EGF相关因子和TGF-β1对人颗粒细胞排卵相关基因表达及相关信号通路的调控作用研究

发布时间：2018-06-06 20:42

本文选题：人颗粒细胞 + EGF相关生长因子　；参考：《郑州大学》2015年博士论文

【摘要】：第一部分研究背景排卵障碍是导致不孕症发生的一个重要因素。虽然很多动物实验已经证明表皮生长因子(Epidermal growth factor,EGF)相关生长因子,特指双调蛋白(Amphiregulin,AREG)、β细胞素(Betacellulin,BTC)和表皮调节素(Epiregulin,EREG),通过调控环氧合酶-2(Cyclooxygenase-2,COX-2)及其作用下的前列腺素E2(Prostaglandin E2,PGE2)的生成调节排卵的发生,但在人颗粒细胞未见相关研究。研究目的研究AREG、BTC和EREG在人颗粒细胞是否促进COX-2的表达及其作用下的PGE2的生成。研究方法采用SVOG细胞系研究AREG、BTC和EREG在人颗粒细胞排卵过程中的调节作用。SVOG细胞系是一株使用SV40T抗原转染人颗粒细胞得到的细胞系。分别采用RT-q PCR和western blotting进行m RNA和蛋白的检测。通过酶联免疫法(Enzyme-linked immunosorbent assay,ELISA)检测培养液中的PGE2水平。结果外源性给予LH促进AREG、BTC、EREG、COX-2和PGE2的表达上调;使用EGF受体(EGF Receptor,EGFR)小干扰RNA(Small interfering RNA,si RNA)转染方法敲除EGFR阻断LH上调的COX-2表达及其作用下的PGE2的生成;AREG、BTC、EREG通过活化ERK1/2信号通路促进COX-2表达及其作用下的PGE2的生成,使用EGFR抑制剂或EGFR si RNA抑制EGFR活性或敲除内源性EGFR表达可以阻断这种上调作用。结论AREG,BTC和EREG通过活化ERK1/2信号通路促进人颗粒细胞COX-2的表达及其作用下的PGE2生成。第二部分研究背景COX-2及其作用下的PGE2是调控排卵发生的关键因素。转移生长因子-β(Transforming growth factor-beta,TGF-β)超家族不仅在卵巢组织分布广泛,而且在调控卵巢各种生理和病理过程中也具有重要作用。TGF-β1是TGF-β超家族的重要成员之一。虽然TGF-β1及其受体在人颗粒细胞上具有表达,而且颗粒细胞是调控排卵发生的重要部位,但截至目前,未见TGF-β1在人颗粒细胞是否促进COX-2表达及PGE2生成的相关报道。研究目的研究TGF-β1在人颗粒细胞是否促进COX-2的表达及其作用下的PGE2的生成。研究方法采用SVOG细胞系研究TGF-β1在人颗粒细胞排卵过程中的调节作用。SVOG细胞系是一株使用SV40T抗原转染人颗粒细胞得到的细胞系。分别采用RT-q PCR和western blotting进行m RNA和蛋白的检测。通过ELISA检测培养液中的PGE2水平。结果TGF-β1促进COX-2的表达和PGE2的生成。使用TβRI抑制剂或TβRI si RNA抑制TβRI活性或敲除内源性TβRI表达,可以阻断TGF-β1对COX-2表达和PGE2生成的促进作用。TGF-β1激活Smad2和Smad3信号通路,使用Smad2 si RNA或Smad3 si RNA抑制内源性Smad2和Smad3表达同样阻断TGF-β1对COX-2表达和PGE2生成的促进作用。结论TGF-β1激活Smad2/3信号通路促进人颗粒细胞COX-2的表达及其作用下的PGE2生成。第三部分研究背景卵巢颗粒细胞参与调控孕激素的生成,在卵巢类固醇甾体激素生成的过程中具有重要意义。类固醇激素合成急性调节蛋白(Steroidogenic acute regulatory protein,St AR)是孕激素合成过程的限速酶。很多因子参与St AR的表达,其中TGF-β1是非常重要的一个生长因子,在卵泡发育各个阶段均有表达,在人卵泡液中也可以检测到,并且颗粒细胞上TGF-β1及其受体均有表达,但TGF-研究目的β1在调节St AR及孕激素生成中的作用却未可知。研究目的研究TGF-β1在人颗粒细胞是否调控St AR的表达及孕激素的生成。研究方法研究方法采用SVOG细胞系研究TGF-β1在人颗粒细胞排卵过程中的调节作用。SVOG细胞系是一株使用SV40T抗原转染人颗粒细胞得到的细胞系。分别采用RT-q PCR和western blotting进行m RNA和蛋白的检测。通过ELISA检测培养液中的孕激素水平。结果TGF-β1下调St AR的表达及孕激素的生成。使用TβRI抑制剂或TβRI si RNA转染方法抑制TβRI活性或敲除内源性TβRI表达,可以阻断TGF-β1对St AR和孕激素生成的抑制作用。TGF-β1分别激活Smad2/3信号通路和ERK1/2信号通路,其中Smad3和ERK1/2信号通路参与TGF-β1下调的St AR表达及孕激素的生成。结论TGF-β1通过激活Smad3和ERK1/2信号通路抑制人颗粒细胞St AR的表达及孕激素的生成,参与人卵巢类固醇甾体激素生成的调节过程。
[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.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R711.6

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