SIRT1在多囊卵巢综合征中的作用及机制研究

发布时间：2018-03-07 06:35

本文选题：多囊卵巢综合征(PCOS)　切入点：沉默信息调节因子1(SIRT1)　出处：《郑州大学》2017年硕士论文　论文类型：学位论文

【摘要】：多囊卵巢综合征(PCOS)是一种常见的生殖内分泌紊乱疾病,常表现为卵泡无周期成熟导致慢性无排卵,并常伴发不孕及糖脂代谢紊乱等。其发病机制除与下丘脑-垂体-卵巢轴功能失调有关,还与卵泡微环境失调,卵泡发育、成熟、闭锁的调控失衡有关。卵巢颗粒细胞对卵泡的生长发育及卵泡微环境有重要影响,颗粒细胞凋亡增多破坏卵泡微环境,导致卵泡成熟障碍。SIRT1是一种广泛存在于哺乳动物中的去乙酰化酶,其功能广泛,包括对能量代谢、DNA修复、细胞寿命和凋亡的调控等。SIRT1的表达受多种因子的调控,如P53、FOXO和E2F1形成的自反馈环路。此外,SIRT1参与类固醇激素合成关键酶的表达调控,参与哺乳动物的生殖过程。RSV是至今为止发现的活力最强的特异性SIRT1激动剂,其功能除涉及维持机体能量平衡,并且还具有抗氧化,抗炎和抗肿瘤活性。EX-527属于吲哚类SIRT1抑制剂,是至今发现的活性最强的选择性SIRT1抑制剂。因此,本课题研究中选择RSV和EX527分别作为SIRT1特异性的激活剂和抑制剂。目的:本研究以行IVF-ET或ICSI助孕的PCOS患者为主要研究对象,对其颗粒细胞进行体外培养,使用RSV和EX527处理颗粒细胞,通过qPCR、Western Blot、流式细胞术及化学发光法测定培养液中雌二醇浓度等方法,分析SIRT1表达水平改变对细胞凋亡率以及类固醇激素合成关键酶的影响,初步探讨SIRT1与PCOS的潜在关系。方法:2016年4月1日至2016年12月1日于海南医学院第一附属医院生殖医学中心行常规体外授精-胚胎移植(IVF-ET)或卵泡浆内单精子显微注射(ICSI)助孕的患者共114例被纳入研究,根据纳入标准及排除标准分组:PCOS组(57例)与对照组(57例)。收集PCOS组与对照组患者卵泡液,采用红细胞裂解法提取卵泡液中颗粒细胞,均匀接种于6孔板中,使用含15%FBS的DMEM-F12的培养基作为基础培养基对颗粒细胞进行体外培养,细胞贴壁后使用含100μM RSV和含100μM EX527的培养基换液,分别继续培养12h、24h、48h后收获细胞,同时收集培养液,未经加药处理的细胞作为对照。收获细胞后,用Trizol一步法提取颗粒细胞总RNA,使用RIPA裂解液提取颗粒细胞总蛋白,两组中目的基因和蛋白(SIRT1、StAR、CYP17、CYP19和PPARγ)的表达水平由qPCR和Western Blot进行检测并对比分析;流式细胞术检测各标本细胞凋亡率,Western Blot检测凋亡相关蛋白Bcl-2和Caspase-3蛋白表达,采用化学发光法检测培养液中E2浓度并对比分析。结果:1在两组颗粒细胞中均能检测到SIRT1的表达,PCOS中SIRT1呈低表达(P0.05)。使用100μM RSV处理颗粒细胞后,随作用时间延长,两组颗粒细胞中SIRT1、StAR、CYP17、CYP19表达水平升高,PPARγ表达水平降低(P0.05)。使用100μM EX527处理颗粒细胞后,随作用时间延长,两组颗粒细胞SIRT1、St AR、CYP17、CYP19表现为不同程度的表达水平降低,PPARγ表达水平升高(P0.05)。2 RSV和EX527处理颗粒细胞后,随作用时间延长,对照组与PCOS组颗粒细胞凋亡率均升高,EX527处理颗粒细胞后细胞凋亡率升高更明显,且随时间延长,与RSV处理后相比凋亡率差异更大(P0.05)。RSV处理后,颗粒细胞Bcl-2蛋白表达升高,Caspase-3蛋白表达降低,EX527处理后相反。3 RSV或EX527处理颗粒细胞后,培养液中E2浓度均显示加药后下降,随处理时间延长,浓度又呈上升趋势(P0.05),RSV处理后培养液中E2浓度上升更明显(P0.05)。结论:在人颗粒细胞体外培养中,RSV和EX527介导SIRT1对颗粒细胞凋亡的调控,SIRT1抑制颗粒细胞凋亡,并促进类固醇激素合成关键酶的表达和颗粒细胞中雌二醇激素的合成,参与PCOS发病。
[Abstract]:Polycystic ovary syndrome (PCOS) is a common disease of reproductive endocrine disorders, Changbiaoxianwei follicle cycle without mature leads to chronic anovulation, and is often associated with infertility and lipid metabolic disorder. Its pathogenesis is not only related to hypothalamic pituitary ovarian axis dysfunction, and follicular microenvironment disorders, follicular development mature, relevant regulation, imbalance of atresia. Have an important impact on the growth and follicular granulosa cells of follicles microenvironment, apoptosis of granulosa cell damaged follicles microenvironment, cause.SIRT1 follicle dysmaturity deacetylase is a widely exist in mammals, its wide range of functions, including on energy metabolism, DNA repair the expression of life, cell apoptosis and regulation of.SIRT1 is regulated by many factors, such as P53, FOXO and E2F1 form a self feedback loop. In addition, the expression of SIRT1 involved in steroid hormone synthesis key enzyme Control,.RSV is involved in mammalian reproductive process specific SIRT1 found so far the most dynamic agonist, its function in addition to maintain the body's energy balance, and also has antioxidant, anti-inflammatory and antitumor activity of indole.EX-527 belongs to the SIRT1 inhibitor is a selective inhibitor of SIRT1 activity has been found. Therefore, this topic in the study of RSV and EX527 respectively as SIRT1 specific activators and inhibitors. Objective: in this study, IVF-ET or ICSI help pregnant patients with PCOS as the main research object, the granule cells were cultured in vitro, the use of RSV and EX527 in granulosa cells by qPCR, Western, Blot, flow cytometry was used to cultivate the female the concentration of glycol solution in cell method and chemiluminescence method, the expression level of SIRT1 change on the apoptosis rate and the effect of steroid hormone synthesis key enzyme, preliminary discuss SIRT1 and PCOS The potential relationship between April 1, 2016 to December 1, 2016. Methods: in the First Affiliated Hospital of Hainan Medical University of conventional in vitro fertilization and embryo transfer (IVF-ET) or intracytoplasmic sperm injection (ICSI) help pregnant patients with a total of 114 patients were included in the study according to the inclusion criteria and exclusion criteria group: PCOS group (57 cases) and the control group (57 cases) were collected. The PCOS group and the control group in patients with follicular granulosa cells in follicular fluid, extraction with red blood cell lysis method, inoculated into 6 well plates, medium containing 15%FBS DMEM-F12 as basic culture medium on granulosa cells were cultured in vitro, adherent cells containing 100 M and RSV containing 100 M EX527 culture medium was replaced, were cultured for 12h, 24h, 48h after the cells were harvested, and collect the culture liquid, without adding treated cells as control. The cells were harvested after extraction of particles by Trizol method The total cell RNA extracted granulosa cell total protein lysates using RIPA, two groups of genes and proteins (SIRT1, StAR, CYP17, CYP19 and PPAR gamma) expression levels were detected and analyzed by qPCR and Western Blot; the rate of cell apoptosis were detected by flow cytometry, the expression of Western Blot detection of apoptosis related Bcl-2 protein and Caspase-3 protein, using chemiluminescence detection of medium E2 concentration and comparative analysis. Results: 1 in two groups of granulosa cells were detected in the expression of SIRT1, SIRT1 expression was low in PCOS (P0.05). Using 100 M RSV granule cells, with the prolongation of time, SIRT1. Granulosa cells in the two groups StAR, CYP17, CYP19 expression levels increased, PPAR expression level decreased (P0.05). Using 100 M EX527 granule cells, with the prolongation of time, two groups of granulosa cells SIRT1, St AR, CYP17, CYP19 expressed in different levels of water The reduction of PPAR expression level (P0.05).2 RSV and EX527 in granulosa cells, with the prolongation of time, the control group and the apoptosis of granulosa cells of PCOS group increased EX527 granulosa cell apoptosis rate increased obviously, and the longer the time, compared with the RSV treated the apoptosis rate of greater differences (P0.05) after.RSV treatment, the expression of Bcl-2 protein in granulosa cells increased, Caspase-3 protein expression decreased after treatment with EX527.3 or EX527 RSV instead of granular cells, cultured in E2 concentrations showed that after dosing decreased with time prolonged, and the concentration increased (P0.05), RSV after the treatment of E2 concentration in liquid culture rose more obvious (P0.05). Conclusion: in cultured human granulosa cells in vitro, RSV and EX527 mediated SIRT1 regulation of granulosa cell apoptosis, SIRT1 inhibits granulosa cell apoptosis, and promote granulosa cells and expression of steroid hormone synthesis key enzyme The synthesis of estradiol hormone is involved in the pathogenesis of PCOS.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R711.75

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