Construction method and application of polycystic ovarian syndrome endometrial epithelium organ fibrosis model

Abstract

The invention relates to the technical field of biology, and particularly discloses a construction method and application of an endometrial epithelium organ fibrosis model. According to the method, human endometrial epithelial cells are extracted, and an endometrial epithelial organ is established by adopting a Matrigel three-dimensional culture system. Afterwards, the organoid is induced to form endometrial phenotypes in different physiological cycles through hormone stimulation, and a ferroptosis inducer FIN56 is added in the culture process to induce fibrosis. The expression of GPX4, Fibronectin and p-Smad2 / 3 is detected through Western blot and immunofluorescence, and the successful construction of the fibrosis model is verified. The invention provides an in-vitro model capable of simulating endometrial fibrosis pathological characteristics of a patient with PCOS (polycystic ovarian syndrome), the in-vitro model can be used for mechanism research and drug screening of PCOS-related endometrial lesions, and a new research tool and an intervention strategy are provided for treatment of PCOS endometrial fibrosis.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a method for constructing a fibrosis model of endometrial epithelial organoids in polycystic ovary syndrome and its application. Background Art

[0002] Polycystic ovary syndrome (PCOS) is a complex and heterogeneous endocrine and metabolic disorder syndrome, and is the main cause of anovulatory infertility in women of childbearing age. It usually presents symptoms such as anovulation, hyperandrogenemia, and insulin resistance. These abnormalities may lead to endometrial dysfunction, including problems such as endometrial hyperplasia and progesterone resistance.

[0003] Endometrial epithelial organoids (EOs) are three-dimensional structures formed by the self-organization of endometrial cells, and can simulate the structure and function of the endometrium. According to the cell types included, EOs can be divided into single-cell EOs and multi-cell EOs. These models provide new tools for studying endometrial development and disease mechanisms.

[0004] Existing studies have used endometrial epithelial organoid models to explore endometrial abnormalities related to PCOS. For example, some researchers have established a scaffold-free multi-cell organoid model composed of human endometrial epithelial cells and stromal cells to simulate the effect of elevated androgen on organoids in PCOS, and found that excessive androgen promotes organoid cell proliferation and leads to dysregulation of genes related to proliferation and migration, revealing a new mechanism for the risk of endometrial carcinogenesis related to PCOS.

[0005] The construction of a fibrosis model of endometrial epithelial organoids can be used to study the mechanism of endometrial fibrosis in PCOS patients, which will help develop new treatment strategies and improve the reproductive health of PCOS patients. However, there is currently no report on organoids specifically targeting endometrial fibrosis in PCOS patients. Summary of the Invention

[0006] In order to fill the gap in the existing technology, the present invention provides a method for constructing a fibrosis model of endometrial epithelial organoids in polycystic ovary syndrome and its application. And specifically provides the following technical solutions:

[0007] In the first aspect of the present invention, a method for constructing a fibrosis model of endometrial epithelial organoids is provided, and the method includes the following steps:

[0008] (1) Extract human endometrial epithelial cells and construct endometrial epithelial organoids through a Matrigel three-dimensional culture system;

[0009] (2) Induce the endometrial phenotype of different physiological cycles in organoids through hormonal stimulation;

[0010] (3) Add the ferroptosis inducer FIN56 during the organoid culture process to induce fibrosis in endometrial epithelial cells;

[0011] (4) Verify the formation of organoid fibrosis by detecting the activation of the fibrosis marker Fibronectin and the Smad2 / 3 signaling pathway.

[0012] In one embodiment, the concentration of the ferroptosis inducer FIN56 is 5 - 20 μM, preferably 10 μM.

[0013] In one embodiment, the endometrial epithelial organoids are cultured using a basal expansion medium (Expansion Medium, ExM), and the basal expansion medium contains R-Spondin 1, Noggin, EGF, FGF10, HGF, A83-01, ITS, and N-acetylcysteine.

[0014] In one embodiment, the method for verifying organoid fibrosis includes detecting the protein expression levels of GPX4, Fibronectin, and p-Smad2 / 3 by Western blot, and further verifying their expression and cell distribution by immunofluorescence.

[0015] The second aspect of the present invention provides an endometrial epithelial organoid fibrosis model obtained according to the foregoing method.

[0016] The third aspect of the present invention provides an application of the foregoing endometrial epithelial organoid fibrosis model in the screening of drugs for the treatment of endometrial fibrosis.

[0017] In one embodiment, the drugs for the treatment of endometrial fibrosis include GPX4 inhibitors.

[0018] Compared with the prior art, the present invention has the following remarkable advantages:

[0019] 1. The present invention constructs for the first time a cell model closer to the original tissue state - an endometrial epithelial organoid fibrosis model. Through hormonal stimulation, it is possible to induce the formation of endometrial epithelial organoids in the proliferative phase, early secretory phase, and mid-secretory phase. Applying the ferroptosis inducer FIN56 intervention can induce epithelial cell fibrosis, and this model can be used as an effective tool for preventing and / or treating endometrial fibrosis in different physiological cycles.

[0020] 2. The present invention first reports the role of GPX4 deficiency in the cyclic transformation of the endometrium, indicating that GPX4 deficiency is an important initiating factor leading to fibrosis of endometrial epithelial cells in PCOS. The present invention first reveals that during the transformation of the normal endometrium from the proliferative phase to the secretory phase, it is accompanied by the inhibition of ferroptosis activity and ECM remodeling of endometrial epithelial cells. We used an in vitro organoid model and constructed a fibrotic model of endometrial epithelial cell organoids by applying an inducer FIN56 that activates ferroptosis by inhibiting GPX4 activity. Through experimental verification, it was analyzed that endometrial epithelial cells in PCOS may cause fibrosis by activating the SMAD2 / 3 pathway. These findings contribute to analyzing the mechanism of action of endometrial lesions in PCOS and provide new treatment strategies for improving the clinical pregnancy outcome of PCOS patients and reducing the risk of endometrial lesions.

[0021] 3. It was first revealed that during the transformation of the normal endometrium from the proliferative phase to the secretory phase, it is accompanied by the inhibition of ferroptosis activity and ECM remodeling of endometrial epithelial cells. And it was demonstrated that there are GPX4 deficiency and fibrotic phenotypes in endometrial epithelial cells of PCOS. We used an in vitro organoid model and through experimental verification, it was analyzed that GPX4 deficiency in endometrial epithelial cells of PCOS causes fibrosis by activating the Smad2 / 3 signaling pathway. These findings contribute to analyzing the mechanism of action of endometrial lesions in PCOS and provide new treatment strategies for improving the clinical pregnancy outcome of PCOS patients and reducing the risk of endometrial lesions. Brief Description of the Drawings

[0022] The drawings are used to provide further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation to the present invention. In the drawings:

[0023] Figure 1 : Through single-cell transcriptome analysis and mining of the GSE111976 dataset, the endometrial tissue was classified into non-ciliated epithelial cells, ciliated cells, endothelial cells, lymphocytes, macrophages, smooth muscle cells, and stromal fibroblasts according to cell-specific marker genes.

[0024] Figure 2 : Using the ferroptosis-related gene set WP_FERROPTOSIS and the AUCell package to calculate the ferroptosis pathway activity in each cell. The analysis results show that the expression of ferroptosis-related genes is the most active in non-ciliated epithelial cells.

[0025] Figure 3 : GSEA enrichment analysis found that the ferroptosis signaling pathway was significantly enriched in endometrial epithelial cells in the secretory phase.

[0026] Figure 4: KEGG analysis found that the glutathione pathway, an upstream signaling pathway closely related to ferroptosis, was significantly downregulated in the proliferative-phase endometrium.

[0027] Figure 5 : KEGG analysis found that the ECM receptor interaction pathway, a signaling pathway closely related to the fibrosis process, was significantly upregulated in the proliferative-phase endometrium.

[0028] Figure 6 : Enrichment analysis diagram of KEGG downregulated signaling pathways in the transcriptome sequencing of the proliferative-phase endometrium of PCOS

[0029] Figure 7 : Venn diagram of the intersection analysis results of significantly different genes in the ferroptosis and GSH metabolic pathways in single-cell transcriptomics and transcriptome data.

[0030] Figure 8 : The mRNA expression of GPX4 in the epithelial cells of the proliferative-phase endometrium of PCOS patients was significantly decreased.

[0031] Figure 9 : The protein expression of GPX4 in the epithelial cells of the proliferative-phase endometrium of PCOS patients was significantly decreased.

[0032] Figure 10 : The proliferative-phase endometrium of PCOS patients showed a fibrotic phenotype.

[0033] Figure 11 : Schematic diagram of the EEOs stimulation protocol. In ExM, EEOs were passaged and plated on day 0 (d0); on day 4, the ExM was replaced with different media for 8 days. ExM group: EEOs were continuously cultured in ExM. Prol: EEOs were pretreated with E2 to simulate the proliferative-phase endometrium. Sec: EEOs were pretreated with P4 + cAMP to simulate the secretory-phase endometrium. Mid-sec: EEOs were pretreated with the differentiation medium (P4 + cAMP + hCG + PRL + hPL) to simulate the endometrium during pregnancy.

[0034] Figure 12 : Bright-field image of EEOs: Established EEOs can be amplified every 7 - 10 days at a passage ratio of 1:2 or 1:3.

[0035] Figure 13: GPX4 inhibitor FIN56 was used to intervene in EEOs of normal patients, and western blot verified that the expression of GPX4 was significantly decreased at different times. In addition, compared with the untreated control group of patients, the protein expression of the fibrosis marker Fibronectin in EEOs treated with FIN56 was significantly increased, and the phosphorylation level of Smad2 / 3 was elevated. Therefore, it is suggested that GPX4 deficiency is an important initiating factor mediating the occurrence of EEOs fibrosis, and this effect is caused by activating the phosphorylation of Smad2 / 3.

[0036] Figure 14 : Immunofluorescence verified again that FIN56 treatment could effectively reduce the expression of GPX4 in EEOs at different times.

[0037] Figure 15 : In EEOs of the proliferative-phase endometrium in PCOS, the expression of the fibrosis marker Fibronectin continued to increase, suggesting a significant increase in the fibrosis level in endometrial epithelial cells of PCOS patients. Specific implementation manners

[0038] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0039] Example 1 Activation of ferroptosis in PCOS endometrial epithelial cells promotes the occurrence of fibrosis

[0040] In a prospective study, we re-analyzed the single-cell transcriptomics data of the endometrium of 10 normal menstrual cycles (GSE111976). This dataset used 10x Genomics technology and paired-end reads and sequencing were performed on the Nextseq (Illumina) platform. According to the specific marker genes of different cell subsets, after a strict subpopulation annotation process, seven cell subsets with unique molecular characteristics and functional specialization were finally successfully identified in normal endometrial tissue, including non-ciliated epithelial cells, ciliated epithelial cells, endothelial cells, lymphocytes, macrophages, smooth muscle cells, and stromal fibroblasts ( Figure 1 ).

[0041] The AUCell algorithm was used to calculate the activity of the ferroptosis pathway in each cell. The data analysis results showed that the activity of the ferroptosis pathway was the highest in non-ciliated epithelial cells compared with the other six cell subsets ( Figure 2)。To further explore the key biological events involved in the transition of endometrial epithelial cells from the proliferative phase to the secretory phase, we performed differential analysis of genes in endometrial epithelial cells of the two phases. Pathway enrichment analysis showed that compared with non-ciliated epithelial cells in the secretory phase, the ferroptosis signaling pathway ( Figure 3 ) and its upstream glutathione (GSH) metabolism signaling pathway ( Figure 4 ) were significantly downregulated in non-ciliated endometrial epithelial cells in the proliferative phase, and the key pathway mediating fibrosis, the extracellular matrix (ECM)-receptor interaction pathway, was significantly upregulated ( Figure 5 ), suggesting that during the transition of endometrial epithelial cells from the proliferative phase to the secretory phase, it is necessary to increase the activity of genes related to the ferroptosis pathway while downregulating the activity of the fibrosis pathway.

[0042] To further clarify whether there is abnormal activation of ferroptosis in the proliferative endometrium of PCOS, we performed transcriptomic sequencing on the proliferative endometrium using clinical samples (30 control cases and 20 PCOS cases). Pathway enrichment analysis showed that compared with the control group, the ferroptosis signaling pathway and its upstream glutathione metabolism signaling pathway were significantly downregulated in the PCOS group ( Figure 6 ). Intersection analysis was performed on the significantly differentially expressed genes enriched in the ferroptosis and GSH metabolism pathways in the two groups of data. The analysis results showed that GPX4 was the only common differential gene in the ferroptosis and GSH pathways in the two groups of data ( Figure 7 ).

[0043] Furthermore, we used 10 control cases and 9 PCOS endometrial epithelial cells for qPCR detection and verified that the gene expression of GPX4 was significantly downregulated in PCOS endometrial epithelial cells ( Figure 8 ). Using 5 control cases and 8 PCOS endometrial epithelial cells for Western blot detection, we verified that GPX4 was significantly downregulated in PCOS endometrial epithelial cells ( Figure 9 ). Using 6 control cases and 6 PCOS endometrial tissues for collagen fiber staining, we verified the phenomenon of excessive deposition of collagen fibers in the proliferative endometrium of PCOS ( Figure 10 ).

[0044] Therefore, in a prospective study, we first discovered that the proliferative endometrium of PCOS has a fibrotic phenotype, and this phenotype is related to the downregulation of GPX4 expression. And the following experiments were carried out to verify it.

[0045] Example 2 Construction of an endometrial epithelial cell organoid (EEOs) model

[0046] 1. Inclusion and exclusion of clinical samples

[0047] The clinical sample study was approved by the Ethics Committee of Peking University Third Hospital, and 23 control patients (control group) and 25 female patients with polycystic ovary syndrome (PCOS group) who visited the Reproductive Medicine Center of Peking University Third Hospital from 2022 to 2024 were recruited, aged between 18 and 38 years old.

[0048] Inclusion and exclusion criteria:

[0049] PCOS group: According to the Rotterdam PCOS diagnostic criteria in 2003, the enrolled population was selected, and two of the following three conditions were met: oligomenorrhea and / or amenorrhea, hyperandrogenemia or clinical manifestations of hyperandrogenism (hirsutism, acne, etc.), polycystic ovarian changes under ultrasound; at the same time, other causes of hyperandrogenemia were excluded.

[0050] Control group: Patients who sought treatment due to bilateral fallopian tube obstruction or due to male factors alone, with regular menstrual cycles and no manifestations of hyperandrogenism and polycystic ovaries;

[0051] Exclusion criteria: Pregnancy, endocrine and metabolic related diseases (congenital adrenal hyperplasia, androgen-secreting tumors, hypogonadotropic hypogonadism, thyroid dysfunction, Cushing's syndrome, hyperprolactinemia and premature ovarian failure), ovarian cysts or tumors, and chromosomal abnormalities.

[0052] None of the selected subjects had a history of taking hormones, insulin sensitizers, and lipid-lowering drugs in the past 3 months.

[0053] 2. Construction of endometrial epithelial cell organoid (EEOs) models at different menstrual cycles

[0054] The endometrial epithelial cell organoid (EEOs) model was constructed according to the methods reported in the art. The specific steps in this example are as follows ( Figure 11 ):

[0055] (1) The endometrial samples discarded during hysteroscopy of patients in the control group and the PCOS group were collected separately. After being placed in ice-precooled DPBS solution containing 2% PS without phenol red, they were quickly transferred into a sterile laminar flow hood. The endometrial tissues were washed 3 times with DPBS, and after thoroughly removing blood clots and impurities, they were digested with collagenase IV (Sigma-Aldrich, USA, C4-28) at 37°C for 15 minutes to isolate endometrial epithelial cells from the endometrial tissues. If large cell clumps were observed, additional digestion for 5 - 10 minutes was carried out until most of the clumps became single cells. The digestion was terminated by adding an equal volume of DMEM / F-12 (Gibco, USA, 12634028). The cell suspension was filtered through 100μm (Falcon, USA, 352360) and 40μm (Falcon, USA, 352340) cell filters, and then centrifuged at 300g for 5 minutes to retain the glandular components.

[0056] (2) Prepare the expansion medium (Expansion medium, ExM) and three menstrual cycle differentiation media (Differentiation medium, DM):

[0057] ExM:

[0058] 1% N2, 1% B27, 1% Glutamax, R-Spondin 1, Noggin, 1.25 mM N-acetylcysteine, 10 mM nicotinamide, HGF, EGF, FGF10, 10 μM Y-27632, A83-01, ITS and PS;

[0059] Proliferative phase DM:

[0060] Add 10 nM E2 to ExM;

[0061] Early secretory phase DM:

[0062] Add 1 μM medroxyprogesterone acetate (MPA) + 1 μM 8-bromoadenosine 3',5'-cyclic monophosphate sodium salt (8-cAMP) to the proliferative phase DM;

[0063] Mid-secretory phase DM:

[0064] Add 1 μg / ml hCG, 20 ng / ml hPL and 20 ng / ml PRL to the early secretory phase DM.

[0065] (3) Precool Matrigel on ice. Resuspend the endometrial epithelial cells containing glandular components with 40 μl Matrigel, gently pipette to mix well to avoid generating bubbles, and seed the cells into a 12-well plate.

[0066] (4) Place it in an incubator at 37°C to accelerate solidification for 30 min. Gently adhere to the wall and add 500 μl of ExM. Then place it back in the incubator at 37°C for further culture. Replace the culture medium every 2 - 3 days to obtain EEOs without periodic characteristics. The EEOs are passaged at a ratio of 1:2 - 1:3 every 7 - 10 days and can be stably passaged for more than 10 generations.

[0067] (5) After the EEOs grow in ExM for 4 days and obtain stable morphological structures and physiological characteristics, they are divided into four groups:

[0068] Group (i): Continue to culture in ExM;

[0069] Group (ii): Induce proliferation-phase differentiation with 10 nM E2 to obtain EEOs with a proliferation-phase phenotype;

[0070] Group (iii): After the EEOs in Group (ii) are continuously differentiated for 48 hours, replace the culture medium with early secretory DM to differentiate them into EEOs with an early secretory phenotype;

[0071] Group (iv): After the EEOs in Group (ii) are continuously differentiated for 48 hours, replace the culture medium with mid-secretory DM to differentiate them into EEOs with a mid-secretory phenotype.

[0072] Example 3: Obtaining a model of endometrial organoid fibrosis

[0073] Add 10 μM of FIN56 (a GPX4 inhibitor) to the culture media of the EEOs with different menstrual cycles obtained in Groups (i) - (iv) in Example 2, and change the culture fluid for each group of organoids every 2 days to obtain EEOs with a fibrotic phenotype in different menstrual cycles ( Figure 12 ).

[0074] Example 4: Verification of the endometrial organoid fibrosis model

[0075] 1. Western blot:

[0076] Collect the EEOs of four different menstrual cycle phenotypes obtained in Example 2 and the EEOs of fibrotic phenotypes of different menstrual cycles obtained in Example 3. Add a certain volume of RIPA lysis buffer and protease / phosphatase inhibitors. After sufficient lysis on ice, determine the concentration by the BCA method. According to the protein concentration, add the corresponding volume of the protein sample to be tested, 5x protein loading buffer, and protein lysate to configure a protein loading system with a certain volume of 1 μg / μl. After shaking and mixing evenly, boil at 100 °C for 5 min to fully denature the protein. Add the denatured protein sample and protein marker into the loading wells of the electrophoresis gel. Set the voltage to be constant at 100 V and perform electrophoresis for 85 min. After electrophoresis, perform membrane transfer. Activate the PVDF membrane in methanol for 10 - 15 s in advance, then place it in the order of blackboard - sponge - electrotransfer filter paper - electrophoresis gel - PVDF membrane - electrotransfer filter paper - sponge - whiteboard, and then place it in the electrophoresis tank, add pre-cooled transfer buffer, and set the membrane transfer conditions to 300 mA for 90 min. After membrane transfer, carefully take out the PVDF membrane and place it in the blocking solution (5% BSA for antibodies against phosphorylated proteins, and 5% skim milk for the rest of the antibodies), and block it on a horizontal shaker at room temperature for 1 h. Discard the blocking solution, place the PVDF membrane in an incubation box containing the primary antibody solution, and incubate overnight at 4 °C. After incubation, wash the membrane, wash it 4 times with 1x TBST, 5 min for each time, prepare the secondary antibody (1:5000) with the blocking solution, place the PVDF membrane in an incubation box containing the secondary antibody solution, and incubate on a horizontal shaker at room temperature for 1 h; after incubation, wash the membrane. Place the membrane with the protein side up in the ECL chemiluminescence imager, evenly add the prepared ECL luminescence substrate solution on the membrane and react for 1 min. Set the exposure time gradient and then collect the imaging data.

[0077] The proteins to be detected are: GPX4, Fibronectin, Smas2 / 3, p-Smad2 / 3.

[0078] 2. Immunofluorescence:

[0079] Collect the EEOs of four different menstrual cycle phenotypes obtained in Example 2 and the EEOs of fibrotic phenotypes of different menstrual cycles obtained in Example 3. Fix the organoids with 1 mL of 4% paraformaldehyde (PFA) at room temperature for 1 hour. Carefully remove the PFA, permeabilize with 0.5% Triton X-100 / PBS for 10 minutes, and then block with 3% bovine serum albumin in PBS for 30 minutes. Incubate the organoids overnight with the first antibody and the corresponding second antibody conjugated with Alexa 488 or Alexa 555. Perform nuclear staining with DAPI. Obtain confocal images using a confocal microscope (Zeiss LSM980).

[0080] The detected proteins are: GPX4, Fibronectin, Smas2 / 3, p-Smad2 / 3.

[0081] 3. Detection results:

[0082] As Figures 13 - 15 shown, compared with the untreated control group of patients, the protein expression of the fibrosis marker Fibronectin in EEOs treated with FIN56 (a GPX4 inhibitor) was significantly increased, indicating that the fibrosis construction of endometrial organoids was successful. In the EEOs of the proliferative phase endometrium of PCOS, the expression of the fibrosis marker Fibronectin continued to increase, suggesting a significant increase in the fibrosis level in the endometrial epithelial cells of PCOS patients. In addition, the phosphorylation level of Smad2 / 3 in EEOs treated with FIN56 (a GPX4 inhibitor) was increased, verifying that GPX4 deficiency is an important initiating factor mediating the occurrence of EEOs fibrosis, and this effect is caused by activating the phosphorylation of Smad2 / 3.

[0083] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and its equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A method for constructing an endometrial epithelial organoid fibrosis model, characterized in that: The following steps are involved: (1) Extract human endometrial epithelial cells and construct endometrial epithelial organoids using the Matrigel three-dimensional culture system; (2) Inducing organoids to form endometrial phenotypes of different physiological cycles through hormone stimulation; (3) adding the ferroptosis inducer FIN56 during organoid culture to induce fibrosis in endometrial epithelial cells; (4) The formation of organoid fibrosis was verified by detecting the activation of fibrosis markers Fibronectin and Smad2 / 3 signaling pathway.

2. The method according to claim 1, characterized in that The concentration of the ferroptosis inducer FIN56 is 5-20 μM, preferably 10 μM.

3. The method according to claim 1, characterized in that The endometrial epithelial organoids are cultured in a basic expansion medium (Expansion Medium, ExM), which contains R-Spondin 1, Noggin, EGF, FGF10, HGF, A83-01, ITS and N-acetylcysteine.

4. The method according to claim 1, characterized in that: The verification method of organoid fibrosis includes detecting the protein expression levels of GPX4, Fibronectin and p-Smad2 / 3 by Western blot, and further verifying their expression and cell distribution by immunofluorescence.

5. An endometrial epithelial organoid fibrosis model obtained by the method according to any one of claims 1-4.

6. Use of the endometrial epithelial organoid fibrosis model according to claim 5 in the screening of therapeutic drugs for endometrial fibrosis.

7. The use according to claim 6, characterized in that: The endometrial fibrosis treatment drug includes a GPX4 inhibitor.

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