Method for constructing human endometrial organ aging model

Abstract

The invention provides a construction method of a human endometrial organ aging model, and belongs to the technical field of biological models. On the basis of a human endometrial organ, hydrogen peroxide (H2O2) is utilized to stimulate and induce to obtain the human endometrial organ aging model. The human endometrial organoid senescence model can clearly present various cell senescence phenotypes such as slow down of organoid growth speed, reduction of cell proliferation index (Ki67), enhancement of SA-beta-gal activity, up-regulation of expression of cell cycle inhibition proteins P16 and P21, significant up-regulation of gene expression level of senescence-related secretion phenotype (SASP) components, and the like. And an effective tool can be provided for female anti-aging drug screening, high-throughput and high-content screening, individualized treatment strategy development and mechanism research.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological models, and particularly relates to a construction method of a human endometrium organoid aging model. BACKGROUND

[0002] The female reproductive capacity decreases significantly with age. Previous studies have focused on age-related ovarian dysfunction, especially the decline in egg quality and quantity. However, in recent years, more and more studies have shown that uterine physiology aging is a key factor affecting pregnancy outcome. As the key site for embryo implantation and maintenance of pregnancy, the functional status of the endometrium directly determines the success or failure of pregnancy. Studies have shown that the endometrium of older women will undergo a series of changes at the molecular and cellular levels, including decreased cell proliferation, accumulation of cell aging markers (such as SA-β-gal, P16, P21), increased senescence-associated secretory phenotype (SASP), increased oxidative stress, weakened DNA damage repair ability, and decreased responsiveness to sex hormones. These aging characteristics can lead to decreased endometrial receptivity, which in turn manifests as frequent implantation failure, recurrent miscarriage and adverse pregnancy outcomes in older pregnancies, among which endometrial decidualization defects are considered to be one of the important pathophysiological mechanisms. Decidualization is the process of morphological and functional transformation of endometrial stromal cells under the action of progesterone and local signals, which provides a suitable microenvironment for embryo implantation and placenta formation. The aging of the endometrium of older women can impair its normal decidualization ability, thereby affecting the establishment and maintenance of pregnancy.

[0003] For a long time, the challenge of studying endometrial aging and its impact on pregnancy has been the lack of an ideal in vitro model. Traditional two-dimensional cell culture systems cannot fully simulate the complex cell composition, three-dimensional structure and physiological microenvironment of the endometrium, and cannot truly reflect the aging process at the tissue level. Organoid technology, as a revolutionary three-dimensional cell culture method, has emerged as the times require. Organoids can form miniature organs with similar in vivo tissue organ structure, cell composition and partial physiological function through self-organization process. However, although research on human endometrial organoids and organoid aging models has made progress, combining the two to construct a model that can accurately simulate human endometrial aging and be used for research on decidualization defects in older pregnancies is still in the blank. In particular, the mechanism of the decline in decidualization ability of the endometrium of older women during the aging process, and the screening of treatment strategies that can reverse or delay endometrial aging, lack effective in vitro tools. SUMMARY

[0004] Therefore, the present application aims to provide a method for constructing a human endometrium organoid aging model, which simulates the accelerated cell aging process by treating human endometrium organoids with hydrogen peroxide (H2O2) inducer, and the obtained aging model can simulate the aging characteristics of old endometrium in terms of morphology, cell proliferation, expression of aging markers, secretion of SASP, and response to hormones, etc.

[0005] The present application provides a method for constructing a human endometrium organoid aging model, comprising the following steps: constructing a human endometrium organoid; treating the human endometrium organoid with H2O2 to obtain a human endometrium organoid aging model.

[0006] Preferably, the treatment concentration of H2O2 is 45-55 nM.

[0007] Preferably, the treatment concentration of H2O2 is 50 nM.

[0008] Preferably, the treatment time of H2O2 is 110-125 h.

[0009] Preferably, during the treatment of H2O2, the human endometrium organoid is cultured in a culture medium containing H2O2. The culture medium containing H2O2 is replaced every 48 h. The culture density of the human endometrium organoid is 100-200 organoids / 20 μL.

[0010] Preferably, the method for constructing the human endometrium organoid comprises the following steps: pretreating the human endometrium tissue to remove impurities, performing enzyme digestion, and performing solid-liquid separation and purification on the obtained enzyme solution to obtain human endometrium single cells or gland cell clusters. The human endometrium single cells or gland cell clusters are cultured in three dimensions using extracellular matrix glue as a medium to obtain human endometrium organoids.

[0011] Preferably, during the three-dimensional culture, the number of human endometrium single cells or gland cell clusters contained in each 20 μL or 50 μL of extracellular matrix glue is 1×10 4 ~5×10 4 .

[0012] Preferably, the enzyme solution used in the enzyme digestion contains 0.5-2 mg / mL dispersin, 0.05-0.1 mg / mL DNase I, and 0.5-2 mg / mL collagenase type I or collagenase type II in RPMI 1640 culture solution containing 10% FBS by volume percentage.

[0013] The present application provides a method for constructing a human endometrial organoid aging model. The present application first constructs a human endometrial organoid that maintains a three-dimensional structure; the human endometrial organoid is stimulated with H2O2 to induce an aging model, so that the human endometrial organoid exhibits various cell aging phenotypes, including a slowed organoid growth rate, a reduced cell proliferation index (Ki67), enhanced SA-β-gal activity, up-regulated expression of cell cycle inhibitors P16 and P21, and significantly up-regulated gene expression levels of senescence-associated secretory phenotype (SASP) components (such as IL-6, CCL-2, etc.), and other core aging characteristics, so that the aging model becomes a powerful tool for studying endometrial aging mechanisms. It can be seen that the present application provides a rapid, controllable and repeatable in vitro induction of aging method based on H2O2 stimulation, while ensuring a multi-dimensional aging phenotype evaluation system, ensuring comprehensive, objective and quantitative evaluation of the aging state. At the same time, the aging model constructed by the present application is based on a human endometrial organoid, which can provide an in vitro model that is closer to the in vivo physiological environment, to further analyze the molecular and cellular level changes in the aging process of the endometrium of elderly women, especially its role in the mechanism of defective decidualization in elderly pregnant women. In addition, the aging model can serve as an efficient screening platform for testing and evaluating potential anti-aging drugs or treatment regimens, accelerating the development of personalized intervention strategies for reproductive aging-related diseases and geriatric syndromes. Furthermore, the construction method provided by the present application opens up new avenues for the study of biological aging, particularly reproductive system aging, and helps to discover new aging biomarkers, predict adverse outcomes during the aging process, and provide new possibilities for future precision medicine and individualized treatment. It can be seen that the present application constructs a highly biomimetic, functional and accurate simulation of aging human endometrial organoid model, and combines advanced analysis techniques to effectively overcome the limitations of existing research models in simulating the complexity of human physiological and pathological conditions, evaluating drug effects and conducting high-throughput screening, providing an unprecedented, reliable and efficient tool for the mechanism research of endometrial aging and decidualization defects caused by aging during pregnancy, female anti-aging drug screening and the development of treatment regimens, which has significant scientific value and clinical application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Figure 1 shows representative morphological images of human endometrial organoids, wherein A is a morphological image of the organoid at P3 generation culture for 3 days, wherein the left scale bar is 500 μm and the right scale bar is 200 μm; B is a morphological image of the organoid at P3 generation culture for 6 days, wherein the left scale bar is 500 μm and the right scale bar is 200 μm; C is a HE staining of the human endometrial organoid showing that it has a typical gland lumen-like three-dimensional structure; left: 40 ×, right: 200 ×; Figure 2 Figure 2. Immunofluorescence staining results of human endometrium tissue and human endometrium organoid epithelial cell markers. A. Immunofluorescence staining of human endometrium organoid showing co-expression of CK7 (green) and E-cad (red) in endometrium tissue (top panel) and human endometrium organoid (bottom panel). Note: DAPI (blue) was used for nuclear counterstaining. Scale bar: 50 pm (top panel), 250 pm (bottom panel). B. Immunofluorescence staining of human endometrium organoid showing co-expression of LAM (green) and EPCAM (red) in endometrium tissue (top panel) and human endometrium organoid (bottom panel). Note: DAPI (blue) was used for nuclear counterstaining. Scale bar: 50 pm (top panel), 250 pm (bottom panel). Figure 3 Figure 3. Morphological observation results of human endometrium organoids treated with different concentrations of H2O2. A. Bright field images of human endometrium organoids exposed to different concentrations of H2O2. Scale bar: 500 pm. B. Bright field images of human endometrium organoids in CTR and SEN groups at 48 h, 72 h, 96 h and 120 h after adding H2O2-containing medium. Scale bar: 1000 pm. Figure 4 Figure 4. Results of Ki67 immunofluorescence staining to evaluate the proliferation capacity of organoids. A. DAPI (blue), Ki67 (red), CK7 (green) staining images of CTR group organoids (100 pm) (top panel) and SEN group organoids (100 pm) (bottom panel). B. Column chart of quantitative analysis of the proportion of Ki67 positive cells, showing mean ± SD. p <0.01; Figure 5 Figure 5. Results of SA-β-gal staining to evaluate the senescence level of organoids. A. SA-β-gal staining images of CTR organoids (100 pm) (top panel) and SEN organoids (100 pm) (bottom panel). B. Column chart of quantitative analysis of the proportion of SA-β-gal positive cells, showing mean ± SD. p <0.05; Figure 6 Figure 6. Results of expression level analysis of cell cycle inhibitor proteins and SASP factors. Relative expression amounts of P16 (A), P21 (B), IL-6 (C) and CCL-2 (D) mRNA detected by RT-qPCR. Note: Column chart shows mean ± SD. p <0.05, *** p <0.001; DETAILED DESCRIPTION

[0015] The application provides a method for constructing a human endometrial organoid aging model, comprising the following steps: constructing a human endometrial organoid; treating the human endometrial organoid with H2O2 to obtain a human endometrial organoid aging model.

[0016] The application first constructs a human endometrial organoid.

[0017] In the application, the method for constructing the human endometrial organoid preferably comprises the following steps: pretreating human endometrial tissue to remove impurities, performing enzyme digestion on the pretreated human endometrial tissue, and performing solid-liquid separation and purification on the obtained enzymatic solution to obtain human endometrial single cells or gland cell clusters. The human endometrial single cells or gland cell clusters are cultured in three dimensions with extracellular matrix glue as a medium to obtain a human endometrial organoid.

[0018] In the application, the method for pretreating the human endometrial tissue to remove impurities preferably comprises the following steps: removing excess adipose tissue, muscle layer and fibrous connective tissue in the human endometrial tissue, retaining only endometrial functional layer and / or basal layer tissue having organoid formation potential, and further crushing the tissue into uniform fragments. The particle size of the fragments is preferably 1-2 mm3.

[0019] In the application, the enzyme solution used in the enzyme digestion preferably comprises 0.5-2 mg / mL dispase II, 0.05-0.1 mg / mL DNase I and 0.5-2 mg / mL type I collagenase or type II collagenase in RPMI 1640 culture solution containing 10% FBS by volume percentage, or 1-1.5 mg / mL dispase, 0.06-0.08 mg / mL DNase I and 1-1.5 mg / mL type I collagenase or type II collagenase in RPMI 1640 culture solution containing 10% FBS by volume percentage. The temperature of the enzyme digestion is preferably 36-38℃, and can be 37℃. The time of the enzyme digestion is preferably 30-90 min, and can be 45-80 min, or 50-60 min. The enzyme digestion is preferably accompanied by oscillation. The enzyme digestion is preferably performed in a constant-temperature shaker or a water bath.

[0020] In the present application, the method for separating the obtained enzymatic hydrolysate into solid and liquid is preferably filtration. The method for filtration is preferably accomplished by using a cell screen. The cell screen is preferably passed through a screen with a pore size of 70 μm first, and then through a screen with a pore size of 40 μm. After the filtration, the cell suspension is preferably collected for purification. The method for purification preferably includes red blood cell lysate treatment or differential centrifugation to remove red blood cells and dead cell debris. After obtaining the human endometrial single cells or glandular cell clusters, the precise cell count is preferably performed by using a hemocytometer or an automatic cell counter, and / or the cell viability and purity are detected by using trypan blue staining.

[0021] After obtaining the human endometrial single cells or glandular cell clusters, the present application performs three-dimensional culture of the human endometrial single cells or glandular cell clusters in extracellular matrix glue as a medium to obtain human endometrial organoids.

[0022] In the present application, during the three-dimensional culture, the number of the endometrial single cells or glandular cell clusters contained in each 20 μL or 50 μL of extracellular matrix glue is preferably 1×10 4 ~5×10 4 , can be 2×10 4 ~4×10 4 , or can be 3×10 4The extracellular matrix gel (Matrigel) provides the necessary physical support and biochemical signals for the organoids to simulate the extracellular matrix environment in vivo. The extracellular matrix gel is preferably a commercial growth factor-reduced Matrigel, purchased from Corning Company, with the item number 356231. The method of three-dimensional culture preferably involves mixing and incubating the endometrial single cells or glandular cell clusters with the extracellular matrix gel, and then placing the mixture in complete culture medium after the extracellular matrix gel solidifies. The mixing and incubation conditions are preferably 15-30 min at 36-38°C in a 5% CO2 environment, which can be 20-25 min at 37°C in a 5% CO2 environment. The complete culture medium is preferably based on Advanced DMEM / F12 medium, and also includes the following components: B27 supplement (1:50 dilution), N-2 supplement (1:100 dilution), antibiotic Primocin (100 μg / mL), antioxidant N-Acetyl-L-cysteine (1.25 mM), amino acid L-glutamine (2 mM), epidermal growth factor (EGF, 50 ng / mL), fibroblast growth factor 10 (FGF10, 100 ng / mL), R-spondin 1 (500 ng / mL), Noggin (100 ng / mL), HGF (50 ng / mL), A83-01 (ALK5 inhibitor, 500 nM), and Nicotinamide (10 mM). All medium components are of cell culture grade, and are strictly prepared and filtered (0.22 μm filter) under a clean bench. The complete culture medium can successfully culture human endometrial organoids and maintain their long-term viability. The culture environment of the human endometrial organoids is preferably in a 37°C, 5% CO2 humidified incubator, and the fresh complete culture medium is replaced every 2-3 days. During the culture period, the human endometrial organoids are passaged when they grow to a diameter of about 300-500 μL, or when the density of the organoids in the culture well is too high, in order to maintain their long-term stable growth and expand the culture scale. The method of passaging the human endometrial organoids preferably includes mechanical passaging and / or enzymatic digestion passaging. The method of mechanical passaging preferably involves repeatedly and gently mechanically blowing the organoid mass with a sterile pipette tip to disperse it into small fragments or small cell clusters, and then re-embedding it in the extracellular matrix gel at a ratio of 1:6 for culture.The method for enzymatic digestion passage, preferably the organoids are separated from the Matrigel using Cell Recovery Solution (Corning, 354253), and digestion is performed using TrypLE Express dissociation enzyme (Invitrogen, 12604-013) at 37℃ for 5 min to completely dissociate into single cells or small cell clusters. Subsequently, the cells are collected by centrifugation and re-embedded in fresh Matrigel for culture as needed. Through the above-mentioned fine and standardized method for constructing and long-term maintaining human endometrial organoids, the present application can stably and efficiently obtain human endometrial organoids with good morphology, structure and function, which lays a solid and reliable foundation for the subsequent construction of human endometrial aging models and their application in the research of decidualization defects in advanced pregnancy and drug screening.

[0023] In the present application, the treatment concentration of H2O2 is preferably 45-55 nM, which can be 50 nM. The treatment time of H2O2 is preferably 110-125 h, which can be 115-120 h. When the H2O2 treatment is performed, the human endometrial organoids are preferably cultured in a culture medium containing H2O2. The culture medium containing H2O2 is preferably replaced every 48 h. The culture density of the human endometrial organoids is preferably 100-200 organoids / 20 μL.

[0024] In the present application, the construction method takes human endometrial organoids as a model, and the cells are derived from epithelial cells (especially glandular cells) in human endometrial tissue. These cells can self-organize into three-dimensional organoids with a structure similar to in vivo endometrial gland structure in a specific three-dimensional culture scaffold through fine separation and culture techniques. Specifically, the organoids have a hollow spherical shape and contain at least one cell type that can express epithelial cell markers such as E-cadherin, CK7, LAM, and EPCAM. This organoid system can achieve long-term stable in vitro passage culture, maintain its basic morphological structure and functional stability, and lay a solid foundation for subsequent aging induction and functional research.

[0025] The human endometrium organoid aging model constructed by the construction method exhibits one or more clear cell aging phenotypes. These phenotypes include, but are not limited to: significant slowing of the overall growth rate of the organoid, lagging volume growth, significantly reduced cell proliferation index (Ki67), significantly enhanced SA-beta-gal activity, significantly up-regulated mRNA expression levels of cell cycle inhibitory proteins P16 and P21, significantly up-regulated gene expression levels of senescence-associated secretory phenotype (SASP) components (such as IL-6, CCL-2, etc.), and decreased mitochondrial membrane potential. Macroscopically, the cell volume in the aging organoid may increase, the morphology may be flattened, and the overall structure of the organoid may be disordered or degenerated.

[0026] The application provides an application of the human endometrium organoid aging model constructed by the construction method in the development, screening or evaluation of high-age pregnancy uterine decidualization treatment drugs, uterine aging drugs or habitual abortion treatment drugs.

[0027] In the present application, the construction method constructs a highly biomimetic, functional and accurate simulation of aging human endometrium organoid model, and combines advanced analysis techniques to effectively overcome the limitations of existing research models in simulating the complexity of human physiology and pathology, evaluating drug effects and developing high-throughput screening, providing an unprecedented, reliable and efficient tool for the mechanism research of endometrial aging and decidualization defects caused by it in high-age pregnancy, which has significant scientific value and clinical application prospect.

[0028] The construction method of the human endometrium organoid aging model provided by the present application will be described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the present application.

[0029] Embodiment 1. Construction method of human endometrium organoid The establishment of the human endometrium organoid aging model is based on the construction of a stable, long-term survival and hormone-responsive human endometrium organoid. The construction method aims to efficiently and reliably isolate and culture a three-dimensional structure and part of the physiological function of the organoid from human endometrium tissue, providing a solid in vitro experimental platform for subsequent aging model construction and drug safety research.

[0030] 1.1 Acquisition of human endometrium tissue samples and cell preparation method ① The acquisition of endometrium tissue samples must strictly follow the ethical norms and procedures. The samples are derived from individuals who have obtained the full informed consent of the patient and have been approved by the ethics committee. After the sample is obtained, it is immediately placed in ice-cold sterile phosphate buffered saline (PBS, preferably without calcium and magnesium ions) or antibiotic-containing basal medium, and is transported to the laboratory as soon as possible for subsequent processing.

[0031] 2. Detailed pretreatment and enzymatic digestion of endometrial tissue in a biological safety cabinet. First, remove the excess adipose tissue, muscle layer and fibrous connective tissue from the tissue sample to ensure that only the functional layer and / or basal layer of the endometrium with the potential for organoid formation is retained. Then, cut the purified endometrial tissue into small pieces of uniform size (e.g., 1-2 mm3) and transfer them to a digestion solution containing a mixture of digestive enzymes. The digestion enzyme mixture contains 2 mg / mL collagenase type I and 2 mg / mL dispase II in RPMI 1640 medium containing 10% FBS. The enzymatic digestion is performed in a constant temperature shaker or water bath at 37°C for 60 min, during which the digestion is observed every 20 min until the tissue pieces are completely dissociated into a single cell suspension or a mixture containing glandular structures.

[0032] 3. Filtration and purification of the digestion product. First, filter the cell suspension after digestion through a cell strainer (preferably first through a 70 μm strainer and then through a 40 μm strainer). Then, transfer the filtered cell suspension to a centrifuge tube and centrifuge it at a relative centrifugal force of 600 g for 6 min to collect the cell pellet. To further remove red blood cells and dead cell fragments, cell purification can be performed using a red blood cell lysis solution or differential centrifugation as needed. Finally, resuspend the purified cell pellet in an appropriate amount of basal medium and use a hemocytometer or an automatic cell counter to accurately count the live cells obtained, while evaluating the viability and purity of the cells by trypan blue staining and other methods.

[0033] 1.2 Three-dimensional culture and long-term maintenance of endometrial organoids The three-dimensional culture of endometrial organoids is the core step of the present application. First, the purified endometrial cells (or glandular cell clusters) are seeded at an optimized cell density (usually 5 x 105cells / cm2) in a suitable three-dimensional culture medium (e.g., Matrigel) in a suitable culture vessel (e.g., a 24-well plate). The culture vessel is then incubated in a constant temperature incubator at 37°C and 5% CO2for 1-2 weeks, during which the cells are allowed to form organoids. During the culture period, the culture medium is replaced every 2-3 days to maintain the optimal growth conditions for the organoids. After the organoids are formed, they can be passaged and maintained in a suitable three-dimensional culture medium (e.g., Matrigel) in a suitable culture vessel (e.g., a 24-well plate) for long-term maintenance. 4Live cells were resuspended in ice-cold, high-concentration Matrigel (commercially available growth factor-reduced Matrigel, e.g., Corning, 356231) at a concentration of 1 x 105cells per 50 μL Matrigel. The cell-Matrigel mixture was then carefully and uniformly pipetted into the bottom of pre-cooled low-attachment culture plates (typically 50 μL per well) under sterile conditions using pre-cooled pipette tips, and care was taken to avoid air bubbles. Immediately after pipetting, the plates were transferred to a cell culture incubator at 37 °C, 5% CO2for 20 min to allow the Matrigel to solidify completely. After the Matrigel solidified, pre-warmed complete endometrial organoid culture medium was immediately added to each well. The complete medium was based on Advanced DMEM / F12 and included the following components: B27 supplement (1:50 dilution), N-2 supplement (1:100 dilution), antibiotic Primocin (100 μg / mL), antioxidant N-Acetyl-L-cysteine (1.25 mM), amino acid L-glutamine (2 mM), epidermal growth factor (EGF, 50 ng / mL), fibroblast growth factor 10 (FGF10, 100 ng / mL), R-spondin 1 (500 ng / mL), Noggin (100 ng / mL), HGF (50 ng / mL), A83-01 (ALK5 inhibitor, 500 nM), and Nicotinamide (10 mM). All medium components were of cell culture grade and were prepared and filtered (0.22 μm filter) under aseptic conditions in a clean bench.

[0034] Daily culture of the organoids was performed in a humidified incubator at 37 °C, 5% CO2. Freshly prepared complete endometrial organoid culture medium was carefully replaced every 2-3 days. During the culture period, the growth morphology, size, structure, budding, and contamination of the organoids were observed periodically under an inverted microscope, and the growth curve was recorded. When the organoids grew to a diameter of about 300-500 μm or the density of the organoids in the culture well was too high, the organoids were passaged to maintain their long-term stable growth and to scale up the culture. Mechanical passaging: the organoid clusters were dispersed into small fragments or small cell clusters by repeated and gentle mechanical pipetting using sterile pipette tips. The dispersed organoid fragments were then re-embedded in fresh Matrigel at a ratio of 1:3 to 1:6 for culture.

[0035] Enzymatic digestion and passage: Organoids were isolated from the matrix gel using Cell Recovery Solution (Cell Recovery Solution, Corning, 354253) and digested using TrypLE Express dissociation enzyme (Invitrogen, 12604-013) at 37°C for 5 min to completely dissociate them into single cells or small cell clusters. Cells were then collected by centrifugation and, as needed, re-embedded in fresh matrix gel for culture.

[0036] 2. Methods for constructing an endometrial organoid aging model The H2O2-induced aging model was constructed using young, well-grown, and cultured endometrial organoids (e.g., passaged 3-5 times). Before H2O2 treatment, the organoids were removed from the matrix gel and resuspended at an appropriate density (e.g., 150 organoids / 20 μL) in the gel and seeded into 48-well plates. After the endometrial organoids had stabilized (2-3 days), different concentrations of H2O2 (e.g., 10 nM, 50 nM, 100 nM, 200 nM, 400 nM) were added to the complete culture medium, while a control group (without H2O2 treatment) was also included. The endometrial organoids were cultured in H2O2-containing medium for 120 h, with medium changes every 48 h during the culture period. After H2O2 treatment, the induced organoids were comprehensively evaluated at different time points.

[0037] After determining the optimal H2O2 stimulation concentration, endometrial organoids were cultured in a medium containing the optimal H2O2 concentration for different times (e.g., 48 h, 72 h, 96 h, and 120 h) to comprehensively evaluate the induced organoids. A control group was also established, consisting of endometrial organoids cultured in a medium without H2O2 for the aforementioned different times.

[0038] 3. Characterization of the hydrogen peroxide (H2O2)-induced aging model A control group (CTR) and an aging model group (SEN) were set up. SEN was the endometrial organoid aging model constructed in step 2 above, and CTR consisted of endometrial organoids treated according to the "Method for Constructing the Endometrial Organoid Aging Model". Both groups of organoids were cultured under the same basic culture conditions. Organoid samples were collected at predetermined time points for subsequent phenotypic analysis.

[0039] 3.1 Methods for assessing aging phenotypes: ①Observation of organoid growth status and morphology: Growth rate and volume change monitoring: After H2O2 treatment, bright field images of each group of organoids were observed and taken under an inverted microscope every 24 h. The overall structure of the organoids was observed by inverted microscope to see if it was complete, the edge was clear, there were no disintegration or degradation phenomena. At the same time, the morphology of the cells in the organoids was observed, and whether there were cell volume enlargement, morphological flattening and other aging-related changes was noted.

[0040] 2. Cell proliferation index detection (Ki67): After fixing and permeabilizing the collected organoids, immunofluorescence staining was performed using Ki67 antibody. At the same time, DAPI was used for nuclear counterstaining, and endometrial epithelial marker (such as E-cad) can be used for co-staining to confirm cell type. Under a fluorescence microscope, multiple fields were randomly selected for imaging, and the number of Ki67 positive cells and total nuclei were counted to calculate the proportion of Ki67 positive cells.

[0041] 3. Detection of senescence-associated β-galactosidase (SA-β-gal) activity: The fixed endometrial organoids were stained using a commercially available SA-β-gal staining kit. Under the condition of pH 6.0, β-galactosidase in senescent cells can hydrolyze X-gal substrate to produce blue product. After staining, the proportion of blue positive (senescent) cells was observed and counted under a light microscope.

[0042] 4. Detection of cell cycle inhibitor (P16, P21) mRNA expression level: Total RNA was extracted from the organoids collected from each group, and cDNA was synthesized by reverse transcription reaction. Using cDNA as a template, primers specific for human P16 (CDKN2A) and P21 (CDKN1A) genes, and primers for internal reference genes (such as GAPDH), real-time fluorescent quantitative PCR (RT-qPCR) was performed. The relative expression level of P16 and P21 mRNA was calculated by comparing the Ct value and using the 2 -ΔΔCt Method. Total RNA of endometrial organoids was extracted with RNeasy Mini Kit. Buffer RLT containing 1% B-mercaptoethanol was added to each group, and it was mixed well by blowing to fully lyse, then an equal volume of 70% anhydrous ethanol was added, and after mixing well by blowing, it was transferred to the RNA binding column for purification. The purified RNA was detected for RNA content and quality using a microspectrophotometer such as NanoDrop. The A 260 / A 280The absorbance ratio range was 1.8-2.1. Then, 20 μL of reverse transcription system was prepared according to the instructions of HiFiScript cDNA Synthesis Kit, including 4.0 μL of 5× RT Buffer, 4.0 μL of dNTP Mix, 2.0 μL of Primer Mix, 2.0 μL of DTT, 1.0 μL of HiFiScript and 7.0 μL of RNA sample. After mixing thoroughly, the reaction program of 42℃ incubation for 15 min and 85℃ incubation for 5 min was set to perform reverse transcription. The cDNA sample obtained by reverse transcription was aliquoted and stored at -20℃ for standby. HieffUNICON ® qPCR SYBR Green Master Mix kit was used to perform qRT-PCR reaction on the cDNA sample. 20 μL of reaction system was prepared according to the instructions, containing 2.0 μL of cDNA template, 7.2 μL of DEPC water, 10 M of forward primer 0.4 μL, 10 M of reverse primer 0.4 μL and 10 μL of SYBR Green PCR Master Mix. After mixing thoroughly, the above reaction system was placed in a PCR instrument, incubated at 95℃ for 2 min, and then cycled for 40 times by two-step method of 95℃ for 10 s and 60℃ for 30 s. The relative expression amount of the target gene was calculated by 2 -ΔΔCt The primer sequences are shown in Table 1.

[0043] Table 1 Primer sequences for qRT-PCR experiment

[0044] ⑤ Expression level detection of senescence-associated secretory phenotype (SASP) component genes: RNA was extracted and RT-qPCR analysis was performed by using the method similar to that for detecting cell cycle inhibitor mRNA. Specific primers for key components of SASP (IL-6, CCL-2) were selected to detect the relative expression level of their mRNA.

[0045] 4. Construction and identification results of human endometrial organoids In this example, cells were successfully isolated from human endometrial tissue and endometrial organoids with stable morphology and function were constructed.

[0046] 4.1 Morphological characteristics of organoids The results are shown in Figure 1 . The morphology of the organoids on the third day of P3 generation culture, under an optical microscope, the endometrial organoids had a cavity with a transparent appearance, surrounded by a hollow spherical structure formed by a single layer of columnar cells arranged tightly (see Figure 1Figure 4. Morphology of endometrial organoids. A) Morphology of endometrial organoids at day 6 of P3 generation. The organoids showed no change in morphology but increased in volume under light microscopy. Some organoids reached a diameter of 200 pm (see Figure 1 Figure 4. Morphology of endometrial organoids. A) Morphology of endometrial organoids at day 6 of P3 generation. The organoids showed no change in morphology but increased in volume under light microscopy. Some organoids reached a diameter of 200 pm (see Figure 1 Figure 4. Morphology of endometrial organoids. A) Morphology of endometrial organoids at day 6 of P3 generation. The organoids showed no change in morphology but increased in volume under light microscopy. Some organoids reached a diameter of 200 pm (see

[0047] 4.2 Cell composition and marker expression of organoids To identify the cell types of the constructed organoids, immunofluorescence staining was performed to detect the expression of markers of endometrial epithelial cells. The results are shown in Figure 2 The results showed that the organoids could mimic the expression of epithelial cell markers Cytokeratin 7 (CK7), E-cadherin (E-cad), Epithelial cell adhesion molecule (EPCAM) and basement membrane marker Laminin (LAM) of endometrial tissue in vivo. These cells were arranged regularly and formed the main cystic structure of the organoids. This result suggested that the organoids were mainly of epithelial origin.

[0048] 4.3 Construction and characterization of senescence model of endometrial organoids 4.3.1 Construction and characterization of H2O2-induced senescence model Normal endometrial organoids were treated with different concentrations of H2O2 (e.g. 10 nM, 50 nM, 100 nM, 200 nM, 400 nM). The results are shown in Figure 3Figure 6A. Compared with CTR, 10 nM H202 treatment, the organoids started to appear black groups; 50 nM H202 treatment, the organoids morphology further shrunk, volume decreased, organoids proliferation significantly weakened; while in 100 nM, 200 nM and 400 nM H202 treatment, the organoids died and could not grow, so the best exposure concentration was determined to be 50 nM; then, using 50 nM H202 to treat normal endometrial organoids for different times (such as 48 h, 72 h, 96 h and 120 h), the best induction of aging conditions was finally determined: 50 nM H202 treated organoids for 120 h, with liquid change every 48 h. Under this condition, the organoids treated with H202 showed a typical aging phenotype.

[0049] ①The early apoptosis of organoids increased: 50 nM H202 treated endometrial organoids for 120 h, with liquid change every 48 h, and the morphological changes of organoids were photographed every 24 h. The results are shown in Figure 6B. Figure 3 Figure 6B. Compared with CTR, H202 treated for 48 h, the growth state of organoid cells had no significant change; H202 treated for 72 h, the volume of organoids increased, suggesting normal proliferation, H202 treated for 96 h, organoids gradually appeared to group, fold, volume shrinkage, suggesting that organoid proliferation began to weaken; H202 treated for 120 h, the morphology of organoids further deteriorated, cell death and aggregation were more serious (blue arrow), and the structural integrity of organoids decreased significantly. While the CTR group of endometrial organoids maintained good morphology and structural integrity during the entire observation period, without obvious cell death or morphological changes, indicating that without hydrogen peroxide treatment, the organoids could maintain normal growth and development state.

[0050] ②The cell proliferation ability decreased: The cell proliferation index was evaluated by Ki67 immunofluorescence staining, and the results showed that the proportion of Ki67 positive cells in SEN group organoids was significantly lower than that in CTR (Figure 6C). Figure 4 Figures 6A and B show that the cell proliferation ability of SEN group organoids was significantly impaired.

[0051] ③SA-β-gal activity increased: SA-β-gal staining results showed that the proportion of SA-β-gal positive (blue staining) cells in SEN group endometrial organoids was significantly higher than that in CTR group, which is a classic biomarker of cell aging (Figures 6D and E). Figure 5 Figures 6A and B show that the cell proliferation ability of SEN group organoids was significantly impaired.

[0052] ④Cell cycle inhibitory proteins were up-regulated and SASP factors were increased: RT-qPCR experimental results (see Figure 6F) showed that the expression of cell cycle inhibitory proteins p21 and p16 in SEN group organoids was significantly higher than that in CTR group, and the expression of SASP factors IL-6, IL-8 and CXCL1 in SEN group organoids was also significantly higher than that in CTR group. Figure 6) The mRNA expression levels of cell cycle inhibitor P16, P21 and senescence-associated secretory phenotype (SASP) factors IL-6, CCL2 in the SEN group of organoids were significantly higher than those in the CTR group of organoids. The above results show that the SEN group of organoids has up-regulation of cell cycle inhibitors and increased secretion of SASP factors compared with the CTR group, indicating that the cell senescence state is more significant.

[0053] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the scope of protection of the present application.

Claims

1. A method for constructing a human endometrial organoid aging model, characterized in that, Includes the following steps: Constructing human endometrial organoids; The human endometrial organoids were treated with H2O2 to obtain an aging model of human endometrial organoids.

2. The construction method according to claim 1, characterized in that, The concentration of H2O2 used in the treatment is 45~55 nM.

3. The construction method according to claim 2, characterized in that, The concentration of H2O2 used in the treatment was 50 nM.

4. The construction method according to claim 1, characterized in that, The H2O2 treatment time is 110~125 h.

5. The construction method according to claim 1, characterized in that, During the H2O2 treatment, the human endometrial organoids are cultured in a culture medium containing H2O2. The culture medium containing H2O2 was changed every 48 hours; The culture density of the human endometrial organoids is 100-200 organoids / 20 μL.

6. The construction method according to any one of claims 1 to 5, characterized in that, The method for constructing human endometrial organoids involves enzymatic digestion of pretreated human endometrial tissue, followed by solid-liquid separation and purification of the resulting enzymatic hydrolysate to obtain single-cell or glandular cell clusters of human endometrial tissue. Human endometrial single cells or glandular cell clusters were cultured in three dimensions using extracellular matrix gel as a medium to obtain human endometrial organoids.

7. The construction method according to claim 6, characterized in that, During the three-dimensional culture, the number of human endometrial single cells or glandular cell clusters contained in each 20 μL or 50 μL extracellular matrix gel was 1 × 10⁻⁶. 4 ~5×10 4 .

8. The construction method according to claim 6, characterized in that, The enzyme solution used for the enzyme digestion is RPMI 1640 culture medium containing 10% FBS at a volume percentage concentration of 0.5-2 mg / mL dispersant, 0.05-0.1 mg / mL DNase I, and 0.5-2 mg / mL type I or type II collagenase.

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