CN-120888486-B - Construction method of human endometrium organoid aging model

Abstract

The invention provides a construction method of a human endometrium organoid aging model, belonging to the technical field of biological models. The invention is based on human endometrium organoids, and the human endometrium organoid aging model is obtained by utilizing hydrogen peroxide (H 2 O 2 ) stimulation and induction. The human endometrium organoid senescence model can clearly show various cell senescence phenotypes such as slow organoid growth speed, reduced cell proliferation index (Ki 67), enhanced SA-beta-gal activity, up-regulated expression of cell cycle inhibitor protein P16 and P21, obviously up-regulated gene expression level of senescence-associated secretion phenotype (SASP) components and the like, and can provide effective tools for female anti-aging drug screening, high-throughput and high-content screening, individualized treatment strategy development and mechanism research.

Inventors

• LI LU
• ZHANG SONGYING
• FAN XIAOHUI
• XIE HONGLIANG
• LIU YING
• YANG CUIYU

Assignees

• 浙江大学

Dates

Publication Date

20260505

Application Date

20251010

Claims (4)

1. 1. A method for constructing a human endometrium organoid aging model, which is characterized by comprising the following steps: The method for constructing the human endometrium organoid comprises the steps of performing enzyme digestion on human endometrium tissue subjected to impurity removal pretreatment, and performing solid-liquid separation and purification on the obtained enzymolysis liquid to obtain human endometrium single cells or gland cell clusters; carrying out three-dimensional culture on the humanized endometrial single cells or glandular cell clusters by taking extracellular matrigel as a medium to obtain a humanized endometrial organoid; treating the human endometrium organoid with H 2 O 2 to obtain a human endometrium organoid aging model; Placing the human endometrium organoid in a culture medium containing H 2 O 2 for culture; The culture medium containing H 2 O 2 is subjected to liquid exchange once every 48: 48H, the treatment concentration of H 2 O 2 is 45-55 nM, and the treatment time of H 2 O 2 is 110-125H; the culture density of the human endometrium organoids is 100-200 organoids/20 mu L.
2. 2. The method of claim 1, wherein the treatment concentration of H 2 O 2 is 50 nM.
3. 3. The method according to claim 1, wherein the number of the human endometrial single cells or glandular cell clusters contained per 20. Mu.L or 50. Mu.L of the extracellular matrix gel is 1X 10 4 ~5×10 4 at the time of the three-dimensional culture.
4. 4. The construction method according to claim 1, wherein the enzyme solution used in the enzyme digestion is an RPMI 1640 culture solution containing 10% FBS by volume percentage concentration, wherein the RPMI 1640 culture solution contains 0.5-2 mg/mL of the dispersing enzyme, 0.05-0.1 mg/mL of the DNase I and 0.5-2 mg/mL of the type I collagenase or the type II collagenase.

Description

Construction method of human endometrium organoid aging model Technical Field The invention belongs to the technical field of biological models, and particularly relates to a construction method of a human endometrium organoid aging model. Background Female fertility decreases significantly with age, and previous studies have focused on age-related ovarian dysfunction, especially deterioration of ovum quality and number. However, more and more studies in recent years have shown that uterine physiological ageing is a key factor affecting pregnancy outcome. Endometrium is used as a key part of embryo implantation and pregnancy maintenance, and its functional state directly determines the success or failure of pregnancy. Studies have shown that a range of changes in the endometrium of elderly women occur at the molecular and cellular level, including decreased cell proliferation capacity, accumulation of cell senescence markers (e.g., SA- β -gal, P16, P21), increased senescence-associated secretory phenotypes (SASP), increased levels of oxidative stress, decreased DNA damage repair capacity, decreased responsiveness to sex hormones, and the like. These aging characteristics may lead to a decrease in endometrial receptivity, which in turn is manifested by implantation failure, recurrent abortion and bad pregnancy outcome common in advanced gestations, where the defect of decidua of the endometrium is considered to be one of the important pathophysiological mechanisms. Deciduation is the process of morphological and functional transformation of endometrial mesenchymal cells under the action of progestogen and local signals, and provides a suitable microenvironment for embryo implantation and placenta formation. Aging of advanced endometrium may impair its normal deciduation capacity, thereby affecting the establishment and maintenance of pregnancy. For a long time, the challenge in studying endometrial aging and its effects on pregnancy has been the lack of an ideal in vitro model. The traditional two-dimensional cell culture system cannot fully simulate complex cell composition, three-dimensional structure and physiological microenvironment of endometrium, and is difficult to truly reflect the aging process of a tissue layer. Organoid technology has emerged as a revolutionary three-dimensional cell culture method. Organoids are capable of forming micro-organs with structures, cellular components and partial physiological functions that resemble tissue organs in the body through self-organizing processes. However, despite advances in the study of both human endometrial organoids and organoid aging models, the combination of both to construct a model that accurately mimics human endometrial aging and is useful in advanced pregnancy decidua defect studies is currently left blank. In particular to a mechanism for reducing the deciduation capacity of the endometrium of an elderly female in the aging process and a treatment strategy for screening the endometrium aging reversing or delaying, which lacks effective in vitro tools. Disclosure of Invention In view of the above, the present invention aims to provide a method for constructing a model of human endometrial organoids, which simulates accelerated cellular aging processes by treatment with hydrogen peroxide (H 2O2) inducers, and which is capable of simulating advanced endometrial aging characteristics in morphology, cell proliferation, aging marker expression, SASP secretion, and responsiveness to hormones. The invention provides a construction method of a human endometrium organoid aging model, which comprises the following steps: constructing a human endometrium organoid; And (3) treating the human endometrium organoid with H 2O2 to obtain a human endometrium organoid aging model. Preferably, the treatment concentration of the H 2O2 is 45-55 nM. Preferably, the treatment concentration of H 2O2 is 50 nM. Preferably, the treatment time of the H 2O2 is 110-125 hours. Preferably, said human endometrium organoid is incubated in a medium comprising H 2O2 during said H 2O2 treatment; The culture medium containing H 2O2 is changed once every 48: 48H; the culture density of the human endometrium organoids is 100-200 organoids/20 mu L. Preferably, in the construction method of the human endometrium organoid, enzyme digestion is carried out on human endometrium tissue subjected to impurity removal pretreatment, and solid-liquid separation and purification are carried out on the obtained enzymolysis liquid to obtain human endometrium single cells or glandular cell clusters; and carrying out three-dimensional culture on the humanized endometrial single cells or glandular cell clusters by taking extracellular matrigel as a medium to obtain the humanized endometrial organoids. Preferably, the number of the human endometrial single cells or glandular cell clusters is 1×10 4~5×104 per 20 μl or 50 μl of extracellular matrix gel during the three-dimensional culture. Preferably,