促卵泡刺激素通过调控颗粒细胞分泌干细胞因子促进卵泡发育成熟的研究
发布时间：2018-11-04 08:18
【摘要】：目的研究成熟卵泡与未成熟卵泡中促卵泡刺激素受体(FSHR)及干细胞因子(SCF)的表达情况,探究颗粒细胞中FSHR及SCF的表达与卵泡成熟是否有关;研究不同浓度的重组促卵泡刺激素(rFSH)对体外培养的人卵巢颗粒细胞的SCF信使RNA(mRNA)及蛋白表达是否有影响;探讨人卵巢颗粒细胞中促卵泡刺激素调节干细胞因子分泌的机制。方法实验性研究。收集行卵胞浆内单精子显微注射技术(ICSI)患者(均为输卵管因素或男方因素)的颗粒细胞,以成熟卵泡及未成熟卵泡来源分组,分别检测两组颗粒细胞中FSHR及SCF的表达情况;对行体外受精-胚胎移植技术(IVF-ET)后的卵泡液来源的颗粒细胞进行不同浓度的rFSH(果纳芬;10IU/L,25 IU/L,50 IU/L,100 IU/L,200IU/L)干预培养,与空白对照组(正常培养)一同检测FSHR及SCF的表达情况;用含有rFSH、环磷酸腺苷激动剂(cAMP)及PKA抑制剂(H-89)的培养基培养IVF卵泡液来源的颗粒细胞,将细胞分为6组:空白对照组,rFSH单独培养组,H-89单独培养组,cAMP单独培养组,H-89联合cAMP培养组,H-89联合rFSH培养组。用Real-time PCR,细胞免疫荧光技术及Western blot检测人颗粒细胞中FSHR及SCF的蛋白和mRNA水平。结果未成熟卵泡来源的颗粒细胞中的FSHR及SCF的表达比成熟卵泡来源颗粒细胞中较低;用不同剂量的rFSH干预培养颗粒细胞后,FSHR及SCF mRNA及蛋白呈现浓度依赖性升高而后降低;FSH可能通过cAMP/PKA途径影响颗粒细胞中SCF的mRNA及蛋白分泌。结论在人卵巢颗粒细胞中,FSH可能通过上调FSHR表达,激活cAMP/PKA信号传导通路,从而增加SCF分泌进一步促进卵泡及卵母细胞的发育成熟。
[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.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R714.8
，
本文编号：2309264
[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.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R714.8
，
本文编号：2309264
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