Bim蛋白对促排卵过早黄素化颗粒细胞促凋亡作用的研究

Apoptosis, the physiological process of cell death, is critical for modelling tissues and maintaining homeostasis in multicellular organisms. The mechanism of this intrinsic suicide programme is under intense scrutiny. The executioners are a set of cysteine proteinases, termed caspases, that degrade critical cellular substrates. The regulatory machinery that governs the activation of the caspases is less well understood, but a central role is played by the Bcl-2 family. Bcl-2 itself was the first intracellular regulator of apoptosis to be identified, and high levels enhance cell survival under diverse cytotoxic conditions. . The homology between members of the Bcl-2 family is greatest within four small segments, designated Bcl-2 homology (BH) regions, some of which contribute to the interactions between Bcl-2 family members. The importance of the BH3 region for facilitating apoptosis has been underscored by the discovery of several BH3- containing proteins: Bik/Nbk , Bid and Hrk/DP5, which are otherwise unrelated to the Bcl-2 family but are potent activators of apoptosis when overexpressed. To search for additional regulators of apoptosis, we have screened a cDNA expression library with a Bcl-2 protein probe. This interaction screen has yielded a novel BH3-containing protein, which we have denoted Bim. Three Bim isoforms all promote apoptosis but differ in potency. Bim interacts with some but not all Bcl-2 family members that promote cell survival, and only those prosurvival relatives that bind to it can neutralize its cytotoxicity. Bim therefore appears to represent a death ligand with a specificity restricted to certain pro-survival members.of the Bcl-2 family.. A premature serum progesterone(P)elevation untimely before HCG administration during controlled ovarian hyperstimulation(COH)and the concomitant process of granulosa cells differentiate into luteal cells is usually called premature luteinization(PL),which could adversely affect clinical outcomes in IVF-ET．However,the molecular mechanisms of PL are not completely understood．. Apoptosis can be triggered by members of the Bcl-2 protein family, such as Bim, that may have effect between premature luteinizattion and granulosa cells apoptasis.The role of Bim in apoptosis signaling was investigated by comparing the sensitivity of luteinized granulosa cells in different treatment. Luteinized granulosa cells was impacted of FSH, LH and P, that may have more role in apoptosis be triggered by Bim, focus some key factor .

探讨控制性超排过程中Bim在卵泡发育中促凋亡的作用，以及促排卵后FSH、P、LH在调控细胞Bim表达中的分子机制。本研究：①对筛选出过早黄素化的颗粒细胞样本，采用TUNEL 和免疫组化等技术确认Bim的表达，并通过体外培养，检测其凋亡以及Bim基因的表达水平。②添加不同剂量的FSH、P、LH和P13K、MAPK、ERK等激酶通路抑制剂对未过早黄素化颗粒细胞进行培养，检测其凋亡以及Bim基因的表达水平，筛选出FSH、P、LH、对Bim调控的关键因子，再结合基因超表达、RNA干扰以及Western Blot等技术，对该关键因子进行验证。③采用免疫组化技术观察调控因子表达水平与颗粒细胞和卵泡闭锁键的关系，为建立控制性促排卵过程中卵泡凋亡提供新的理论依据。④研究中发现的关键因子期望为控制哺乳动物卵泡发育的药物研究提供新的方向。

目标：卵子老化能够影响卵子质量、胚胎质量、卵子利用率、妊娠率、胎儿。Bim基因是一种关键的促凋亡因子，颗粒细胞过早黄素化会可能使Bim基因过表达导致卵子老化。方法：564个长方案纳入选择，排除其它因素，93个周期按照HCG日孕酮分组，114个周期按雌激素分成两组。结果：本项目发现，ART促排卵过程中，HCG日高孕酮（P>1.3）和高雌激素(P>1.3)下出现颗粒细胞过早黄素化，在HCG日高孕酮患者；卵子成熟率（60.27 vs 86.88；P﹤0.05）显著低于对照组；胚胎利用率（57.90 vs 79.55；P﹤0.05）显著低于对照组；妊娠率低于对照组（27.3 vs 52.46; P<0.05）；BIM基因的相对蛋白含量Westblot结果（2.46 VS 0.31）高于对照组，Bim基因的甲基化（6.29 vs13.95 ）相对不活跃但显著低于于对照组，AMH基因的相对蛋白含量（0.973 vs 0.896 ）与对照组相比差异不显著，但AMH基因甲基化(87.39 vs 76.15;P<0.05)活跃，表明AMH不能正常形式其功能。高雌激素患者（E2>4500），卵子成熟率（73.19 vs 92.17；P﹤0.01）显著低于对照组；受精率（68.43 vs 81.56；P﹤0.05）形成率显著低于对照组，胚胎利用率（57.92 vs 66.44；P﹥0.05）低于对照组，，妊娠率于对照组（45.95 vs 44.04；P﹥0.05）差异不显著；RT-PCR检测的BIM基因的相对蛋白含量(2.373 VS 0.52，P<0.05)；Bim基因的Westblot结果（1 VS 0.31 ）高于对照组，Bim基因的甲基化（2.86 vs 14.17;P<0.05）相对不活跃但显著低于对照组，AMH基因的相对蛋白含量(2.54 vs 0.538)与对照组相比显著升高，但AMH基因甲基化活跃（83.71 vs 79.83），表明AMH不能正常形式其功能。结论：ART时HCG日高孕酮和雌激素下会出现过早黄素化，过早黄素化颗粒细胞中Bim基因过表达，Bim基因的促凋亡作用是颗粒细胞加速凋亡，颗粒细胞细胞的大量凋亡加速了卵子老化，可能造成卵子成熟、受精、胚胎质量的下降、妊娠率下降。而AMH对胚胎质量的影响需要进一步研究。