發(fā)布時間：2018-03-07 06:35

  本文選題：多囊卵巢綜合征(PCOS)　切入點：沉默信息調(diào)節(jié)因子1(SIRT1)　出處：《鄭州大學》2017年碩士論文　論文類型：學位論文

【摘要】：多囊卵巢綜合征(PCOS)是一種常見的生殖內(nèi)分泌紊亂疾病,常表現(xiàn)為卵泡無周期成熟導致慢性無排卵,并常伴發(fā)不孕及糖脂代謝紊亂等。其發(fā)病機制除與下丘腦-垂體-卵巢軸功能失調(diào)有關,還與卵泡微環(huán)境失調(diào),卵泡發(fā)育、成熟、閉鎖的調(diào)控失衡有關。卵巢顆粒細胞對卵泡的生長發(fā)育及卵泡微環(huán)境有重要影響,顆粒細胞凋亡增多破壞卵泡微環(huán)境,導致卵泡成熟障礙。SIRT1是一種廣泛存在于哺乳動物中的去乙�；�,其功能廣泛,包括對能量代謝、DNA修復、細胞壽命和凋亡的調(diào)控等。SIRT1的表達受多種因子的調(diào)控,如P53、FOXO和E2F1形成的自反饋環(huán)路。此外,SIRT1參與類固醇激素合成關鍵酶的表達調(diào)控,參與哺乳動物的生殖過程。RSV是至今為止發(fā)現(xiàn)的活力最強的特異性SIRT1激動劑,其功能除涉及維持機體能量平衡,并且還具有抗氧化,抗炎和抗腫瘤活性。EX-527屬于吲哚類SIRT1抑制劑,是至今發(fā)現(xiàn)的活性最強的選擇性SIRT1抑制劑。因此,本課題研究中選擇RSV和EX527分別作為SIRT1特異性的激活劑和抑制劑。目的:本研究以行IVF-ET或ICSI助孕的PCOS患者為主要研究對象,對其顆粒細胞進行體外培養(yǎng),使用RSV和EX527處理顆粒細胞,通過qPCR、Western Blot、流式細胞術及化學發(fā)光法測定培養(yǎng)液中雌二醇濃度等方法,分析SIRT1表達水平改變對細胞凋亡率以及類固醇激素合成關鍵酶的影響,初步探討SIRT1與PCOS的潛在關系。方法:2016年4月1日至2016年12月1日于海南醫(yī)學院第一附屬醫(yī)院生殖醫(yī)學中心行常規(guī)體外授精-胚胎移植(IVF-ET)或卵泡漿內(nèi)單精子顯微注射(ICSI)助孕的患者共114例被納入研究,根據(jù)納入標準及排除標準分組:PCOS組(57例)與對照組(57例)。收集PCOS組與對照組患者卵泡液,采用紅細胞裂解法提取卵泡液中顆粒細胞,均勻接種于6孔板中,使用含15%FBS的DMEM-F12的培養(yǎng)基作為基礎培養(yǎng)基對顆粒細胞進行體外培養(yǎng),細胞貼壁后使用含100μM RSV和含100μM EX527的培養(yǎng)基換液,分別繼續(xù)培養(yǎng)12h、24h、48h后收獲細胞,同時收集培養(yǎng)液,未經(jīng)加藥處理的細胞作為對照。收獲細胞后,用Trizol一步法提取顆粒細胞總RNA,使用RIPA裂解液提取顆粒細胞總蛋白,兩組中目的基因和蛋白(SIRT1、StAR、CYP17、CYP19和PPARγ)的表達水平由qPCR和Western Blot進行檢測并對比分析;流式細胞術檢測各標本細胞凋亡率,Western Blot檢測凋亡相關蛋白Bcl-2和Caspase-3蛋白表達,采用化學發(fā)光法檢測培養(yǎng)液中E2濃度并對比分析。結果:1在兩組顆粒細胞中均能檢測到SIRT1的表達,PCOS中SIRT1呈低表達(P0.05)。使用100μM RSV處理顆粒細胞后,隨作用時間延長,兩組顆粒細胞中SIRT1、StAR、CYP17、CYP19表達水平升高,PPARγ表達水平降低(P0.05)。使用100μM EX527處理顆粒細胞后,隨作用時間延長,兩組顆粒細胞SIRT1、St AR、CYP17、CYP19表現(xiàn)為不同程度的表達水平降低,PPARγ表達水平升高(P0.05)。2 RSV和EX527處理顆粒細胞后,隨作用時間延長,對照組與PCOS組顆粒細胞凋亡率均升高,EX527處理顆粒細胞后細胞凋亡率升高更明顯,且隨時間延長,與RSV處理后相比凋亡率差異更大(P0.05)。RSV處理后,顆粒細胞Bcl-2蛋白表達升高,Caspase-3蛋白表達降低,EX527處理后相反。3 RSV或EX527處理顆粒細胞后,培養(yǎng)液中E2濃度均顯示加藥后下降,隨處理時間延長,濃度又呈上升趨勢(P0.05),RSV處理后培養(yǎng)液中E2濃度上升更明顯(P0.05)。結論:在人顆粒細胞體外培養(yǎng)中,RSV和EX527介導SIRT1對顆粒細胞凋亡的調(diào)控,SIRT1抑制顆粒細胞凋亡,并促進類固醇激素合成關鍵酶的表達和顆粒細胞中雌二醇激素的合成,參與PCOS發(fā)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.

【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R711.75

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