Establishment and application of human ovarian ectopic inner membrane cell strain based on SV40T gene transfection

Abstract

The invention discloses establishment and application of a human ovarian ectopic inner membrane cell strain (HOEEC) based on SV40T gene transfection, and relates to the technical field of biology, and the establishment comprises the following steps: S1, collecting a specimen; s2, isolated culture of human ovarian ectopic inner membrane primary cells; s3, amplification and extraction of plasmids; s4, performing lentivirus transfection to establish a human ovarian ectopic inner membrane cell strain; the method comprises the following steps: firstly, extracting and separating to obtain primary cells of the human ectopic ovarian endometrium, then establishing the human ectopic ovarian endometrium cell strain by transfecting SV40T, comparing the differences between the HOEEC and the primary cells in the aspects of cellular morphology, passage condition, multiplication capacity, invasion capacity, hormone receptor expression and estrogen reaction, and detecting the number and morphology of HOEEC chromosomes and the existence of in-vivo tumorigenicity. A new human ovarian ectopic endometrial cell strain is established by transfecting the SV40T gene for the first time, and an in-vitro model is provided for studying the pathogenesis of ovarian endometriosis.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to the establishment and application of a human ovarian ectopic endometrial cell strain based on SV40T gene transfection. Background Art

[0002] Endometriosis (EMS) refers to a disease caused by the ectopic location of active endometrial tissue outside the uterine cavity. It is one of the main causes of pelvic pain and infertility in women, and about 6-12% of women of childbearing age suffer from this disease. EMS mainly occurs in the ovaries, pelvic peritoneum and fallopian tubes, among which ovarian EMS is one of the most common types, accounting for about 40% of EMS. It has attracted much attention due to its high recurrence rate and potential risk of malignant transformation.

[0003] Primary ectopic endometrial cells have the disadvantages of being difficult to separate and purify, growing slowly, and having a short survival time, which limits their application. At present, there are few human ovarian ectopic endometrial cell lines, and only two of them are established by transfecting telomerase. However, transfection of telomerase can cause unbalanced translocation between the long arms of cell chromosomes, and the success rate is low. Therefore, it is urgent to establish new human ovarian ectopic endometrial cell lines. SV40T is one of the widely used immortalization genes. It interacts with cell cycle regulatory proteins to prevent apoptosis induced by cell cycle progression and immortalize cells. More importantly, compared with telomerase, transfection of SV40T has higher gene expression efficiency and transfection success rate. At present, there is no human ovarian ectopic endometrial cell line established using SV40T.

[0004] In view of this, the present invention provides a method for establishing a human ovarian ectopic endometrial cell line based on SV40T gene transfection and its application to overcome the above problems. Summary of the invention

[0005] The purpose of the present invention is to provide a human ovarian ectopic endometrial cell line based on SV40T gene transfection and its application, so as to solve the problems raised in the above background technology.

[0006] In order to solve the above technical problems, the present invention provides a method for establishing a human ovarian ectopic endometrial cell line based on SV40T gene transfection, which is characterized by comprising the following steps:

[0007] S1. Specimen collection: Obtain fresh ovarian endometriosis clinical specimens from patients diagnosed with left ovarian endometriosis stage IV. The patients had not received any hormone treatment 3 months before surgery.

[0008] S2. Isolation and culture of primary human ovarian ectopic endometrial cells: The clinical specimens were rinsed with PBS to remove blood clots and tissue fragments, and cut into 1-2 mm 3The tissue blocks were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, 0.1% type I collagenase was added and digested in a 37°C water bath for 90 min, with continuous shaking during the period. The digested tissue blocks were first filtered through a 100-mesh sterile sieve, the filtrate was collected, and then the epithelial cells and mesenchymal cells were separated through a 400-mesh sterile sieve. Finally, the cell suspension was placed in DMEM / F12 culture medium containing 10% fetal bovine serum and double antibodies, and placed in an incubator at 37°C and 5% CO2 for static culture. The medium was changed after 24 hours;

[0009] S3. Amplification and extraction of plasmids: Add 2 μL of SSR69 plasmid containing SV40T gene and lentiviral vector pCL-Ampho plasmid to 100 μL competent E. coli, mix gently, let stand on ice for 20 min, heat the centrifuge tube in a 42°C water bath for 90 s, quickly cool on ice for 2 min after heating, add 1 mL of Luria-Bertani to the centrifuge tube, shake at 37°C, 150 rpm for 1 h, until the liquid becomes slightly turbid, take 20, 40, 60, 80, and 100 μL of the above bacterial solution and spread them on solid culture plates containing antibiotics, first positive Place the tube upside down for 30 minutes, then invert and culture at 37°C for 12 hours, take a single colony and mix it with Luria-Bertani liquid medium, take 5 mL of the mixture after culturing for 12 hours, add it to a centrifuge tube, centrifuge at 4000 rpm for 5 minutes to collect bacteria, transfer the bacteria to a 1.5 mL centrifuge tube, add sterile sodium acetate and anhydrous ethanol in sequence, centrifuge at 4°C, 12000 rpm for 5 minutes, discard the supernatant, add 80% ethanol to wash the precipitate twice, dry it at room temperature for 5 minutes, then add 25 μL of enzyme-free water, and finally measure the purity and concentration of the plasmid with a spectrophotometer;

[0010] S4. Establishment of human ovarian ectopic endometrial cell line by lentiviral transfection: set up the transfection system, add 250 μL Opti-MEM, 1 μg lentiviral packaging plasmid pCL-Ampho, 2 μg plasmid SSR69 containing SV40T gene and 15 μL Lipofectamine2000 in sequence, vortex mix for 10 seconds, leave at room temperature for 20 minutes, and transfect after HEK293T cells adhere to the wall and grow to 70%. Collect the virus supernatant at 36, 60, 84 and 108 hours after transfection, centrifuge at 1200 rpm for 5 minutes, and filter out impurities with a 0.45 μm microporous filter membrane to obtain the lentiviral solution containing SV40T gene. Infect primary cells with the lentiviral solution. After 72 hours of infection, use 150 μg mL -1Primary cells were selected for hygromycin B resistance for 14 days, and then monoclonal cells were obtained by limiting dilution. The surviving resistant clones were subcultured and amplified to establish a human ovarian ectopic endometrial cell line.

[0011] Furthermore, the established HOEEC cells were cryopreserved, revived and cultured, and the specific steps were as follows:

[0012] Collect logarithmic growth phase cells with good growth status and no contamination, wash the cells twice with PBS, add 0.25% trypsin to digest the cells for 2-3 minutes, centrifuge at 1000rpm for 5 minutes, discard the supernatant, add the prepared cell freezing solution (FBS: DMSO = 9: 1), divide into 1mL cryotubes, seal with sealing film, place at 4℃ for 1h, transfer to -20℃ for 8h, and finally store at -80℃ or liquid nitrogen; take out the cell lines from -80℃ or liquid nitrogen and immediately Place in a 37°C water bath to thaw, add the cell suspension to 5mL of culture medium, centrifuge at 1000rpm for 5min, discard the supernatant, re-add 1mL of culture medium to resuspend the cells, transfer to a culture flask, and culture in an incubator at 37°C and 5% CO2. Replace the culture medium the next day; primary cells and HOEEC were cultured in DMEM / F12 medium containing 10% fetal bovine serum and double antibodies, and HEK293T and HEC1-B cells were cultured in MEM medium containing 10% fetal bovine serum and double antibodies.

[0013] Furthermore, HOEEC and primary cells were tested in many aspects through cell counting, immunofluorescence, DNA extraction and amplification, Transwell chamber experiment, MTT method, in vitro decidualization experiment, RNA extraction and RT-qPCR method, karyotype analysis, in vivo tumor formation experiment and HE staining method.

[0014] Furthermore, the cell counting method is to wipe the coverslip and the cell counter clean, take an appropriate amount of the mixed cell suspension, drop it on the edge of the coverslip, and observe and count under a microscope. The number of cells contained in each 1mL of culture medium = the total number of cells in 4 large squares / 4×10 4 .

[0015] Furthermore, the immunofluorescence method was performed by isolating cells at 2×10 4 / well were inoculated in a 24-well plate, and the cells were taken out when the cell density reached 70-80%, washed twice with PBS, fixed with 4% paraformaldehyde at room temperature for 10 minutes, permeabilized and fixed with 0.25% TritonX-100 for 30 minutes, and then blocked with 10% goat serum at room temperature for 1 hour; then primary antibodies were added: mouse anti-human vimentin, keratin (1:200 dilution), rabbit anti-human forkhead box protein A2 (1:200 dilution), rabbit anti-human serum binding globulin (1:300 dilution), mouse anti-human SV40T (1:50 dilution), incubated overnight at 4°C, washed three times with PBS, and then goat anti-rabbit FITC (1:100 dilution) and goat anti-mouse rhodamine (1:20 dilution) secondary antibodies were added, incubated at room temperature for 1 hour in the dark, washed three times with PBS, and sealed with anti-fluorescence quenching sealing agent (containing DAPI), observed under laser confocal microscope, and photographed.

[0016] Furthermore, the karyotype analysis method is to subculture cells with good growth status for 6 days and add 0.2 μg mL -1 Colchicine solution was continued to be cultured in the incubator for 5-6 hours; the cells were digested and collected in a 15 mL centrifuge tube, centrifuged at 1200 rpm for 10 minutes, and then 5 mL of 0.075 mol·L -1 The KCL hypotonic solution was pipetted evenly, placed in an incubator for incubation for 15-20 minutes, 1 mL of fixative solution (methanol: glacial acetic acid = 3:1) was slowly added along the tube wall, and allowed to stand at room temperature for 5 minutes. The centrifugation operation was performed at 1200 rpm and continued for 10 minutes. The supernatant was discarded, and 3 mL of fixative solution was added, pipetted evenly, and allowed to stand at room temperature for 30 minutes. The centrifugation operation was performed at 1200 rpm and continued for 10 minutes. The supernatant was discarded, and 3 mL of fixative solution was added again, pipetted evenly, and allowed to stand at room temperature. 30min, 1200rpm, centrifuge for 10min, discard the supernatant, then add 1mL of fixative, blow thoroughly until the cells turn milky white, use a dropper to absorb the cell suspension, drop it from a height of 50cm onto a frozen glass slide precooled at 4℃, immediately fix it by burning it 3 times under an alcohol lamp, place it at room temperature, and after drying, stain it with Giemsa for 10min, rinse the stain with water, decolorize it with 75% alcohol after drying, seal it with neutral resin, and examine it under a microscope at 100 times with cedar oil.

[0017] An application of the human ovarian ectopic endometrial cell line established based on SV40T gene transfection as described in any one of claims 1 to 6 in studying the pathogenesis of ovarian EMS.

[0018] Furthermore, human ovarian ectopic endometrial cell lines can be used to study the therapeutic mechanism of endometriosis on ovarian EMS, including inhibiting cell proliferation by downregulating the Notch pathway to inhibit the expression of proliferation markers.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] Primary human ovarian ectopic endometrial cells were isolated and extracted, and the purity of interstitial cells reached 99%. At the same time, identification results showed that the primary cells originated from the uterus rather than the ovaries; HOEEC carrying the SV40T gene was successfully established. This cell line maintained the interstitial phenotype of the primary cells and could be sustainably passaged. There were no significant differences in its growth morphology, proliferation ability, invasion ability, hormone receptor expression and estrogen response compared with the primary cells. At the same time, HOEEC did not show abnormalities in chromosome number and morphology, and did not have in vivo tumorigenicity.

[0021] A human ovarian ectopic endometrial cell line carrying the SV40T gene was successfully established. The cell line maintained the mesenchymal phenotype of the primary cells and continued to grow after the 35th generation. There were no significant differences in its cell morphology, proliferation ability, invasion ability, hormone receptor expression and estrogen response compared with the primary cells. The number and morphology of its chromosomes were normal, and it did not have in vivo tumorigenicity, so it can be used to study the mechanism of ovarian EMS.

[0022] Endotropin can significantly inhibit the proliferation and plate clone formation of human ovarian ectopic endometrial cell lines, and reduce the expression of proliferation indicators Ki67, PCNA, CyclinD1 and c-Myc. Endotropin can downregulate the Notch pathway, and antagonize the Notch pathway activator VPA to activate the Notch pathway, thereby promoting proliferation. It can also cooperate with the Notch pathway inhibitor DAPT to downregulate the Notcht pathway, thereby inhibiting proliferation.

[0023] For the first time, a new human ovarian ectopic endometrial cell line was established by transfecting the SV40T gene, providing an in vitro model for studying the pathogenesis of ovarian EMS; for the first time, the role of endometriosis in inhibiting the proliferation of human ovarian ectopic endometrial cell lines was explored, and its mechanism was achieved by downregulating the Notch pathway and inhibiting the expression of proliferation markers Ki67, PCNA, CyclinD1 and c-Myc, providing a theoretical basis for the treatment of ovarian EMS with endometriosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The invention shows the establishment of a human ovarian ectopic endometrial cell line based on SV40T gene transfection and the growth morphology of human ovarian ectopic endometrial primary cells in its application;

[0025] Figure 2 The invention provides a human ovarian ectopic endometrial cell line based on SV40T gene transfection and immunofluorescence expression of vimentin, keratin, serum binding globulin and forkhead box protein A2 in primary human ovarian ectopic endometrial cells in its application;

[0026] Figure 3 The invention provides a human ovarian ectopic endometrial cell line based on SV40T gene transfection and the expression of SV40T in HOEEC and primary cells detected by PCR in its application;

[0027] Figure 4 The invention provides a human ovarian ectopic endometrial cell line based on SV40T gene transfection and an immunofluorescence method for detecting the expression of SV40T in HOEEC.

[0028] Figure 5 The present invention is a human ovarian ectopic endometrial cell line based on SV40T gene transfection and an immunofluorescence method for identifying the expression of vimentin and keratin in HOEEC in its application;

[0029] Figure 6 The invention provides a human ovarian ectopic endometrial cell line based on SV40T gene transfection and the morphology and growth cycle diagram of primary cells and HOEEC in its application;

[0030] Figure 7 The figure is a diagram of the establishment of a human ovarian ectopic endometrial cell line based on SV40T gene transfection and the proliferation of primary cells and HOEEC in its application;

[0031] Figure 8 The invention shows the establishment of a human ovarian ectopic endometrial cell line based on SV40T gene transfection and the detection of the invasion ability of primary cells and HOEEC using a Transwell chamber in its application;

[0032] Fig. 9 The present invention is a human ovarian ectopic endometrial cell line based on SV40T gene transfection, and the expression diagram of estrogen receptors ER-α, ER-β and progesterone PR in primary cells and HOEEC in its application;

[0033] Fig.10 The figure is a diagram showing the establishment of a human ovarian ectopic endometrial cell line based on SV40T gene transfection and the effect of estradiol on the proliferation of primary cells and HOEEC in its application;

[0034] Fig.11 The figure is a diagram of the establishment of a human ovarian ectopic endometrial cell line based on SV40T gene transfection and the effect of estradiol on the expression of decidual marker genes in primary cells and HOEEC in its application;

[0035] Fig.12 The invention provides a human ovarian ectopic endometrial cell line based on SV40T gene transfection and a HOEEC chromosome karyotype analysis diagram in its application;

[0036] Fig.13 This is a diagram of the establishment of a human ovarian ectopic endometrial cell line based on SV40T gene transfection and the tumorigenicity experiment in nude mice used in the application to detect the tumorigenicity of HOEEC. DETAILED DESCRIPTION

[0037] 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.

[0038] Primary ectopic endometrial cells have the disadvantages of being difficult to separate and purify, growing slowly, and having a short survival time, which limits their application. At present, there are few human ovarian ectopic endometrial cell lines, and only two of them are established by transfecting telomerase. However, transfection of telomerase can cause unbalanced translocation between the long arms of cell chromosomes, and the success rate is low. Therefore, it is urgent to establish new human ovarian ectopic endometrial cell lines. SV40T is one of the widely used immortalization genes. It interacts with cell cycle regulatory proteins to prevent apoptosis induced by cell cycle progression and immortalize cells. More importantly, compared with telomerase, transfection of SV40T has higher gene expression efficiency and transfection success rate. At present, there is no human ovarian ectopic endometrial cell line established using SV40T.

[0039] The pathogenesis of ovarian EMS is still unclear. Sampson reported the first case of ovarian EMS in 1921 and proposed the classic implantation theory, which believes that active endometrial tissue is attached to sites outside the uterine cavity (such as ovaries, pelvic cavity, peritoneum, etc.), and then proliferates to cause EMS. Ki67 is a nuclear antigen associated with cell proliferation, PCNA is a proliferation marker protein that only appears in proliferating cells, CyclinD1 is a cyclin that regulates key sites of the cell cycle, and c-Myc is a multifunctional transcription factor that regulates genes related to cell proliferation and angiogenesis. All four are considered important markers for evaluating cell proliferation activity. Studies have found that Ki67, PCNA, c-Myc, and CyclinD1 are highly expressed in ectopic lesions, and inhibiting their expression can achieve the effect of treating EMS, but the specific mechanism of regulating proliferation has not yet been elucidated.

[0040] The Notch pathway is an important pathway for regulating cell development. After the ligand binds to the receptor Notch1 or Notch2, Notch1 / 2 is cleaved to form the intracellular cleavage body NICD. NICD is transferred to the nucleus to activate the expression of downstream target genes Hey1 and Hes1. In EMS, compared with normal and eutopic endometrium, the Notch pathway is highly expressed in ectopic endometrium. Its pathway inhibitor can inhibit the proliferation of ectopic endometrial epithelial cells, but the specific mechanism of action has not yet been elucidated. The Notch pathway also plays a key role in the proliferation of mouse osteosarcoma cells and embryonic stem cells and rat tracheal epithelial cells. When the Notch pathway is inactivated, the expression of Ki67, PCNA, c-Myc and CyclinD1 is downregulated, inhibiting cell proliferation. When the Notch pathway is activated, the expression of Ki67, PCNA, c-Myc and CyclinD1 is upregulated, promoting cell proliferation. However, it is still unclear whether the Notch pathway can be affected in ovarian EMS to regulate the expression of Ki67, PCNA, c-Myc and CyclinD1, thereby inhibiting the proliferation of ectopic endometrium.

[0041] See also Figure 1-Figure 13 , as shown, the establishment and application of a human ovarian ectopic endometrial cell line based on SV40T gene transfection:

[0042] Establishment of HOEEC:

[0043] Immortalized cell lines can overcome the shortcomings of primary cells such as short survival time and slow growth rate, and have become an important in vitro model for EMS research. Currently, there is a lack of human ovarian ectopic endometrial cell lines, and SV40T is a common transfection gene used to establish immortalized cell lines. In this chapter, human ovarian ectopic endometrial primary cells were obtained by separation and purification, and the SV40T gene was introduced into primary cells using retroviral vectors to establish HOEEC. The cell morphology, passage, proliferation ability, invasion ability, hormone receptor expression and estrogen response of primary cells and HOEEC were then tested, as well as the chromosome number and morphology of HOEEC, and whether it has tumorigenicity in vivo.

[0044] Experimental Materials:

[0045] Specimen collection:

[0046] Endometriosis tissue specimens were obtained from the First Affiliated Hospital of Chongqing Medical University. The patient was a 25-year-old female diagnosed with left ovarian endometriosis stage IV. She had not received any hormone therapy 3 months before surgery and had signed an informed consent form. This experiment was approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University, with the ethics review number: 2019-059.

[0047] Cell lines:

[0048] Cell lines used in this experiment: human embryonic kidney HEK293T (gift from Professor Xu Xiaoyu of the School of Pharmacy, Southwest University), endometrial cancer cell line HEC1-B (purchased from the China Center for Type Culture Collection).

[0049] Experimental animals:

[0050] SPF-grade female nude mice, weighing 16 - 18 g, were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. The production license number of experimental animals: SCXK (Xiang) 2020 - 0006, certificate number:

[0051] No.430727210102423286. The nude mice were housed in the Experimental Animal Center of the School of Pharmacy, Southwest University, with appropriate temperature and humidity, and free access to food and water. The animal use license number: SYXK (Yu) 2020 - 0006. The experimental procedures complied with the "Regulations on the Administration of Experimental Animals" of China and the relevant regulations of the Experimental Animal Ethics Committee of Southwest University. The ethical review number: IACUC - 20201110 - 02.

[0052] Reagents and consumables:

[0053] Experimental reagents:

[0054] Newborn calf serum;

[0055] Type I collagenase;

[0056] Sijiqing fetal bovine serum;

[0057] pCL - Ampho plasmid;

[0058] SSR69 plasmid;

[0059] Ampicillin (AB0028 - 25g);

[0060] Competent Escherichia coli (B528413 - 0020);

[0061] Lipofectamine2000 (11668 - 027);

[0062] Opti - MEM (31985 - 070);

[0063] Plasmid miniprep kit (27104);

[0064] Hygromycin B;

[0065] HiPure Tissue DNA MicroKit (D3125 - 02) DMSO; MTT;

[0066] Crystal violet;

[0067] Trizol Reagent (Cat. No. 15596018);

[0068] Chloroform;

[0069] Isopropyl alcohol;

[0070] DEPC water;

[0071] ProTaqHS premixed qPCR Kit;

[0072] EvoM-MLV reverse transcription premix kit;

[0073] 17β-estradiol (E8140);

[0074] Colchicine;

[0075] PCR eight-tube strip;

[0076] Transwell plates;

[0077] Cell culture flasks and plates;

[0078] Vimentin antibody (60330-1-lg);

[0079] Cytokeratin antibody (66483-1-lg);

[0080] FOXA2 antibody (22474-1-AP);

[0081] HP antibody (DF6467);

[0082] SV40T antibody (SC-147);

[0083] FITC goat anti-rabbit secondary antibody (A0562);

[0084] Rhodamine antibody goat anti-mouse secondary antibody (SA00007-1);

[0085] Experimental instruments (consumables):

[0086] Main experimental instruments:

[0087] Cell culture incubator (model 6000);

[0088] Biological safety cabinet (model: HR40-ⅡA2);

[0089] Inverted fluorescence microscope (model: DFC310FX);

[0090] Laser confocal microscope (model: LSM780);

[0091] Vernier caliper;

[0092] ELISA reader (model: Elx800);

[0093] Desktop low-temperature centrifuge (model: 5417R);

[0094] Pipette (0.1μL-5mL);

[0095] High pressure steam sterilizer (model: CL-32L);

[0096] Vertical ultra-low temperature refrigerator (model: UF3410);

[0097] Micro vortex mixer (model: WH-2);

[0098] Palm type centrifuge (model: LX-100);

[0099] pH meter (model: FE20);

[0100] Roller mixer (model: SRT-202);

[0101] CFX96 Real-Time PCR instrument;

[0102] Micro-amount nucleic acid quantifier;

[0103] Electric constant temperature water bath (model: HWS-24);

[0104] Electronic balance (model: EL2046);

[0105] Ultrapure water system (model: MilliporeSynergy);

[0106] Ice maker (model: XB70);

[0107] Primer sequences:

[0108] The primers used in this experiment were synthesized by Shanghai Sangon Biotechnology Co., Ltd. The primer sequences are as follows:

[0109]

[0110]

[0111] Experimental methods:

[0112] Isolation and culture of primary human ovarian ectopic endometrial cells:

[0113] Fresh ovarian endometriosis clinical specimens obtained during surgery were rinsed with PBS to remove blood clots and tissue fragments, and then cut into tissue blocks of about 1-2 mm3, placed in a centrifuge, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and 0.1% type I collagenase was added to digest in a 37°C water bath for 90 minutes with continuous shaking during the period. The digested tissue blocks were first filtered through a 100-mesh sterile sieve, the filtrate was collected, and then the epithelial cells and interstitial cells were separated through a 400-mesh sterile sieve. Finally, the cell suspension was placed in DMEM / F12 culture medium containing 10% fetal bovine serum and double antibodies, and placed in an incubator at 37°C and 5% CO2 for static culture to observe the growth of primary cells. The medium was changed after 24 hours.

[0114] Plasmid amplification and extraction:

[0115] Add 2 μL of SSR69 plasmid containing SV40T gene and lentiviral vector pCL-Ampho plasmid to 100 μL competent E. coli, mix gently, let stand on ice for 20 min, put the centrifuge tube in a 42°C water bath and heat for 90 s. After heating, quickly put it on ice to cool for 2 min, then add 1 mL of Luria-Bertani to the centrifuge tube, shake at 37°C, 150 rpm for 1 h until the liquid becomes slightly turbid, take 20, 40, 60, 80, and 100 μL of the above bacterial solution and spread it on the solid culture plate containing antibiotics, first place the front side for 30 min. in, then invert and culture at 37°C for 12h, take a single colony and mix it with Luria-Bertani liquid medium, after culturing for 12h, take 5mL of the mixture and add it to a centrifuge tube, centrifuge at 4000rpm for 5min to collect the bacteria, transfer the bacteria to a 1.5mL centrifuge tube, add sterile sodium acetate and anhydrous ethanol in turn, centrifuge at 4°C, 12000rpm for 5min, discard the supernatant, add 80% ethanol to wash the precipitate twice, dry it at room temperature for 5min, then add 25μL of enzyme-free water, and finally measure the purity and concentration of the plasmid with a spectrophotometer.

[0116] Establishment of human ovarian ectopic endometrial cell line by lentiviral transfection:

[0117] Set up the transfection system: add 250 μL Opti-MEM, 1 μg of lentiviral packaging plasmid pCL-Ampho, 2 μg of plasmid SSR69 containing SV40T gene and 15 μL Lipofectamine2000 in sequence, vortex mix for 10 seconds, leave at room temperature for 20 minutes, and transfect after HEK293T cells grow to 70% of the cell adherence. Collect the virus supernatant at 36, 60, 84 and 108 hours after transfection, centrifuge at 1200 rpm for 5 minutes, and filter out impurities with a 0.45 μm microporous filter membrane to obtain the lentiviral solution containing the SV40T gene. Infect primary cells with the lentiviral solution. After 72 hours of infection, screen the primary cells with 150 μg·mL-1 hygromycin B resistance for 14 days, and then use the limiting dilution method to obtain monoclonal cells, and subculture and amplify the surviving resistant clones.

[0118] Cryopreservation of cells:

[0119] Collect logarithmic growth phase cells that are in good growth condition and uncontaminated, wash the cells twice with PBS, add 0.25% trypsin to digest the cells for 2-3 minutes, centrifuge at 1000rpm for 5 minutes, discard the supernatant, add the prepared cell freezing solution (FBS: DMSO = 9: 1), divide into 1 mL cryovials, seal with sealing film, place at 4°C for 1 hour, transfer to -20°C for 8 hours, and finally store at -80°C or in liquid nitrogen.

[0120] Cell recovery and culture:

[0121] The cell lines were taken out from -80℃ or liquid nitrogen, immediately placed in a 37℃ water bath to thaw, the cell suspension was added to 5mL of culture medium, centrifuged at 1000rpm for 5min, the supernatant was discarded, 1mL of culture medium was added to resuspend the cells, and then transferred to a culture bottle, placed in an incubator at 37℃ and 5% CO2, and the culture medium was changed the next day. Primary cells and HOEEC were cultured in DMEM / F12 medium containing 10% fetal bovine serum (Hyclone, USA) and double antibodies, and HEK293T and HEC1-B cells were cultured in MEM medium containing 10% fetal bovine serum (Sijiqing, China) and double antibodies.

[0122] Cell counting:

[0123] Wipe the coverslip and cell counter clean, pipette an appropriate amount of the mixed cell suspension and drop it onto the edge of the coverslip to avoid bubbles. Observe and count under a microscope. The number of cells contained in 1 mL of culture medium = the total number of cells in 4 large squares / 4×104.

[0124] Immunofluorescence

[0125] The cells were seeded in a 24-well plate at a density of 2×104 / well, and taken out when the cell density reached 70-80%. The cells were washed twice with PBS, fixed with 4% paraformaldehyde at room temperature for 10 minutes, permeabilized and fixed with 0.25% TritonX-100 for 30 minutes, and then blocked with 10% goat serum at room temperature for 1 hour. Then, primary antibodies were added: mouse anti-human vimentin, keratin (1:200 dilution), rabbit anti-human forkhead box protein A2 (1:200 dilution), rabbit anti-human serum binding globulin (1:300 dilution), mouse anti-human SV40T (1:50 dilution), incubated overnight at 4°C, washed 3 times with PBS, and then goat anti-rabbit FITC (1:100 dilution) and goat anti-mouse rhodamine (1:20 dilution) secondary antibodies were added, incubated at room temperature for 1 hour in the dark, washed 3 times with PBS, and sealed with anti-fluorescence quenching sealing agent (containing DAPI), observed under laser confocal microscope, and photographed.

[0126] DNA extraction and amplification:

[0127] DNA extraction:

[0128] After the cells have grown to 80%-90%, they are taken out, digested, collected and counted, and the total number of cells is adjusted to 3-5×106. Cell lysis solution and digestion solution are added to the cells, shaken to mix, and then isopropanol is added. The centrifugation operation is carried out at 12000rpm for 5 minutes. Finally, the eluent preheated at 37°C is added, and the cells are allowed to stand at room temperature for 5 minutes. The centrifugation operation is carried out at 12000rpm for 2 minutes. The extracted DNA is stored at -20°C.

[0129] PCR amplification:

[0130] According to the DNA enzyme manual, prepare the corresponding reaction solution in 23 μL system for PCR amplification.

[0131] Set up the reaction system:

[0132] Reaction components Volume (μL) 2XHighFidelityMasterMix 12.5 10 μM upstream primer 1 10 μM downstream primer 1 DNA samples 2 ddH2O 8.5

[0133] Amplification program: ①95℃×5min②95℃×30s③58℃×30s④72℃×30s⑤72℃×30s, cycle ②-④ steps 40 times, and finally keep at 4℃.

[0134] Agarose gel electrophoresis and imaging:

[0135] Weigh 2g agarose, add 100mL 1×TAE liquid, microwave medium-high heat for 4min, add 5μL nucleic acid dye, immediately pour into the gel plate and insert the comb, pull out the comb after cooling and solidification, put it into the electrophoresis tank and start adding samples, add 14μL sample (including 4μL loading buffer) to each well, add 5μL DNA marker on both sides, use 1×TAE liquid for electrophoresis buffer, and set the voltage to 110V. Use a gel imaging system to observe the position of each band after electrophoresis under a UV detector, and take photos for preservation.

[0136] Transwell chamber experiment:

[0137] Place the chamber in a 24-well plate, add 250 μL of serum-free culture medium to the upper chamber, equilibrate at 37°C for 25 minutes, hydrate the filter membrane, aspirate the remaining culture medium, wash with PBS 1-2 times, resuspend the cells with serum-free culture medium, adjust the cell density to 3×104 cells / well, add 200 μL of cell suspension to the upper chamber, add 700 μL of culture medium containing 5% fetal bovine serum to the lower chamber, set 3 replicates for each group, put it in the incubator for 24 hours, then take out the chamber, wash it 3 times with PBS, wipe the cells in the inner layer of the upper chamber with a cotton swab, fix it with 4% paraformaldehyde for 15 minutes, and stain it with 0.1% crystal violet for 15 minutes. Observe and count the cells moved to the outer layer of the microporous membrane under an inverted microscope, and randomly count 3-5 100x fields of view for each sample.

[0138] MTT method:

[0139] Collect and count the cells with good growth status, inoculate them in a 96-well plate at 6×103 / well, mix them evenly, set up 3 replicate wells, and after the cells adhere to the wall, take out one plate after 1, 2, 3, 4, 5 and 6 days, add 20μL of MTT solution (5mg·mL-1) to each well, incubate for 4h in the dark, put the plate on the filter paper, tap it gently to make all the liquid flow out, add 150μL DMSO, shake it in the dark for 10min, and measure the absorbance value of each well at 490nm with an enzyme marker. Calculate the average value of the 3 wells, and draw a growth curve with time as the horizontal axis and absorbance value as the vertical axis.

[0140] In vitro decidualization experiment:

[0141] The cells in good growth state were collected and counted, and seeded in a 12-well plate at a density of 2×105 cells / well. When the cell density reached 70-80%, the culture medium was aspirated. The drug-treated group was 200 nM 17β-estradiol, and 0.1% DMSO was used as the control group. The RNA of the cells was collected 24 hours after the drug administration for RT-qPCR detection.

[0142] RNA extraction:

[0143] According to the ratio of 1-5×107 cells, 1 mL of Trizol reagent was added and mixed. The lysate containing the cells was transferred to a centrifuge tube and allowed to stand at room temperature for 5 min. 0.2 mL of chloroform was added to the above lysate, shaken for 15 s, allowed to stand at room temperature for 5 min, and centrifuged at 4°C, 12000 g for 15 min. The supernatant was transferred to another new centrifuge tube, 0.5 mL of isopropanol was added, mixed thoroughly, allowed to stand at room temperature for 10 min, and centrifuged at 4°C, 12000 g for 10 min. The supernatant was discarded, 1 mL of 80% ethanol (pre-cooled at -20°C) was added to the precipitate, the precipitate was washed, and centrifuged at 4°C, 12000 g for 5 min. Dry at room temperature for 5 min, and 25 μL of enzyme-free water was added to dissolve the RNA precipitate. Use enzyme-free water to calibrate the micro-nucleic acid quantifier to 0.001, add 1.3 μL of sample during measurement, and when the A260 / 280 value is between 1.8-2.0, the sample purity is considered qualified, and the rest is frozen at -80°C.

[0144] RT-qPCR method:

[0145] Remove genomic DNA. Prepare the reaction solution according to the table below and perform the genomic DNA removal reaction. The reaction conditions are 42℃×2min;

[0146] Component name Addition amount 5×gDNAclean Clean Reaction Mix 2.0μL Total RNA 1μg / Total RNA concentration Total RNA up to 10.0μL

[0147] (2) Reverse transcription reaction. Prepare RNA template solution and place in PCR instrument for denaturation and annealing reaction. The reaction conditions are 37℃×15min, 85℃×5s, and 4℃;

[0148] Component name Addition amount The reaction solution prepared in the above table 10.0μL 5×Evo M-MLV RT Reaction Mix 4.0μL RNase free water 6.0μL Total 20.0μL

[0149] (3) Quantitative PCR analysis. The cDNA obtained by the above reverse transcription process can be directly subjected to quantitative PCR analysis. The RT-qPCR reaction conditions are as follows: ① 95℃×30s ② 95℃×5s ③ 60℃×30s, ②-③ steps are cycled 40 times in total, using 2 -△△CT Methods Calculate the relative expression of target genes;

[0150] Component name Addition amount 2×SYBR Green Pro Taq HS Premix 5.0μL Primer F 0.2μL Primer R 0.2μL RNase free water 3.6μL cDNA 1.0μL Total 10.0μL

[0151] Karyotype analysis:

[0152] After subculturing cells with good growth status for 6 days, add 0.2μg·mL-1 colchicine solution to the cells and continue to culture in the incubator for 5-6h. Digest and collect the cells in a 15mL centrifuge tube, centrifuge at 1200rpm for 10min, then add 5mL of 0.075mol·L-1 KCL hypotonic solution, blow evenly, place in the incubator for incubation for 15-20min, slowly add 1mL of fixative (methanol: glacial acetic acid = 3:1) along the tube wall, let stand at room temperature for 5min, centrifuge at 1200rpm for 10min, discard the supernatant, add 3mL of fixative, blow evenly, let stand at room temperature for 30min, centrifuge at 1200rpm for Incubate for 10 minutes, discard the supernatant, add 3 mL of fixative again, pipette evenly, let stand at room temperature for 30 minutes, centrifuge at 1200 rpm for 10 minutes, discard the supernatant, then add 1 mL of fixative, pipette thoroughly until the cells turn milky white, use a dropper to draw up the cell suspension, drop it from a height of 50 cm onto a frozen glass slide precooled at 4°C, immediately fix it by burning it three times under an alcohol lamp, let it stand at room temperature, and after drying, stain it with Giemsa for 10 minutes, rinse the stain with clean water, decolorize it with 75% alcohol after drying, seal the slide with neutral resin, and examine it under a microscope at 100 times with cedar oil.

[0153] In vivo tumorigenesis experiment:

[0154] 12 nude mice were randomly divided into 3 groups, 4 in each group. The cell density was adjusted to 1-5×107 / mL, and the cells were mixed thoroughly. 100 μL of primary cells, HOEEC and HEC1-B cell suspensions were injected into the armpits of nude mice respectively. The tumor size was measured with a vernier caliper every 3-4 days. Tumor volume = 0.52×tumor length (mm)×tumor width (mm)×tumor height (mm). After 28 days of inoculation, the nude mice were anesthetized and killed for photos.

[0155] HE staining:

[0156] Tumor tissues were fixed in 4% formaldehyde for 24 h, embedded in paraffin and sliced. The slices were dewaxed in xylene for 10 min, hydrated with gradient ethanol, stained with hematoxylin-eosin and observed under an optical microscope and photographed.

[0157] Statistical analysis:

[0158] All data were expressed as mean ± standard deviation (x ± SD). One-way analysis of variance was used to compare the data among the groups. SPSS statistical software was used for analysis. The statistical results showed that P < 0.05 indicated a significant difference, and P < 0.01 indicated an extremely significant difference.

[0159] Experimental results:

[0160] Isolation of primary human ovarian ectopic endometrial cells:

[0161] Human ovarian ectopic endometrial primary cells were isolated and grew on the wall 12 hours later, showing a long spindle-shaped, fibrous appearance. They were passaged every 5-6 days, and the cells grew well after passage (see Figure 1 ; Bar=200μm). The purity and origin of primary cells were detected by immunofluorescence double labeling. The results showed that the mesenchymal cell marker vimentin was positively expressed, and the epithelial cell marker keratin was negatively expressed, indicating that the purity of mesenchymal cells reached 99%. At the same time, the endometrial specific markers serum binding globulin and forkhead box protein A2 were positively expressed, indicating that the primary cells originated from the endometrium rather than the ovary. Figure 2 (Bar=100 μm. Vimentin is a mesenchymal cell marker, vimentin; Cytokeratin is an epithelial cell marker, keratin; HP is serum binding globulin; FOXA2 is forkhead box protein A2).

[0162] Successfully established HOEEC carrying SV40T:

[0163] DNA from primary cells and HOEEC was extracted for amplification and electrophoresis. Untransfected primary cells were used as negative controls to observe the expression of the SV40T gene. The PCR results showed that HOEEC had an SV40T-specific amplification band at 97 bp, but not in primary cells (see Figure 3 HOEEC is a human ovarian ectopic endometrial cell line; M is a DNA marker). HOEEC with good growth status were taken for immunofluorescence detection. The results showed that SV40T protein was positively expressed in HOEEC. The above showed that SV40T was successfully transferred into HOEEC (see Figure 4 , Bar=100 μm. HOEEC, human ovarian ectopic endometrial cell line).

[0164] HOEEC is a mesenchymal cell line, and its growth cycle is longer than that of primary cells:

[0165] HOEECs with good growth status were taken for immunofluorescence detection. The results showed that the mesenchymal cell marker vimentin was positively expressed and the epithelial cell marker keratin was negatively expressed, proving that HOEECs maintained the mesenchymal phenotype of primary cells (see Figure 5, Bar=100μm. HOEEC, human ovarian ectopic endometrial cell line; Vimentin, vimentin; Cytokeratin, keratin). The morphological changes and passages of HOEEC and primary cells were observed under a microscope. HOEEC and primary cells had similar morphology, adherent growth, and a long spindle-shaped, fibrous appearance. Currently, HOEEC has grown to the 35th generation and is in good growth condition. Primary cells fell off and died after growing to the 9th generation (see Figure 6 ; In the figure, (A) shows the cell morphology of primary cells and HOEEC at different growth generations, (B) shows the growth cycle of primary cells and HOEEC, Bar = 200 μm; HOEEC is a human ovarian ectopic endometrial cell line).

[0166] The proliferation and invasion abilities of HOEEC are similar to those of primary cells:

[0167] The proliferation and invasion abilities of primary cells and HOEEC were tested by MTT assay and Transwell chamber assay. Primary cells and HOEEC were cultured for 1-6 days. The results of MTT assay showed that the cell number of HOEEC did not change significantly compared with that of primary cells (see Figure 7 ; The proliferation of primary cells and HOEEC was detected by MTT assay after 1-6 days of culture; HOEEC is a human ovarian ectopic endometrial cell line). The results of Transwell chamber experiments showed that there was no significant difference between the number of HOEEC cells and the number of primary cells that passed through the semipermeable membrane (see Figure 8 A, B; Bar=100 μm;). The above results showed that there was no significant difference in the proliferation and invasion ability between HOEEC and primary cells.

[0168] Hormone receptor expression and estrogen response of HOEECs are similar to primary cells:

[0169] DNA was extracted from HOEEC and primary cells for amplification and electrophoresis. The PCR results showed positive expression of estrogen receptors ER-α, ER-β and progesterone receptor PR in HOEEC and primary cells (see Fig. 9 ; M, DNA Marker).

[0170] Primary cells and HOEEC were treated with 17β-estradiol for 24 h. The results of MTT assay showed that the number of cells in the estradiol group increased significantly compared with the blank group (P<0.05) (see Fig.10 A, B; compared with the blank control group, * P<0.05, **P<0.01, x±SD, n=3). RT-qPCR results showed that in primary cells, the mRNA expressions of prolactin and human insulin-like growth factor binding protein 1, markers of decidual cells, in the estradiol group were increased compared with those in the blank control group (P<0.05) (refer to Fig.11 A), in HOEEC, compared with the blank control group, the mRNA level of prolactin was upregulated (P<0.05), and the gene expression of human insulin-like growth factor binding protein 1 was increased (P<0.01) (refer to Fig.11 B; PRL, prolactin; IGFBP1, human insulin-like growth factor binding protein 1). The above results indicate that both primary cells and HOEEC have normal estrogen response.

[0171] HOEEC has a normal diploid karyotype:

[0172] The 20th generation HOEEC was subjected to karyotype analysis, and the results showed that HOEEC had 23 pairs of chromosomes, all of which were normal diploid karyotypes (see Fig.12 ; A: photographing the chromosomes scattered on the slide, B: numbering and aligning the chromosomes), there was no abnormality in the number and morphology of chromosomes in HOEEC, and no chromosome mutation occurred.

[0173] HOEEC are not tumorigenic in vivo:

[0174] In order to test whether HOEEC has tumorigenicity in vivo, primary cells, HOEEC and HEC1-B were inoculated subcutaneously into nude mice and fed for 4 weeks. The experimental results showed that the nude mice injected with primary cells and HOEEC grew well, and no lumps were formed at the inoculation site until the 28th day. The 4 nude mice injected with HEC1-B had visible lumps on the 4th day after inoculation (see Fig.13 AC; A and B show the tumor formation in nude mice on the 28th day after inoculation of primary cells, HOEEC and HEC1-B, and C shows the changes in tumor volume of primary cells, HOEEC and HEC1-B cells in nude mice. The nude mouse masses in the HEC1-B group were taken for HE staining and pathological analysis. The results showed that the nucleus-cytoplasm ratio of the nude mouse mass tissue injected with HEC1-B was high, and nuclear division images were often seen, indicating that it was tumor tissue (see Fig.13 D; D is the structure of HEC1-B cell tumor tissue, Bar=100 μm; HEC1-B is an endometrial cancer cell line).

[0175] In summary:

[0176] Primary human ovarian ectopic endometrial cells were isolated and extracted, and the purity of interstitial cells reached 99%. At the same time, the identification results showed that the primary cells originated from the uterus rather than the ovaries.

[0177] HOEEC carrying the SV40T gene was successfully established. This cell line maintained the mesenchymal phenotype of the primary cells and could be sustainably subcultured. There were no significant differences in its growth morphology, proliferation ability, invasion ability, hormone receptor expression and estrogen response compared with the primary cells. At the same time, HOEEC did not show abnormalities in chromosome number and morphology and did not have in vivo tumorigenicity.

[0178] Summarize:

[0179] A human ovarian ectopic endometrial cell line carrying the SV40T gene was successfully established. The cell line maintained the mesenchymal phenotype of the primary cells and continued to grow after the 35th generation. There were no significant differences in its cell morphology, proliferation ability, invasion ability, hormone receptor expression and estrogen response compared with the primary cells. The number and morphology of its chromosomes were normal, and it did not have in vivo tumorigenicity, so it can be used to study the mechanism of ovarian EMS.

[0180] Neiyixiao can significantly inhibit the proliferation and plate clone formation ability of human ovarian ectopic endometrial cell lines, and reduce the expression of proliferation indicators Ki67, PCNA, CyclinD1 and c-Myc.

[0181] Endoquinolones can downregulate the Notch pathway and antagonize the Notch pathway activator VPA to activate the Notch pathway, thereby promoting proliferation. It can also cooperate with the Notch pathway inhibitor DAPT to downregulate the Notch pathway, thereby inhibiting proliferation.

[0182] For the first time, a new human ovarian ectopic endometrial cell line was established by transfecting the SV40T gene, providing an in vitro model for studying the pathogenesis of ovarian EMS;

[0183] For the first time, the role of endoixifen in inhibiting the proliferation of human ovarian ectopic endometrial cell lines was explored. Its mechanism is achieved by downregulating the Notch pathway and inhibiting the expression of proliferation markers Ki67, PCNA, CyclinD1 and c-Myc, providing a theoretical basis for the treatment of ovarian EMS with endoixifen.

[0184] In the past, human ovarian ectopic endometrial cell lines were mostly established by transfecting telomerase, but there were disadvantages of unbalanced translocation between the long arms of cell chromosomes after transfection and a low success rate. The present invention uses SV40T gene for transfection to establish cell lines. SV40T is one of the widely used immortalization genes, which interacts with cell cycle regulatory proteins, can prevent apoptosis induced by cell cycle progression, and immortalize cells. Compared with telomerase, transfection of SV40T has higher gene expression efficiency and transfection success rate. At present, there is no human ovarian ectopic endometrial cell line established using SV40T.

[0185] Cell phenotype and growth characteristics: The established HOEEC cell line maintains the mesenchymal phenotype of the primary cells and can be continuously subcultured. It can still grow well after the 35th generation. There is no significant difference in its cell morphology, proliferation ability, invasion ability, hormone receptor expression and estrogen response compared with the primary cells. This is in contrast to the existing cell lines in terms of these biological characteristics, demonstrating its unique advantages.

[0186] Cytogenetic characteristics: Karyotype analysis showed that HOEEC had no abnormalities in chromosome number and morphology and did not have in vivo tumorigenicity, which is an important feature that distinguishes it from other cell lines, meaning that this cell line is more reliable and stable in subsequent applications in the study of the pathogenesis of ovarian endometriosis.

[0187] When using the established cell lines to conduct research related to endometriosis, a new mechanism of action was discovered, namely, the inhibitory effect of endometriosis on cell line proliferation and the regulatory mechanism of the Notch pathway.

Claims

1. Establishment of a human ovarian ectopic endometrial cell line based on SV40T gene transfection, characterized in that: The following steps are involved: S1. Specimen collection: Fresh ovarian endometriosis clinical specimens were obtained from patients diagnosed with left ovarian endometriosis stage IV. The patients had not received any hormone treatment 3 months before surgery. S2. Isolation and culture of primary human ovarian ectopic endometrial cells: The clinical specimens were rinsed with PBS to remove blood clots and tissue fragments, and cut into 1-2 mm 3 The tissue blocks were placed in a centrifuge and rotated at 1000 rpm for 5 min, the supernatant was discarded, 0.1% type I collagenase was added and digested in a 37°C water bath for 90 min, with continuous shaking during the period, the digested tissue blocks were first filtered through a 100-mesh sterile sieve, the filtrate was collected, and then the epithelial cells and mesenchymal cells were separated through a 400-mesh sterile sieve, and finally the cell suspension was placed in DMEM / F12 culture medium containing 10% fetal bovine serum and double antibodies, and placed in a 37°C, 5% CO2 incubator for static culture, and the medium was changed after 24 hours; S3. Amplification and extraction of plasmids: Add 2 μL of SSR69 plasmid containing SV40T gene and lentiviral vector pCL-Ampho plasmid to 100 μL competent E. coli, mix gently, let stand on ice for 20 min, heat the centrifuge tube in a 42°C water bath for 90 s, quickly cool on ice for 2 min after heating, add 1 mL of Luria-Bertani to the centrifuge tube, shake at 37°C, 150 rpm for 1 h, until the liquid becomes slightly turbid, take 20, 40, 60, 80, and 100 μL of the above bacterial solution and spread them on solid culture plates containing antibiotics, first positive Place the tube upside down for 30 minutes, then invert and culture at 37°C for 12 hours, take a single colony and mix it with Luria-Bertani liquid medium, take 5 mL of the mixture after culturing for 12 hours, add it to a centrifuge tube, centrifuge at 4000 rpm for 5 minutes to collect bacteria, transfer the bacteria to a 1.5 mL centrifuge tube, add sterile sodium acetate and anhydrous ethanol in sequence, centrifuge at 4°C, 12000 rpm for 5 minutes, discard the supernatant, add 80% ethanol to wash the precipitate twice, dry it at room temperature for 5 minutes, then add 25 μL of enzyme-free water, and finally measure the purity and concentration of the plasmid with a spectrophotometer; S4. Establishment of human ovarian ectopic endometrial cell line by lentiviral transfection: set up the transfection system, add 250 μL Opti-MEM, 1 μg lentiviral packaging plasmid pCL-Ampho, 2 μg plasmid SSR69 containing SV40T gene and 15 μL Lipofectamine2000 in sequence, vortex mix for 10 seconds, leave at room temperature for 20 minutes, and transfect after HEK293T cells adhere to the wall and grow to 70%. Collect the virus supernatant at 36, 60, 84 and 108 hours after transfection, centrifuge at 1200 rpm for 5 minutes, and filter out impurities with a 0.45 μm microporous filter membrane to obtain the lentiviral solution containing SV40T gene. Infect primary cells with the lentiviral solution. After 72 hours of infection, use 150 μg mL -1 Primary cells were selected for hygromycin B resistance for 14 days, and then monoclonal cells were obtained by limiting dilution. The surviving resistant clones were subcultured and amplified to establish a human ovarian ectopic endometrial cell line.

2. The establishment of a human ovarian ectopic endometrial cell line based on SV40T gene transfection as claimed in claim 1, characterized in that: The established HOEEC cells were cryopreserved, revived and cultured. The specific steps are as follows: Collect logarithmic growth phase cells with good growth status and no contamination, wash the cells twice with PBS, add 0.25% trypsin to digest the cells for 2-3 minutes, centrifuge at 1000rpm for 5 minutes, discard the supernatant, add the prepared cell freezing solution (FBS: DMSO = 9: 1), divide into 1mL cryotubes, seal with sealing film, place at 4℃ for 1h, transfer to -20℃ for 8h, and finally store at -80℃ or liquid nitrogen; take out the cell lines from -80℃ or liquid nitrogen and immediately Place in a 37°C water bath to thaw, add the cell suspension to 5mL of culture medium, centrifuge at 1000rpm for 5min, discard the supernatant, re-add 1mL of culture medium to resuspend the cells, transfer to a culture flask, and culture in an incubator at 37°C and 5% CO2. Replace the culture medium the next day; primary cells and HOEEC were cultured in DMEM / F12 medium containing 10% fetal bovine serum and double antibodies, and HEK293T and HEC1-B cells were cultured in MEM medium containing 10% fetal bovine serum and double antibodies.

3. The establishment of a human ovarian ectopic endometrial cell line based on SV40T gene transfection as claimed in claim 1, characterized in that: HOEEC and primary cells were tested in many aspects, including cell counting, immunofluorescence, DNA extraction and amplification, Transwell chamber experiment, MTT method, in vitro decidualization experiment, RNA extraction and RT-qPCR method, karyotype analysis, in vivo tumor formation experiment and HE staining.

4. The establishment of a human ovarian ectopic endometrial cell line based on SV40T gene transfection as claimed in claim 3, characterized in that: The cell counting method is to wipe the coverslip and cell counter clean, take an appropriate amount of mixed cell suspension, drop it on the edge of the coverslip, and observe and count under a microscope. The number of cells contained in each 1mL of culture medium = the total number of cells in 4 large squares / 4×10 4 .

5. The establishment of a human ovarian ectopic endometrial cell line based on SV40T gene transfection as claimed in claim 3, characterized in that: Immunofluorescence analysis was performed by centrifuging 2×10 4 / well were inoculated in a 24-well plate, and the cells were taken out when the cell density reached 70-80%, washed twice with PBS, fixed with 4% paraformaldehyde at room temperature for 10 minutes, permeabilized and fixed with 0.25% TritonX-100 for 30 minutes, and then blocked with 10% goat serum at room temperature for 1 hour; then primary antibodies were added: mouse anti-human vimentin, keratin (1:200 dilution), rabbit anti-human forkhead box protein A2 (1:200 dilution), rabbit anti-human serum binding globulin (1:300 dilution), mouse anti-human SV40T (1:50 dilution), incubated overnight at 4°C, washed three times with PBS, and then goat anti-rabbit FITC (1:100 dilution) and goat anti-mouse rhodamine (1:20 dilution) secondary antibodies were added, incubated at room temperature for 1 hour in the dark, washed three times with PBS, and sealed with anti-fluorescence quenching sealing agent (containing DAPI), observed under laser confocal microscope, and photographed.

6. The establishment of a human ovarian ectopic endometrial cell line based on SV40T gene transfection as claimed in claim 3, characterized in that: The karyotype analysis method is to subculture cells in good growth state for 6 days and add 0.2 μg mL -1 Colchicine solution was continued to be cultured in the incubator for 5-6 hours; the cells were digested and collected in a 15 mL centrifuge tube, centrifuged at 1200 rpm for 10 minutes, and then 5 mL of 0.075 mol·L -1 The KCL hypotonic solution was pipetted evenly, placed in an incubator for incubation for 15-20 minutes, 1 mL of fixative solution (methanol: glacial acetic acid = 3:1) was slowly added along the tube wall, and allowed to stand at room temperature for 5 minutes. The centrifugation operation was performed at 1200 rpm and continued for 10 minutes. The supernatant was discarded, and 3 mL of fixative solution was added, pipetted evenly, and allowed to stand at room temperature for 30 minutes. The centrifugation operation was performed at 1200 rpm and continued for 10 minutes. The supernatant was discarded, and 3 mL of fixative solution was added again, pipetted evenly, and allowed to stand at room temperature. 30min, 1200rpm, centrifuge for 10min, discard the supernatant, then add 1mL of fixative, blow thoroughly until the cells turn milky white, use a dropper to absorb the cell suspension, drop it from a height of 50cm onto a frozen glass slide precooled at 4℃, immediately fix it by burning it 3 times under an alcohol lamp, place it at room temperature, and after drying, stain it with Giemsa for 10min, rinse the stain with water, decolorize it with 75% alcohol after drying, seal it with neutral resin, and examine it under a microscope at 100 times with cedar oil.

7. An application of the human ovarian ectopic endometrial cell line based on SV40T gene transfection as described in any one of claims 1 to 6 in studying the pathogenesis of ovarian endometriosis.

8. The use of a human ovarian ectopic endometrial cell line based on SV40T gene transfection as claimed in claim 7, characterized in that: Human ovarian ectopic endometrial cell lines can be used to study the therapeutic mechanism of endometriosis, including the inhibition of cell proliferation by downregulating the Notch pathway to inhibit the expression of proliferation markers.

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