【摘要】：目的研究成熟卵泡與未成熟卵泡中促卵泡刺激素受體(FSHR)及干細(xì)胞因子(SCF)的表達(dá)情況,探究顆粒細(xì)胞中FSHR及SCF的表達(dá)與卵泡成熟是否有關(guān);研究不同濃度的重組促卵泡刺激素(rFSH)對(duì)體外培養(yǎng)的人卵巢顆粒細(xì)胞的SCF信使RNA(mRNA)及蛋白表達(dá)是否有影響;探討人卵巢顆粒細(xì)胞中促卵泡刺激素調(diào)節(jié)干細(xì)胞因子分泌的機(jī)制。方法實(shí)驗(yàn)性研究。收集行卵胞漿內(nèi)單精子顯微注射技術(shù)(ICSI)患者(均為輸卵管因素或男方因素)的顆粒細(xì)胞,以成熟卵泡及未成熟卵泡來源分組,分別檢測(cè)兩組顆粒細(xì)胞中FSHR及SCF的表達(dá)情況;對(duì)行體外受精-胚胎移植技術(shù)(IVF-ET)后的卵泡液來源的顆粒細(xì)胞進(jìn)行不同濃度的rFSH(果納芬;10IU/L,25 IU/L,50 IU/L,100 IU/L,200IU/L)干預(yù)培養(yǎng),與空白對(duì)照組(正常培養(yǎng))一同檢測(cè)FSHR及SCF的表達(dá)情況;用含有rFSH、環(huán)磷酸腺苷激動(dòng)劑(cAMP)及PKA抑制劑(H-89)的培養(yǎng)基培養(yǎng)IVF卵泡液來源的顆粒細(xì)胞,將細(xì)胞分為6組:空白對(duì)照組,rFSH單獨(dú)培養(yǎng)組,H-89單獨(dú)培養(yǎng)組,cAMP單獨(dú)培養(yǎng)組,H-89聯(lián)合cAMP培養(yǎng)組,H-89聯(lián)合rFSH培養(yǎng)組。用Real-time PCR,細(xì)胞免疫熒光技術(shù)及Western blot檢測(cè)人顆粒細(xì)胞中FSHR及SCF的蛋白和mRNA水平。結(jié)果未成熟卵泡來源的顆粒細(xì)胞中的FSHR及SCF的表達(dá)比成熟卵泡來源顆粒細(xì)胞中較低;用不同劑量的rFSH干預(yù)培養(yǎng)顆粒細(xì)胞后,FSHR及SCF mRNA及蛋白呈現(xiàn)濃度依賴性升高而后降低;FSH可能通過cAMP/PKA途徑影響顆粒細(xì)胞中SCF的mRNA及蛋白分泌。結(jié)論在人卵巢顆粒細(xì)胞中,FSH可能通過上調(diào)FSHR表達(dá),激活cAMP/PKA信號(hào)傳導(dǎo)通路,從而增加SCF分泌進(jìn)一步促進(jìn)卵泡及卵母細(xì)胞的發(fā)育成熟。
[Abstract]:Objective to study the expression of follicle stimulating hormone receptor (FSHR) and stem cell factor (SCF) in mature and immature follicles, and to explore whether the expression of FSHR and SCF in granulosa cells is related to follicular maturation. To investigate the effects of different concentrations of recombinant follicle-stimulating hormone (rFSH) on the expression of SCF messenger RNA (mRNA) and protein in cultured human ovarian granulosa cells. To investigate the mechanism of follicle stimulating hormone (FSH) regulating the secretion of stem cell factor in human ovarian granulosa cells. Methods Experimental study. Granulosa cells were collected from (ICSI) patients with intracytoplasmic sperm microinjection (both oviduct factor and male factor). The granulosa cells were divided into mature follicles and immature follicles to detect the expression of FSHR and SCF in granulosa cells of the two groups. Granulosa cells derived from follicular fluid after in vitro fertilization and embryo transfer (IVF-ET) were treated with different concentrations of rFSH (gonafen). The expression of FSHR and SCF were detected in 10 IUU / L 25 IU/L,50 IU/L,100 IU/L,200IU/L culture group and control group (normal culture group). Granulosa cells derived from IVF follicle fluid were cultured on the culture medium containing rFSH, cyclic adenosine monophosphate agonist (cAMP) and PKA inhibitor (H-89). The cells were divided into six groups: blank control group, rFSH alone culture group, H-89 single culture group. CAMP alone group, H 89 combined with cAMP culture group, H 89 combined with rFSH culture group. The protein and mRNA levels of FSHR and SCF in human granulosa cells were detected by Real-time PCR, cell immunofluorescence and Western blot. Results the expression of FSHR and SCF in granulosa cells from immature follicles was lower than that in granulosa cells derived from mature follicles, and the expression of FSHR, SCF mRNA and protein in granulosa cells cultured with different doses of rFSH increased in a concentration-dependent manner and then decreased. FSH may influence the secretion of mRNA and protein of SCF in granulosa cells via cAMP/PKA pathway. Conclusion in human ovarian granulosa cells, FSH may promote follicle and oocyte maturation by up-regulating FSHR expression and activating cAMP/PKA signal transduction pathway.
【學(xué)位授予單位】：寧夏醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R714.8
，

本文編號(hào)：2309264