【作者】 马黔红；

【导师】 彭芝兰； 李尚为；

【作者基本信息】 四川大学 ， 妇产科学， 2007， 博士

【摘要】 背景与研究目的：颗粒细胞(granulosa cell，GC)是组成卵泡的主要体细胞成分，它在卵泡生长和发育中有重要的作用，与生殖细胞的生长、发育相互依赖、相互协助。一些研究者认为在体外授精．胚胎移植(in vitro fertilization-embryo transfer，IVF-ET)中，黄素化GC中某些超微结构的特征和凋亡可以作为预测IVF结局的指标。但是到目前为止，对影响黄素化GC凋亡的因子尚不完全清楚。本研究以IVF中排卵前卵泡作为研究对象，研究不同浓度的促黄体素(luteinized hormone，LH)是否会对卵泡的卵泡液和黄素化颗粒细胞的凋亡相关因子及细胞间的缝隙连接蛋白的表达产生不同的影响，以期寻找能对IVF妊娠结局有较强预测性的一种或几种因子。材料与方法本研究收集、分离了从体外受精-胚胎移植病人排卵前卵泡中获得的黄素化颗粒细胞，进行原代培养，3天后分别加入促卵泡素(follicle-stimulating hormone，FSH) 3IU/ml(对照组)、LH 1IU/ml(LHI组)、LH 3IU/ml(LH2组)和LH 6IU/ml(LH3组)，培养24小时，分别测定黄素化GC中凋亡效应子半胱氨酸蛋白酶caspase-3、caspase-7和缝隙连接蛋白Cx43的mRNA和蛋白质的表达。并且在IVF取卵时收集排卵前卵泡的卵泡液，测定卵泡液中可溶性Fas(sFas)、sFasL和LH的浓度，分析在妊娠组和未妊娠组中这三个指标之间的差别，以及sFas、sFasL和LH与妊娠的相关性，应用Logistic回归分析sFas、sFasL和LH之间的相关性。结果：1、黄素化颗粒细胞体外原代培养是一个很好的估计卵巢功能及卵细胞质量的系统。原代细胞培养的优势在于最大程度地保存了细胞的特性，使体外实验结果更符合于体内的情况。2、在黄素化颗粒细胞中，加入FSH和不同浓度的LH后，高浓度的LH3组的Caspase-3的mRNA和蛋白质的相对表达量高于其余各组，其中mRNA的表达在各组中有统计学差异，而蛋白的表达无显著统计学差异；而且随着外源性给予的LH浓度的增加，Caspase-3的mRNA和蛋白质相对表达量也有逐渐升高的趋势。Caspase-7的mRNA和蛋白质的相对表达也随着外源性给予的LH的增加也逐渐增高，其中LH3组的表达最高，且在mRNA的表达中LH3组显著高于LH1组和LH2组：而蛋白质的表达中各组间都没有统计学差异。4、FSH组、LH1组和LH2组之间Cx43的mRNA和蛋白质的相对表达量没有差异，但是LH3组明显下降，与FSH组、LH1组和LH2组分别相比较，其中mRNA的表达有统计学的显著性差异，蛋白质的表达无统计学差异。5、在IVF中，妊娠组排卵前卵泡液中的LH和sFasL水平都明显高于未妊娠组，两组之间的sFas水平没有明显差异。6、通过logistic回归分析，卵泡液中与妊娠有统计学相关的因素仅为LH，OR=4.117，虽然sFasL在妊娠组和未妊娠组中有显著性差异，但经过回归分析后，它和sFas一样，都与IVF中病人是否妊娠没有相关性。7、应用Spearman相关分析，发现卵泡液中的LH和sFasL的水平呈明显正相关关系，而卵泡液中的sFas和LH之间并没有相关性。结论：1、低剂量的LH(1IU/ml和3IU/ml)可以对黄素化GC发挥类似FSH抗凋亡的作用，在低剂量的LH作用下，细胞中Caspase-3、Caspase-7和Cx43的mRNA和蛋白质的表达与FSH组中的表达无明显差异；但高剂量的LH(6IU/ml)会使凋亡相关因子表达增加，引起细胞凋亡，影响缝隙连接蛋白Cx43的表达，并且有可能通过异常表达的Cx43导致细胞间连接失耦联，影响卵泡中的正常信息传导，影响GC的存活，导致GC的凋亡增加，从而间接影响卵母细胞的发育、成熟。2、IVF妊娠组中排卵前卵泡卵泡液中的LH和sFasL明显高于未妊娠组；但是经过相关性分析和logistic回归分析后，发现只有卵泡液中的LH水平与妊娠有明显相关性。LH可能是通过某些其他途径对卵泡液中sFasL产生影响，并且LH可能对IVF的结局有很高的预测价值。更多还原

【Abstract】 Background and objectives:Granulosa cells(GC) are the main somatic cells in follicles, which are very important during the follicular growth and development. The ovarian oocyte-granulosa cell interactions are essential for normal follicular development. The characteristics of ultrastructure and apoptosis of luteinized granulosa cells obtained from in vitro fertilization-embryo transfer (IVF-ET) maybe the good indicators for IVF prognosis. But it is unclear on some factors that influencing the apoptosis of luteinized GC. To explore the effects of different concentration of luteinizing hormone (LH) on the apoptosis-associated factors and gap junction (GJ) in the follicle fluid and luteinized GC , we have studied the preovulatoty follicles during IVF, so that we would find one or more factors to indicate the IVF result.Materials and methods:All luteinized GC were obtained from women who undergoing IVF at IVF center, West China Second University Hospital. After 3 days primary cultures, all cells were grouped as adding FSH 3IU/ml (control),LH HU/ml(LHl group), LH 3IU/ml(LH2 group) and LH 6IU/mL(LH3 group)for 24 hours. We analyzed the mRNA and protein expression levels of caspase-3, caspase-7 and Connexin 43. We also detected the LH, soluble Fas (sFas) and the soluble Fas ligand (sFasL) in the follicular fluid to analyzed relevance between these factors and pregnancy results, as well as between the LH and sFas -sFasL system.Results:1、The advantage in primary culture of luteinized GC is to preserve the its determined properties , which making the experiments in vitro more likely with the environment in vivo.2、After adding FSH and different dosage of LH, the relative mRNA and protein expressions in LH 3 group are higher than other groups, the relative mRNA expressions have statistical difference, but relative protein expressions have not statistical difference. Moreover, the expression of mRNA and protein of caspase-3 are gradually increased along with the increased dosage of exogenous LH. The expression of mRNA and protein of caspase-7 are also gradually increased along with the increased dosage of exogenous LH, and the mRNA expression in LH 3 group are statistical higher than that in LH 1 and LH 2 group, but there are no statistical difference in protein expression between these four groups.4、The relative expressions of mRNA and protein of Cx43 in control group、LH1 group and LH2 group have no difference. However, the relative expression of mRNA in LH 3 group is lower than in other three groups, and no difference on protein expression.5、The levels of LH and sFasL in the preovulatory follicle fluid are higher inpregnancy group than that in non pregnancy group, and in these twogroups ,there is no difference in sFas level.6、Using logistic regression analysis method, we found that LH in follicle fluidhad statistical relationship with the pregnancy result, and the odds ratio was4.117. Although there were statistical difference in sFasL between pregnancygroup and non pregnancy group by using the matched t-test, there were norelevance between sFasL and pregnancy result by using logistic regressionanalysis.7、The LH level in follicular fluid had positive relevant relationship withsFasL, but no relationship with sFas, by using Spearman relevanceanalysis .Conclusions:1、Low dose of LH (1IU/ml, 3IU/ml) can mimic the function of FSH on luteinized GC, that making the expression of mRNA and protein of caspase-3and caspase-7 increased, Cx43 decreased . But the high dose of LH(6IU/ml)would make the caspase-3 and caspase-7 increasing, and induce luteinized GC apoptosis, meanwhile it also influence the expression of Cx43, which may destroy the gap junctional coupling between GCs, influence the normal message conduction within follicles and induce the apoptosis of luteinized GC,and as a result,indirectly influence the oocyte development and maturation.2、In the preovulatory follicle fluid, the levels of LH and sFasL in pregnancy group are significantly higher than that in non-pregnancy group, but through relevance analysis and logistic regression analysis, only LH level in follicle fluid was associated with pregnancy. LH may influence sFasL level in follicle fluid through other pathway, and it maybe a good indicator for IVF prognosis.更多还原