【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)及干細胞因子(SCF)的表達情況,探究顆粒細胞中FSHR及SCF的表達與卵泡成熟是否有關;研究不同濃度的重組促卵泡刺激素(rFSH)對體外培養(yǎng)的人卵巢顆粒細胞的SCF信使RNA(mRNA)及蛋白表達是否有影響;探討人卵巢顆粒細胞中促卵泡刺激素調節(jié)干細胞因子分泌的機制。方法實驗性研究。收集行卵胞漿內單精子顯微注射技術(ICSI)患者(均為輸卵管因素或男方因素)的顆粒細胞,以成熟卵泡及未成熟卵泡來源分組,分別檢測兩組顆粒細胞中FSHR及SCF的表達情況;對行體外受精-胚胎移植技術(IVF-ET)后的卵泡液來源的顆粒細胞進行不同濃度的rFSH(果納芬;10IU/L,25 IU/L,50 IU/L,100 IU/L,200IU/L)干預培養(yǎng),與空白對照組(正常培養(yǎng))一同檢測FSHR及SCF的表達情況;用含有rFSH、環(huán)磷酸腺苷激動劑(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胞,將細胞分為6組:空白對照組,rFSH單獨培養(yǎng)組,H-89單獨培養(yǎng)組,cAMP單獨培養(yǎng)組,H-89聯合cAMP培養(yǎng)組,H-89聯合rFSH培養(yǎng)組。用Real-time PCR,細胞免疫熒光技術及Western blot檢測人顆粒細胞中FSHR及SCF的蛋白和mRNA水平。結果未成熟卵泡來源的顆粒細胞中的FSHR及SCF的表達比成熟卵泡來源顆粒細胞中較低;用不同劑量的rFSH干預培養(yǎng)顆粒細胞后,FSHR及SCF mRNA及蛋白呈現濃度依賴性升高而后降低;FSH可能通過cAMP/PKA途徑影響顆粒細胞中SCF的mRNA及蛋白分泌。結論在人卵巢顆粒細胞中,FSH可能通過上調FSHR表達,激活cAMP/PKA信號傳導通路,從而增加SCF分泌進一步促進卵泡及卵母細胞的發(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.
【學位授予單位】：寧夏醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R714.8
，

本文編號：2309264