本文選題：人顆粒細胞 + EGF相關生長因子�。� 參考：《鄭州大學》2015年博士論文

【摘要】：第一部分研究背景排卵障礙是導致不孕癥發(fā)生的一個重要因素。雖然很多動物實驗已經(jīng)證明表皮生長因子(Epidermal growth factor,EGF)相關生長因子,特指雙調蛋白(Amphiregulin,AREG)、β細胞素(Betacellulin,BTC)和表皮調節(jié)素(Epiregulin,EREG),通過調控環(huán)氧合酶-2(Cyclooxygenase-2,COX-2)及其作用下的前列腺素E2(Prostaglandin E2,PGE2)的生成調節(jié)排卵的發(fā)生,但在人顆粒細胞未見相關研究。研究目的研究AREG、BTC和EREG在人顆粒細胞是否促進COX-2的表達及其作用下的PGE2的生成。研究方法采用SVOG細胞系研究AREG、BTC和EREG在人顆粒細胞排卵過程中的調節(jié)作用。SVOG細胞系是一株使用SV40T抗原轉染人顆粒細胞得到的細胞系。分別采用RT-q PCR和western blotting進行m RNA和蛋白的檢測。通過酶聯(lián)免疫法(Enzyme-linked immunosorbent assay,ELISA)檢測培養(yǎng)液中的PGE2水平。結果外源性給予LH促進AREG、BTC、EREG、COX-2和PGE2的表達上調;使用EGF受體(EGF Receptor,EGFR)小干擾RNA(Small interfering RNA,si RNA)轉染方法敲除EGFR阻斷LH上調的COX-2表達及其作用下的PGE2的生成;AREG、BTC、EREG通過活化ERK1/2信號通路促進COX-2表達及其作用下的PGE2的生成,使用EGFR抑制劑或EGFR si RNA抑制EGFR活性或敲除內(nèi)源性EGFR表達可以阻斷這種上調作用。結論AREG,BTC和EREG通過活化ERK1/2信號通路促進人顆粒細胞COX-2的表達及其作用下的PGE2生成。第二部分研究背景COX-2及其作用下的PGE2是調控排卵發(fā)生的關鍵因素。轉移生長因子-β(Transforming growth factor-beta,TGF-β)超家族不僅在卵巢組織分布廣泛,而且在調控卵巢各種生理和病理過程中也具有重要作用。TGF-β1是TGF-β超家族的重要成員之一。雖然TGF-β1及其受體在人顆粒細胞上具有表達,而且顆粒細胞是調控排卵發(fā)生的重要部位,但截至目前,未見TGF-β1在人顆粒細胞是否促進COX-2表達及PGE2生成的相關報道。研究目的研究TGF-β1在人顆粒細胞是否促進COX-2的表達及其作用下的PGE2的生成。研究方法采用SVOG細胞系研究TGF-β1在人顆粒細胞排卵過程中的調節(jié)作用。SVOG細胞系是一株使用SV40T抗原轉染人顆粒細胞得到的細胞系。分別采用RT-q PCR和western blotting進行m RNA和蛋白的檢測。通過ELISA檢測培養(yǎng)液中的PGE2水平。結果TGF-β1促進COX-2的表達和PGE2的生成。使用TβRI抑制劑或TβRI si RNA抑制TβRI活性或敲除內(nèi)源性TβRI表達,可以阻斷TGF-β1對COX-2表達和PGE2生成的促進作用。TGF-β1激活Smad2和Smad3信號通路,使用Smad2 si RNA或Smad3 si RNA抑制內(nèi)源性Smad2和Smad3表達同樣阻斷TGF-β1對COX-2表達和PGE2生成的促進作用。結論TGF-β1激活Smad2/3信號通路促進人顆粒細胞COX-2的表達及其作用下的PGE2生成。第三部分研究背景卵巢顆粒細胞參與調控孕激素的生成,在卵巢類固醇甾體激素生成的過程中具有重要意義。類固醇激素合成急性調節(jié)蛋白(Steroidogenic acute regulatory protein,St AR)是孕激素合成過程的限速酶。很多因子參與St AR的表達,其中TGF-β1是非常重要的一個生長因子,在卵泡發(fā)育各個階段均有表達,在人卵泡液中也可以檢測到,并且顆粒細胞上TGF-β1及其受體均有表達,但TGF-研究目的β1在調節(jié)St AR及孕激素生成中的作用卻未可知。研究目的研究TGF-β1在人顆粒細胞是否調控St AR的表達及孕激素的生成。研究方法研究方法采用SVOG細胞系研究TGF-β1在人顆粒細胞排卵過程中的調節(jié)作用。SVOG細胞系是一株使用SV40T抗原轉染人顆粒細胞得到的細胞系。分別采用RT-q PCR和western blotting進行m RNA和蛋白的檢測。通過ELISA檢測培養(yǎng)液中的孕激素水平。結果TGF-β1下調St AR的表達及孕激素的生成。使用TβRI抑制劑或TβRI si RNA轉染方法抑制TβRI活性或敲除內(nèi)源性TβRI表達,可以阻斷TGF-β1對St AR和孕激素生成的抑制作用。TGF-β1分別激活Smad2/3信號通路和ERK1/2信號通路,其中Smad3和ERK1/2信號通路參與TGF-β1下調的St AR表達及孕激素的生成。結論TGF-β1通過激活Smad3和ERK1/2信號通路抑制人顆粒細胞St AR的表達及孕激素的生成,參與人卵巢類固醇甾體激素生成的調節(jié)過程。
[Abstract]:The first part studies background ovulatory disorders is an important factor in the occurrence of infertility. Although many animal experiments have proved Epidermal growth factor, EGF related growth factors, especially Amphiregulin, AREG, Betacellulin, BTC, and epidermodulin (Epiregulin, EREG), through regulation -2 (Cyclooxygenase-2, COX-2) and the production of prostaglandin E2 (Prostaglandin E2, PGE2) under its action regulate the occurrence of ovulation, but there is no related study in human granulosa cells. The purpose of this study is to investigate whether AREG, BTC and EREG in human granulosa cells promote the production of PGE2 under the action of COX-2. Regulation of AREG, BTC and EREG in the process of ovulation in human granulosa cells.SVOG cell line is a cell line obtained by using SV40T antigen to transfect human granulosa cells. RT-q PCR and Western blotting are used to detect m RNA and protein, respectively. Results exogenous LH promotes the expression of AREG, BTC, EREG, COX-2 and PGE2, and the use of EGF receptor (EGF Receptor, EGFR) to interrupt the up up expression and its action. The expression of OX-2 and the formation of PGE2 under the action of EGFR inhibitors or EGFR Si RNA inhibit EGFR activity or knock out endogenous EGFR expression. Conclusion AREG, BTC and EREG can promote the expression of human granulosa cells by activating the ERK1/2 signal pathway and the formation of it under the action. The second part studies background and PGE2 is a key factor in the regulation of ovulation. The superfamily of Transforming growth factor-beta (TGF- beta) is not only widely distributed in the ovarian tissue, but also plays an important role in the regulation of various physiological and pathological processes of the ovary..TGF- beta 1 is one of the important members of the TGF- beta superfamily. Although TGF- beta 1 and its The receptor is expressed on human granulosa cells, and granulosa cells are the important parts to regulate the occurrence of ovulation. But up to now, no TGF- beta 1 has been reported in human granulosa cells to promote the expression of COX-2 and the formation of PGE2. The purpose of this study is to investigate whether TGF- beta 1 promotes the expression of COX-2 in human granulosa cells and the formation of PGE2. The SVOG cell line was used to study the role of TGF- beta 1 in the ovulatory process of human granulosa cells..SVOG cell line was a cell line obtained by using SV40T antigen to transfect human granulosa cells. RT-q PCR and Western blotting were used to detect m RNA and protein respectively. PGE2 level in the culture liquid was detected by ELISA. The expression of COX-2 and the formation of PGE2. Using T beta RI inhibitor or T beta RI Si RNA to inhibit the activity of T beta RI or knock out endogenous T beta RI expression, it can block the promoting effect of beta 1 on the expression and production of the beta 1. The effect of beta 1 on the expression of COX-2 and the formation of PGE2. Conclusion TGF- beta 1 activates the Smad2/3 signaling pathway to promote the expression of COX-2 in human granulosa cells and the formation of PGE2 under its action. The third part of the study is that ovarian granulosa cells are involved in the regulation of the production of progestin, and it is of great significance in the process of steroid hormone production of ovarian steroids. Steroidogenic acute regulatory protein (St AR) is a speed limiting enzyme in the progesterone synthesis process. Many factors are involved in the expression of St AR. TGF- beta 1 is a very important growth factor, expressed in all stages of follicle development, and can also be detected in human follicle fluid, and TGF- in granular cells. Both the beta 1 and its receptor are expressed, but the role of TGF- 1 in regulating the production of St AR and progesterone is unknowable. The purpose of this study is to investigate whether TGF- beta 1 regulates the expression of St AR and the production of progestin in human granulosa cells. Methods study methods used SVOG cell lines to study the regulation of TGF- beta 1 in the process of human granulosa cells ovulation. .SVOG cell line was a cell line obtained by using SV40T antigen to transfect human granulosa cells. RT-q PCR and Western blotting were used to detect m RNA and protein respectively. Progestin levels in the culture liquid were detected by ELISA. Results TGF- beta 1 downregulated the expression of St AR and the production of progestin. Inhibition of T beta RI activity or knockout endogenous T beta RI could block the inhibition of TGF- beta 1 on the formation of St AR and progestin,.TGF- beta 1 activates the Smad2/3 signaling pathway and ERK1/2 signaling pathway respectively. Smad3 and ERK1/2 signaling pathways are involved in the expression of beta 1 and the production of progestin. The signal pathway inhibits the expression of St AR and progesterone in human granulosa cells, and participates in the regulation of steroid hormone production in human ovary.
【學位授予單位】：鄭州大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R711.6

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