Hippo信号通路在调节卵巢生殖干细胞功能和延缓卵巢衰老中的作用

发布时间：2018-06-05 16:19

本文选题：Hippo信号通路 + 卵巢生殖上皮细胞　；参考：《南昌大学》2016年博士论文

【摘要】：立项背景如今,随着老龄化社会的出现,衰老问题逐渐被人们所重视。在女性,卵巢衰老的速度要明显快于机体其它器官。卵巢衰老影响生殖健康,引发绝经期的到来、癌症和多种慢性疾病的发生。然而,随着生存环境的日益恶化、生活压力不断加大及药物治疗副作用等因素对女性卵巢功能的损害,使卵源性不孕症及病理性卵巢功能衰退已成为困扰现代女性的重大社会卫生问题。特别是随着二胎政策的实施,高龄女性对于生活质量以及生育寿命的需求日益强烈。故阐明卵巢功能的生理及病理性减退机制、建立有效的干预手段,已成为医学界刻不容缓的研究课题!近年来卵巢生殖干细胞(ovarian germline stem cells,OGSCs)的发现能为有效地增加原始卵泡池、改善卵巢功能、延缓卵巢衰老带来前所未有的希望!Hippo信号通路是调控成体干细胞增殖分化及功能的重要信号通路,我们前期研究发现Hippo信号通路的主要效应因子能触发原始卵泡的启动。但Hippo信号通路是否参与OGSCs功能的调控以及和生理性及病理性卵巢功能衰老的关系还有待进一步证实。因此本研究中,以KM系小鼠及人卵巢皮层组织为实验对象:(1)分析Hippo信号通路的主要效应因子在卵巢表面上皮(ovarian surface epithelium,OSE)细胞中的表达及动态变化,这种变化与卵巢功能生理性衰老和病理性衰老的关系;(2)离体观察Hippo信号通路的主要效应因子YAP1在OGSCs增殖分化中的作用;(3)在体分析YAP1经卵巢内微注射对卵巢功能生理性衰老和病理性衰老的拯救作用。通过实验为深入探讨成年哺乳动物OGSCs的自我更新、分化及衰老等的生物学过程提供重要材料,为不孕症的治疗、重塑和保护卵巢功能提供技术方法和新的药物作用靶点。第一部分Hippo信号通路主要分子在生理性衰老和病理性衰老卵巢生殖上皮细胞中的表达及动态变化研究目的分析Hippo信号通路的主要分子在卵巢生殖上皮(OSE)细胞中的表达及动态变化,这种变化与卵巢功能生理性衰老和病理性衰老的关系。研究方法选用7日龄(7days,7D)、2月龄(2months,2M)、10月龄(10months,10M)和20月龄(20months,20M)小鼠卵巢,以及生育旺盛期(青春期后-35岁)、中年期(36-50岁)和绝经期(51-60岁)人卵巢皮层样本为生理性衰老卵巢实验标本;通过雷公藤灌胃(Tripterygium Glycosides,TPT)及环磷酰胺/马利兰片(Cyclophosphamide/Busulfan,CY-BUS)腹腔注射方式,构建病理性衰老卵巢模型。分别经HE染色,免疫化学方法及分子生物学方法等技术,比较Hippo信号通路的主要分子(LATS2,MST1与YAP1)在OSE细胞中的表达及动态变化,以及这种变化与卵巢功能生理性衰老和病理性衰老的关系。研究结果(1)在不同年龄小鼠OSE中存在Hippo信号通路相关分子与OGSCs标记因子(MVH与OCT4)共表达。其中,抑癌因子LATS2,MST1与MVH/OCT4的表达随着年龄的增加而逐渐降低;原癌因子YAP1则在2M小鼠OSE中表达量最高,7D龄其次,20M最低;p YAP1表达则为7D最高,且p YAP1/YAP1比值随年龄增加而逐渐降低。(2)在病理性卵巢衰老模型小鼠OSE中也存在Hippo信号通路相关分子与OGSCs标记因子(MVH与OCT4)共表达。其中,YAP1,MST1与MVH/OCT4在雷公藤和环磷酰胺/马利兰片两组药物造成的衰老模型组中均表现为逐渐降低,而LATS2的表达则呈现出相反趋势。p YAP1在两组病理性卵巢衰老小鼠卵巢中均显著降低,而p YAP1/YAP1的比值则均显著升高。(3)在不同年龄阶段人OSE中同样存在Hippo信号通路相关分子与OGSCs标记因子(MVH与OCT4)共表达。其中,MVH/OCT4随着年龄的增长而逐渐下降,LATS2表达为青春期最高,YAP1和p YAP1为中年期最高,而MST1则为老年期最低;p YAP1/YAP1则显示出从青年期到老年期逐渐降低的趋势。结论Hippo信号通路相关分子LATS2,MST1与YAP1和OGSCs标记因子MVH与OCT4共表达于小鼠和人OSE中,两者表达的变化存在明显的时序相关性,且这种变化与卵巢功能的生理性衰老和病理性衰老亦存在明显的时序相关性。表明,Hippo信号通路可能通过调控OGSCs参与了卵巢功能的生理性衰老和病理性衰老过程。第二部分Hippo信号通路在卵巢生殖干细胞增殖和分化中的作用研究目的进一步完善卵巢生殖干细胞(OGSCs)的分离、鉴定与培养方法;探明Hippo信号通路对OGSCs增殖分化的影响。研究方法采用两步酶分离方法及特异性抗体磁珠筛选技术(magnetic activated cell sorting,MASC)对OGSCs进行分离,通过测定MVH,OCT4,Nango,Fraglis,Stella等多种干细胞和生殖细胞特异性标记因子对OGSCs进行鉴定,选用本室改良的方法对OGSCs进行培养;将携带效应因子YAP1的过表达和相应sh RNA的干扰慢病毒载体转染至OGSCs,观察对OGSCs增殖和分化的影响。研究结果(1)经上述方法分离得到的细胞能共表达MVH,OCT4,Nango,Fraglis,Stella等干细胞和生殖细胞特异性标记因子,故而初步鉴定所得细胞为OGSCs。(2)LATS2,MST1及YAP1可在OGSCs上表达;OGSCs在转染YAP1过表达慢病毒载体后,其特异性标记因子MVH,OCT4的表达显著升高,而在转染YAP1sh RNA的干扰慢病毒载体后,MVH,OCT4的表达则显著降低。结论已初步建立了OGSCs的分离、鉴定与培养方法;Hippo信号通路相关分子存在于OGSCs,并能调控OGSCs体外增殖。第三部分Hippo信号通路通过调控卵巢生殖干细胞的功能在卵巢功能重塑中的作用初探研究目的在体进一步验证Hippo信号通路通过调控卵巢生殖干细胞(OGSCs)增殖和分化在卵巢功能重塑中的作用。研究方法选用Hippo信号通路中主要效应因子YAP1为靶基因,通过卵巢内微注射将携带效应因子YAP1的过表达和相应sh RNA的干扰慢病毒载体分别转染至不孕模型和生育旺盛期小鼠卵巢,观察对卵巢生殖功能和内分泌功能的影响。研究结果(1)不孕模型小鼠在转染过表达YAP1后的30-75天期间,可以检测到卵巢内有卵泡再生,原始卵泡启动激活,子代出生率增加,同时伴有血清中E2和FSH水平上升,卵巢OSE细胞中YAP1与p YAP1表达和MVH与OCT4表达均明显增高。(2)生育旺盛期小鼠在转染YAP1的干扰慢病毒载体后的30-75天期间,检测到卵巢内卵泡数量下降,子代出生率减少,同时伴有血清中E2和FSH水平下降,卵巢OSE细胞中YAP1与p YAP1表达和MVH与OCT4表达均明显降低。结论调控Hippo信号通路效应分子YAP1可在卵巢功能重塑中发挥重要作用。
[Abstract]:With the emergence of the aging society, aging problems are becoming more and more important. In women, the aging of the ovary is faster than the other organs of the body. The ovarian senescence affects reproductive health, the arrival of the menopause, the occurrence of cancer and a variety of chronic diseases. However, with the worsening of the living environment, the pressure of life is stressed. The damage of the female ovarian function, the oogenic infertility and the pathological ovarian function decline have become the major social health problems that perplex modern women. Especially with the implementation of the second child policy, the demand for the quality of life and the life expectancy of the older women is increasingly strong. The physiological and pathological degeneration mechanism of ovarian function and the establishment of effective intervention methods have become an urgent research topic in the medical field. In recent years, the discovery of ovarian germline stem cells (OGSCs) can increase the original follicle pool, improve the egg nest function and delay the ovarian senility with unprecedented hope! Hip Po signaling pathway is an important signaling pathway to regulate the proliferation, differentiation and function of adult stem cells. Our previous study found that the main effector factor of the Hippo signaling pathway can trigger the priming of the original follicle. But whether the Hippo signaling pathway is involved in the regulation of OGSCs function and the relationship between the physiological and pathological ovarian functional senescence remains to be advanced. In this study, in this study, KM mice and human ovarian cortical tissues were used as experimental subjects: (1) analysis of the expression and dynamic changes of the main effect factors of the Hippo signaling pathway in the epithelial ovarian epithelial (ovarian surface epithelium, OSE) cells, the relationship between this change and the physiological senescence of the ovary and the pathological senescence of the ovary; (2) in vitro The role of the main effect factor YAP1 of Hippo signaling pathway in OGSCs proliferation and differentiation; (3) in vivo analysis of the effect of YAP1 on ovarian functional physiological senescence and pathological senescence in ovary. The experiment provides important material for the biological processes of self-renewal, differentiation and senescence of adult mammalian OGSCs. Materials to provide technical methods and new drug targets for the treatment of infertility, reshaping and protecting ovarian function. The expression and dynamic changes of the main molecules of Hippo signaling pathway in the physiological senescence and pathological senescence of ovarian reproductive epithelial cells. Objective to analyze the main molecules of the Hippo signaling pathway in the ovarian reproductive epithelium. OSE) expression and dynamic changes in cells, the relationship between the changes in physiological senescence and pathological senescence of the ovarian function. The methods of study were 7 days of age (7days, 7D), 2 month old (2months, 2M), 10 month old (10months, 10M) and 20 month old (20months, 20M) mouse ovaries, and the birth peak (36-50 years of adolescence), middle age (36-50 years) and menopause (5 The human ovarian cortex samples of 1-60 years old were the experimental specimens of physiological senescent ovary. By intraperitoneal injection of Tripterygium Glycosides (TPT) and cyclophosphamide / Maryland tablets (Cyclophosphamide/Busulfan, CY-BUS) intraperitoneally, the ovarian model of pathological senescence was constructed. HE staining, immuno chemical methods and molecular biological methods were used respectively. The expression and dynamic changes of the main molecules of Hippo signaling pathway (LATS2, MST1 and YAP1) in OSE cells, and the relationship between this change and the physiological senescence and pathological senescence of the ovarian function were compared. The results of the study (1) there was a co expression of Hippo signal pathway related molecules and OGSCs marker factors (MVH and OCT4) in OSE of mice of different ages. In addition, the expression of tumor suppressor factor LATS2, MST1 and MVH/OCT4 gradually decreased with the increase of age, and the expression of primary cancer factor YAP1 in 2M mice OSE was the highest, 7D was second, 20M was the lowest, P YAP1 expression was the highest, and P depreciation ratio gradually decreased with age. (2) there was also a letter in the pathological ovarian aging model mice. Among the two groups, YAP1, MST1 and MVH/OCT4 were gradually reduced in the aging model group caused by two groups of Tripterygium Wilfordii and cyclophosphamide / Maryland tablets, while the expression of LATS2 showed the opposite trend of.P YAP1 in the two groups of pathological ovarian senescent mice. The ratio of P YAP1/YAP1 increased significantly. (3) there was also a co expression of Hippo signaling pathway related molecules and OGSCs marker factors (MVH and OCT4) in human OSE at different ages. MVH/OCT4 decreased gradually with age, and LATS2 was the highest in puberty, YAP1 and P YAP1 was the highest in middle age. P YAP1/YAP1 showed a tendency to decrease gradually from youth to old age. Conclusion Hippo signaling pathway related molecules LATS2, MST1 and YAP1 and OGSCs marker factor MVH and OCT4 are co expressed in mice and OSE, and there is a significant temporal correlation between the two expression changes, and this change is related to the physiological senescence and pathology of ovarian function. Hippo signaling pathway may participate in the physiological senescence and pathological senescence of ovarian function by regulating OGSCs signaling pathway. The role of the second part of Hippo signaling pathway in the proliferation and differentiation of ovarian reproductive stem cells is to improve the separation of ovarian reproductive stem cells (OGSCs). Identification and culture methods; the effect of Hippo signaling pathway on the proliferation and differentiation of OGSCs. The methods used two step enzyme separation method and specific antibody magnetic bead screening technique (magnetic activated cell sorting, MASC) to separate OGSCs, and determine a variety of stem cell and germ cell specific markers, such as MVH, OCT4, Nango, Fraglis, and reproductive cells. OGSCs was identified by factors and OGSCs was cultured in this room. The overexpression of the effect factor YAP1 and the interfering lentivirus vector of the corresponding sh RNA were transfected to OGSCs to observe the effect of OGSCs on the proliferation and differentiation of OGSCs. The results (1) the cells separated by the above methods could co express MVH, OCT4, Nango, Fraglis, Stella. OGSCs. (2) LATS2, MST1 and YAP1 can be expressed on OGSCs, and OGSCs in YAP1 overexpression of lentivirus vector, the expression of its specific marker factor MVH, the expression of OCT4 is significantly increased, and the expression of MVH, and the expression of MVH Significant reduction. Conclusion OGSCs isolation, identification and culture methods have been preliminarily established; Hippo signaling pathway related molecules exist in OGSCs, and can regulate the proliferation of OGSCs in vitro. The third part of Hippo signaling pathway in ovarian function remodeling by regulating the function of ovarian reproductive stem cells in ovarian function remodeling, the purpose of further verification of Hippo letter in vivo Through regulating the proliferation and differentiation of ovarian reproductive stem cells (OGSCs) in ovarian function remodeling, the method selected the main effect factor YAP1 in the Hippo signaling pathway as the target gene. Through microinjection of the ovary, the overexpression of the effect factor YAP1 and the corresponding sh RNA interfering lentivirus vector were transfected into the infertility model respectively. The effect of the ovaries on the reproductive function and the endocrine function of the ovary was observed. The results of the study (1) during the 30-75 day after transfection of YAP1, the rats in the infertile model could detect the follicle regeneration in the ovary, the activation of the original follicle, the increase of the birth rate of the progeny, the increase of the level of E2 and FSH in the serum, and the OS of the ovary. The expression of YAP1 and P YAP1 in E cells and the expression of MVH and OCT4 were significantly increased. (2) during the 30-75 day after transfecting YAP1 with the lentivirus vector, the number of follicles in the ovary decreased, the birth rate of the progeny decreased, and the level of E2 and FSH decreased in the serum, and the YAP1 and P expressions in the OSE cells of the ovary were expressed in the ovary OSE cells. Conclusion the regulation of Hippo signaling pathway effector molecule YAP1 can play an important role in ovarian remodeling.
【学位授予单位】：南昌大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R711.75

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