In-vitro human ovarian granular cell pan-apoptosis model and construction method thereof

Abstract

The invention discloses an in-vitro human ovarian granular cell pan-apoptosis model and a construction method thereof, and belongs to the field of reproductive medicine. According to the invention, hungry-treated human ovarian granulosa cells are cultured by using a culture medium containing LPS and IFN-gamma, such that the human ovarian granulosa cell pan-apoptosis model is obtained. The constructed human ovarian granular cell pan-apoptosis model has pan-apoptosis key characteristics such as apoptosis, pyroptosis and necrotic apoptosis at the same time, and can be used as a cell model for researching a pathological mechanism of polycystic ovarian syndrome. The invention establishes a good foundation for researching the pathogenic mechanism of pan-apoptosis on polycystic ovarian syndrome.

Description

Technical Field

[0001] The present invention belongs to the field of reproductive medicine, and in particular relates to an in vitro human ovarian granulosa cell pan-apoptosis model and a construction method thereof. Background Art

[0002] Polycystic ovarian syndrome (PCOS) is the most common reproductive endocrine disorder in women of childbearing age and one of the main causes of ovulatory infertility. Its incidence rate is as high as 15% in women of childbearing age. [1] It is a highly heterogeneous endocrine and metabolic disorder with complex etiology, and its pathogenesis is still unknown. Exploring its molecular mechanism and pathogenic mechanism is very necessary for accurately selecting individualized treatment plans in the future.

[0003] In 2019, American scholar Malireddi first proposed a new type of programmed cell death (PCD), namely PANoptosis. PANoptosis has the key features of pyroptosis, apoptosis, and necroptosis, but it cannot be explained by any of the three PCD pathways alone. [2] PANoptosis is regulated by a cascade of upstream sensors and molecular signals, which can provide a "molecular scaffold" to promote the coupling and binding of proteins and molecules required for pyroptosis (such as NLRP3, ASC and Caspase1), apoptosis (Caspase3 / 8) and necroptosis (RIPK3, RIPK1, MLKL) to assemble into a multimeric complex, the PANoptosis body (PANoptosome). [3,4] It can serve as the "master switch" for the activation of the three PCD pathways and an activation platform for downstream molecules. [5] . So far, four upstream receptor molecules of PANoptosis have been identified, including Z-DNA binding protein 1 (ZBP1), receptor interacting protein kinase 1 (RIPK1), melanoma deficiency factor 2 (AIM2) and Nod-like receptor family pyrin domain-containing protein 12 (NLRP12). They can trigger the assembly of corresponding PANoptosomes in response to specific stimuli, forming four pan-apoptotic bodies with different sensors and regulatory factors, namely ZBP1-PANoptosome, AIM2-PANoptosome, RIPK1-PANoptosome and NLRP12-PANoptosome. [6 ,7,8,9] .

[0004] Studies have found that DHEA-induced PCOS mice have increased LPS in serum, accompanied by increased IFN-γ in serum and ovaries, which plays an important role in the pathogenesis of PCOS through estrogen synthesis dysfunction and granulosa cell apoptosis.

[10] The applicant's previous study first found that the expression level of IFN-γ in granulosa cells of PCOS patients increased and PANoptosis existed, which is one of the important pathological mechanisms of PCOS.

[0005] At present, there is no pan-apoptosis model of granulosa cells. Therefore, constructing an in vitro human ovarian granulosa cell PANoptosis model will help to further study the role of PANoptosis in the pathological mechanism of PCOS, which is undoubtedly of great significance for the study of the pathological mechanism of PCOS.

[0006] References:

[0007] 1.March WA,Moore VM,Willson KJ,Phillips DI,Norman RJ,Davies MJ.Theprevalence of polycystic ovary syndrome in a community sample assessed undercontrasting diagnostic criteria.Hum Reprod.2010;25(2):544–551.

[0008] 2.Christgen S, Zheng M, Kesavardhana S, Karki R, Malireddi RKS, Banoth B, Place DE, Briard B, Sharma BR, Tuladhar S, Samir P, Burton A, KannegantiTD. Identification of the PANoptosome: AMolecular Platform TriggeringPyroptosis, Apoptosis, and Necroptosis (PANoptosis). Front Cell InfectMicrobiol.2020;29;10:237.

[0009] 3. Place DE, Lee S, Kanneganti TD. PANoptosis in microbial infection. CurrOpin Microbiol. 2021; 59:42-49.

[0010] 4.Malireddi R,Kesavardhana S,Karki R,Kancharana B,Burton AR,Kanneganti TD.RIPK1distinctly regulates yersinia-induced inflammatory celldeath,PANoptosis.Immunohorizons,2020,4(12):789-796.

[0011] 5.Samir P,Malireddi RKS,Kanneganti TD.The PANoptosome:a deadlyprotein complex driving pyroptosis,apoptosis,and necroptosis(PANoptosis).Front Cell Infect Microbiol,2020,10:238.

[0012] 6.Lee S,Karki R,Wang Y,Nguyen LN,Kalathur RC,Kanneganti TD.AIM2 formsa complex with pyrin and ZBP1 to drive PANoptosis and hostdefence.Nature.2021;597(7876):415-419.

[0013] 7.Sundaram B,Pandian N,Mall R,Wang Y,Sarkar R,Kim HJ,Malireddi RKS,Karki R,Janke LJ,Vogel P,Kanneganti TD.NLRP12-PANoptosome activatesPANoptosis and pathology in response to heme and PAMPs.Cell.2023;186(13):2783-2801.

[0014] 8. Rostamtabar M, Esmaeilzadeh S, Karkhah A, Amiri M, Rahmani A, Bakouei F, Nouri HR. Elevated expression of IL-18but not IL-1βgene is associated with NALP3 and AIM2inflammasome in Polycystic Ovary Syndrome. Gene. 2020; 731:144352.

[0015] 9. Zheng M, Karki R, Vogel P, Kanneganti TD. Caspase-6Is a Key Regulator of Innate Immunity, Inflammasome Activation, and Host Defense. Cell. 2020; 181(3):674-687.

[0016] 10. Huang J, Chen P, Xiang Y, Liang Q, Wu T, Liu J, Zeng Y, Zeng H, Liang X, Zhou C. Gut microbiota dysbiosis-derived macrophage pyroptosis causes polycystic ovary syndrome via steroidogenesis disturbance and apoptosis of granulosa cells. Int Immunopharmacol. 2022Jun; 107:108717. Summary of the invention

[0017] Since the pathogenesis of PCOS is not yet fully understood, and PANoptosis is one of the important pathological mechanisms of PCOS, the purpose of the present invention is to provide an in vitro human ovarian granulosa cell PANoptosis model and its construction method, so as to build a good foundation for further in-depth study of the role of ovarian granulosa cell PANoptosis in the pathogenesis of PCOS.

[0018] The purpose of the present invention is achieved through the following technical solutions:

[0019] The first aspect of the present invention provides a method for constructing an in vitro human ovarian granulosa cell PANoptosis model, which comprises the following steps: starving human ovarian granulosa cells are cultured using a culture medium containing LPS and IFN-γ to obtain a human ovarian granulosa cell PANoptosis model.

[0020] Furthermore, the method for constructing the in vitro human ovarian granulosa cell PANoptosis model comprises the following steps:

[0021] (1) Human ovarian granulosa cells in the logarithmic growth phase were starved and cultured with medium containing 0.5%-5% serum for 24-72 h;

[0022] (2) Human ovarian granulosa cells that have been starved are cultured in a medium containing LPS, IFN-γ and 10%-20% serum for 12-24 hours to obtain a human ovarian granulosa cell PANoptosis model.

[0023] The human ovarian granulosa cells are preferably KGN cells. The culture medium is preferably DMEM / F12 complete culture medium. The concentration of LPS is preferably 0.2ng / μL-1ng / μL, and the concentration of IFN-γ is preferably 100ng / mL-500ng / mL.

[0024] In step (1), the serum content is preferably 2%, and the culture time is preferably 24 hours.

[0025] In step (2), the LPS content is 1 ng / μL, the IFN-γ content is 100 ng / mL, the serum content is preferably 10%, and the culture time is preferably 24 h.

[0026] The second aspect of the present invention provides a human ovarian granulosa cell PANoptosis model constructed by the above-mentioned construction method, in which the key features of PANoptosis, such as apoptosis, pyroptosis and necroptosis, occur simultaneously in the cell model.

[0027] The third aspect of the present invention provides an application of the above-mentioned human ovarian granulosa cell PANoptosis model as a cell model for studying the pathological mechanism of PCOS.

[0028] Advantages and beneficial effects of the present invention: The present invention provides for the first time an in vitro human ovarian granulosa cell PANoptosis model and a construction method thereof, and the cell model simultaneously has key PANoptosis features such as apoptosis, pyroptosis, and necroptosis. The cell model is specifically characterized by increased expression levels of NLRP3, IL-1β, Caspase1 related to pyroptosis, Caspase3 related to apoptosis, and MLKL mRNA related to necroptosis in the cell model, increased protein levels of Cleaved-Caspase1, N-GSDMD related to pyroptosis, Cleaved-Caspase3 related to apoptosis, and p-MLKL related to necroptosis, increased secretion levels of IL-1β, a pyroptosis-related factor in the cell culture medium supernatant, and increased expression levels of AIM2 mRNA and protein, an upstream receptor molecule of PANoptosis, in the cell model. The cell model provides a good foundation for further in-depth research on the pathogenic mechanism of PANoptosis on PCOS. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 :The expression level of IFN-γ is increased in granulosa cells of PCOS patients.

[0030] (A) IFN-γ mRNA levels in granulosa cells of PCOS patients and control group. (B) IFN-γ secretion levels in granulosa cell culture medium of PCOS patients and control group. ** indicates P < 0.01, *** indicates P < 0.001.

[0031] Figure 2 : The expression levels of PANoptosis-related genes and PANoptosis upstream receptor AIM2mNRA and protein in granulosa cells of PCOS patients are increased.

[0032] (A) mRNA levels of PANoptosis-related molecules (including pyroptosis-related NLRP3, IL-1β, Caspase1, apoptosis-related Caspase3, and necroptosis-related MLKL) in granulosa cells of PCOS and control patients. (B) Protein levels of PANoptosis-related molecules (including pyroptosis-related Caspase1, GSDMD, apoptosis-related Caspase3, and necroptosis-related MLKL) in granulosa cells of PCOS and control patients. (C) Secretion level of pyroptosis-related IL-1β in the culture medium of granulosa cells of PCOS and control patients. (D) mRNA expression level of AIM2, an upstream receptor molecule of PANoptosis, in granulosa cells of PCOS and control patients. (E) Protein expression level of AIM2 in granulosa cells of PCOS and control patients. ** indicates P < 0.01, *** indicates P < 0.001.

[0033] Figure 3 : The expression levels of PANoptosis-related genes and the PANoptosis upstream receptor AIM2 mRNA and protein were increased in KGN cells treated with LPS and IFN-γ.

[0034] (A) mRNA levels of PANoptosis-related molecules (including pyroptosis-related NLRP3, IL-1β, Caspase1, apoptosis-related Caspase3, and necroptosis-related MLKL) in KGN cells after LPS and IFN-γ treatment. (B) Protein levels of PANoptosis-related molecules (including pyroptosis-related Caspase1, GSDMD, apoptosis-related Caspase3, and necroptosis-related MLKL) in KGN cells after LPS and IFN-γ treatment. (C) IL-1β secretion level in the culture supernatant of KGN cells after LPS and IFN-γ treatment. (D) mRNA expression level of AIM2, an upstream sensor molecule of PANoptosis, in KGN cells after LPS and IFN-γ treatment. (E) Protein expression level of AIM2 in KGN cells after LPS and IFN-γ treatment. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0035] Figure 4 : Inhibition of AIM2 expression can inhibit the expression of PANoptosis-related genes at the mRNA and protein levels in KGN cells co-treated with LPS+IFN-γ.

[0036] (A) AIM2 mRNA and protein expression levels after intervention with AIM2 inhibitor A151 in KGN cells co-treated with LPS+IFN-γ. (B) PANoptosis-related molecules (including pyroptosis-related NLRP3, IL-1β, Caspase1, apoptosis-related Caspase3, and necroptosis-related MLKL) mRNA levels after intervention with AIM2 inhibitor A151 in KGN cells co-treated with LPS+IFN-γ. (C) PANoptosis-related molecules (including pyroptosis-related Caspase1, GSDMD, apoptosis-related Caspase3, and necroptosis-related MLKL) protein levels after intervention with AIM2 inhibitor A151 in KGN cells co-treated with LPS+IFN-γ. (D) IL-1β secretion level in the culture medium of KGN cells co-treated with LPS+IFN-γ. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. DETAILED DESCRIPTION

[0037] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0038] Example 1 Increased expression of IFN-γ in granulosa cells of PCOS patients and the presence of PANoptosis

[0039] 1. Follicular fluid was collected from PCOS (n=10, PCOS group) and non-PCOS (n=10, control group) patients who underwent IVF at Renmin Hospital of Wuhan University from April to July 2022. The PCOS group was diagnosed according to the 2003 Rotterdam criteria, that is, at least two of the following three symptoms must be met after excluding other causes: rare ovulation or anovulation; clinical and / or biochemical signs of hyperandrogenism; polycystic ovaries. Non-PCOS patients were female patients with infertility caused by simple male factors (such as oligospermia or asthenozoospermia).

[0040] 2. Granulosa cells were extracted as follows: the follicle aspirate from the patient during oocyte retrieval was centrifuged at 300 g for 20 minutes, part of the supernatant was collected, and granulosa cells were separated from the precipitate by density gradient centrifugation. Granulosa cells were cultured in DMEM / F12 complete medium containing 10% fetal bovine serum at 37°C and 5% CO. 2 Culture for 24h.

[0041] 3. Collect the cultured granulosa cells to extract RNA and protein for RT-qPCR and Western Blot detection respectively, and collect the cell culture medium for ELISA detection.

[0042] 4. The RT-qPCR method was used to detect the IFN-γ mRNA level in granulosa cells, and the ELISA method was used to detect the IFN-γ secretion level in the granulosa cell culture medium.

[0043] Results Figure 1 , the expression level of IFN-γ mRNA in granulosa cells of PCOS group was significantly higher than that of control group (A), and the secretion level of IFN-γ in cell culture medium was also significantly higher than that of control group (B).

[0044] 5. RT-qPCR was used to detect the mRNA levels of PANoptosis-related molecules (including pyroptosis-related NLRP3, IL-1β, Caspase1, apoptosis-related Caspase3, and necroptosis-related MLKL) in granulosa cells.

[0045] Results Figure 2 (A) The mRNA expression levels of NLRP3, IL-1β, Caspase1, Caspase3, and MLKL in granulosa cells of the PCOS group were significantly higher than those of the control group.

[0046] 6. Western Blot was used to detect the protein levels of PANoptosis-related molecules (including pyroptosis-related Caspase1 and GSDMD, apoptosis-related Caspase3, and necroptosis-related MLKL).

[0047] Results Figure 2 (B) The expression levels of Cleaved-Caspase1, N-GSDMD, Cleaved-Caspase3, and p-MLKL proteins in granulosa cells of the PCOS group were significantly higher than those of the control group.

[0048] 7. ELISA method was used to detect the level of pyroptosis-related IL-1β secretion in cell culture medium.

[0049] Results Figure 2 (C) The level of IFN-γ secretion in the culture medium of granulosa cells in the PCOS group was significantly higher than that in the control group.

[0050] 8. RT-qPCR and Western Blot were used to detect the mRNA and protein levels of AIM2, the upstream receptor molecule of PANoptosis in granulosa cells, respectively.

[0051] Results Figure 2 (D and E), the expression levels of AIM2 mRNA (D) and protein (E) in granulosa cells of the PCOS group were significantly higher than those of the control group.

[0052] In summary, the above results confirmed that the expression level of IFN-γ in granulosa cells of PCOS patients was increased, and PANoptosis (including pyroptosis, apoptosis, and necroptosis) was present, and the upstream sensor molecule AIM2 of PANoptosis was activated. Specifically, the mRNA expression levels of NLRP3, IL-1β, Caspase1 related to pyroptosis, Caspase3 related to apoptosis, and MLKL related to necroptosis in granulosa cells increased, the protein levels of Cleaved-Caspase1, N-GSDMD related to pyroptosis, Cleaved-Caspase3 related to apoptosis, and p-MLKL related to necroptosis increased, the secretion level of IL-1β related to pyroptosis in the culture medium increased, and the mRNA and protein expression levels of AIM2, the upstream sensor molecule of PANoptosis, increased.

[0053] Example 2 A method for constructing an in vitro human ovarian granulosa cell PANoptosis model

[0054] 1. Cell Preparation

[0055] 1) In vitro cell culture: KGN cells in good growth condition were cultured in DMEM / F12 containing 10% fetal bovine serum at 37°C and 5% CO 2 Cultured under conventional conditions.

[0056] 2) Cell passaging: When the density of KGN cells reached 80%-90%, they were routinely digested and passaged using trypsin and then inoculated into six-well plates, with 1.2×10^6 cells inoculated per well.

[0057] 3) Starvation treatment: When the cell density of the six-well plate reached 50%-60%, the medium was replaced with DMEM / F12 complete medium containing 2% fetal bovine serum for starvation treatment and culture was continued for 24 hours.

[0058] 2. Drug intervention

[0059] 1) Drug preparation: LPS and IFN-γ drug powders were prepared into drug solutions using 1×PBS as stock solutions and stored at -20°C.

[0060] 2) Drug-treated cells: LPS and IFN-γ stock solutions were diluted to working solutions using DMEM / 12 complete medium containing 10% fetal bovine serum, with concentrations of 1 ng / μL and 100 ng / mL, respectively. After starvation treatment of KGN cells with DMEM / 12 complete medium containing 2% fetal bovine serum for 24 h, the medium was replaced with DMEM / F12 complete medium containing LPS and IFN-γ and cultured for another 24 h to obtain a human ovarian granulosa cell PANoptosis model.

[0061] Example 3 Validation of a human ovarian granulosa cell PANoptosis model

[0062] 1. LPS and IFN-γ treatment of KGN cells

[0063] The six-well plate KGN cells in good growth state were divided into a control group, an LPS group, an IFN-γ group, and an LPS+IFN-γ group. The starved KGN cells were treated with PBS, 1 ng / μL LPS, 100 ng / mL IFN-γ, and 1 ng / μL LPS+100 ng / mL IFN-γ according to the method in Example 2 for 24 h, and the treated KGN cells and culture medium were collected for the following detection.

[0064] 1) RT-qPCR was used to detect the mRNA levels of PANoptosis-related molecules in each group, including pyroptosis-related NLRP3, IL-1β, and Caspase1, apoptosis-related Caspase3, and necroptosis-related MLKL.

[0065] Results Figure 3(A) The mRNA expression levels of NLRP3, IL-1β, Caspase1, Caspase3, and MLKL in the LPS+IFN-γ group were significantly higher than those in the control group.

[0066] 2) Western Blot was used to detect the levels of PANoptosis-related molecular proteins in each group, including pyroptosis-related Caspase1 and GSDMD, apoptosis-related Caspase3, and necroptosis-related MLKL.

[0067] Results Figure 3 (B) The protein expression levels of Cleaved-Caspase1, N-GSDMD, Cleaved-Caspase3, and p-MLKL in the LPS+IFN-γ group were significantly higher than those in the control group.

[0068] 3) ELISA was used to detect the secretion level of IL-1β in the cell culture medium of each group.

[0069] Results Figure 3 (C), the IL-1β secretion level in the culture medium of the LPS+IFN-γ group was significantly higher than that of the control group.

[0070] 4) RT-qPCR and Western Blot were used to detect the mRNA and protein levels of AIM2, the upstream receptor molecule of PANoptosis in each group.

[0071] Results Figure 3 (D and E), the expression levels of AIM2 mRNA (D) and protein (E) in the LPS+IFN-γ group were significantly higher than those in the control group.

[0072] In summary, the above results confirmed that PANoptosis (including pyroptosis, apoptosis, and necroptosis) occurred in KGN cells treated with LPS combined with IFN-γ, and the upstream receptor molecule AIM2 of PANoptosis was activated. Specifically, the expression levels of pyroptosis-related NLRP3, IL-1β, Caspase1, apoptosis-related Caspase3, and necroptosis-related MLKL mRNA increased in KGN cells treated with LPS combined with IFN-γ, the protein levels of pyroptosis-related Cleaved-Caspase1, N-GSDMD, apoptosis-related Cleaved-Caspase3, and necroptosis-related p-MLKL increased, the secretion level of pyroptosis-related IL-1β in the culture medium increased, and the expression levels of PANoptosis upstream receptor molecule AIM2 mRNA and protein increased, which confirmed that the construction of the KGN cell PANoptosis model was successful.

[0073] 2. Use A151, an AIM2 inhibitor, to intervene in KGN cells co-treated with LPS and IFN-γ

[0074] A151 is an AIM2 inhibitory oligonucleotide ODN TTAGGG (A151), the sequence of which is 5'-TTAGGGTTAGGGTTAGGGTTAGGG-3'. It is prepared into a solution using PBS and stored at -20°C.

[0075] The six-well plate KGN cells with good growth status were divided into a control group, an LPS+IFN-γ group, an LPS+IFN-γ+A151 group, and an A151 group. Among them, the control group and the LPS+IFN-γ group were treated with PBS and 1ng / μL LPS+100ng / mL IFN-γ according to the method in Example 2 for 24 hours after starvation treatment of the KGN cells; the LPS+IFN-γ+A151 group and the A151 group were treated with 1μM A151 according to the method in Example 2 for 1 hour after starvation treatment of the KGN cells, and then treated with 1ng / μL LPS+100ng / mL IFN-γ and PBS for 24 hours, respectively, and the treated KGN cells and culture medium were collected for the following detection.

[0076] 1) Detect the mRNA and protein levels of AIM2, the upstream receptor molecule of PANoptosis, in each group using the same method as above.

[0077] Results Figure 4 (A) LPS+IFN-γ treatment in KGN cells increased the expression of AIM2 mRNA and protein levels, while treatment with the AIM2 inhibitor A151 suppressed the LPS+IFN-γ-induced AIM2 high expression.

[0078] 2) The mRNA levels of PANoptosis-related molecules in each group were detected, including pyroptosis-related NLRP3, IL-1β, Caspase1, apoptosis-related Caspase3, and necroptosis-related MLKL, using the same method as above.

[0079] Results Figure 4 (B) LPS+IFN-γ treatment in KGN cells increased the mRNA expression levels of NLRP3, IL-1β, Caspase1, Caspase3, and MLKL, while treatment with the AIM2 inhibitor A151 suppressed the high expression induced by LPS+IFN-γ.

[0080] 3) Detect the levels of PANoptosis-related molecular proteins in each group, including pyroptosis-related Caspase1 and GSDMD, apoptosis-related Caspase3, and necroptosis-related MLKL, using the same method as above.

[0081] Results Figure 4 (C) LPS+IFN-γ treatment in KGN cells increased the expression of Cleaved-Caspase1, N-GSDMD, Cleaved-Caspase3, and p-MLKL protein levels, while treatment with the AIM2 inhibitor A151 suppressed the high expression induced by LPS+IFN-γ.

[0082] 4) Detect the IL-1β secretion level in the KGN culture medium supernatant of each group using the same method as above.

[0083] Results Figure 4 (D) LPS+IFN-γ treatment in KGN cells increased the level of IL-1β secretion in the culture medium, while treatment with the AIM2 inhibitor A151 suppressed LPS+IFN-γ-induced IL-1β secretion.

[0084] In summary, the above results show that inhibiting the expression of PANoptosis upstream sensor molecule AIM2 significantly reduced the expression of PANoptosis-related gene mRNA and protein in the LPS+IFN-γ-induced KGN cell PANoptosis model. This confirms that the LPS+IFN-γ-induced KGN cell PANoptosis model is regulated by the PANoptosis upstream sensor molecule AIM2.

[0085] Table 1. Primer sequences used in RT-qPCR in the above examples

[0086]

[0087]

[0088] The above embodiments are only used to help illustrate the present invention. The implementation methods of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for constructing an in vitro human ovarian granulosa cell pan-apoptosis model, characterized in that: The following steps are involved: Human ovarian granulosa cells that had been starved were cultured in a medium containing LPS and IFN-γ to obtain a pan-apoptosis model of human ovarian granulosa cells.

2. The method for constructing an in vitro human ovarian granulosa cell pan-apoptosis model according to claim 1, characterized in that: The following steps are involved: (1) Human ovarian granulosa cells in the logarithmic growth phase were starved and cultured with medium containing 0.5%-5% serum for 24-72 h; (2) Human ovarian granulosa cells that have been starved are cultured in a medium containing LPS, IFN-γ and 10%-20% serum for 12-24 hours to obtain a human ovarian granulosa cell pan-apoptosis model.

3. The method for constructing an in vitro human ovarian granulosa cell pan-apoptosis model according to claim 1 or 2, characterized in that: The human ovarian granulosa cells are KGN cells.

4. The method for constructing an in vitro human ovarian granulosa cell pan-apoptosis model according to claim 1 or 2, characterized in that: The culture medium is DMEM / F12 complete culture medium.

5. The method for constructing an in vitro human ovarian granulosa cell pan-apoptosis model according to claim 1 or 2, characterized in that: The concentration of LPS is 0.2-1 ng / μL, and the concentration of IFN-γ is 100-500 ng / mL.

6. The method for constructing an in vitro human ovarian granulosa cell pan-apoptosis model according to claim 2, characterized in that: In step (1), the serum content is 2%.

7. The method for constructing an in vitro human ovarian granulosa cell pan-apoptosis model according to claim 2, characterized in that: In step (2), the LPS content is 1 ng / μL, and the IFN-γ content is 100 ng / mL.

8. The method for constructing an in vitro human ovarian granulosa cell pan-apoptosis model according to claim 2, characterized in that: In step (2), the serum content is 10%.

9. A human ovarian granulosa cell pan-apoptosis model, characterized in that: Obtained by the construction method according to any one of claims 1 to 8.

10. Use of the human ovarian granulosa cell pan-apoptosis model according to claim 9 as a cell model for studying the pathological mechanism of polycystic ovary syndrome.

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