometriosis organoid model as described in any one of claims 1 to 8.

10. Application of an endometriosis organoid model to obtain the trend of change towards ovarian endometrioid adenocarcinoma.

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