雌激素受体α的K266位点乙酰化对GnRH神经元棘突发育及激素分泌的影响

The onset puberty is closely related with the morphology and function mature of GnRH neurons. Our previous studies shown that, the acetylation of the estrogen receptor α in GnRH neurons would make the cells show different reactions to 17β-E2. When transfected the plasmid into GT1-7 cells which could express the K266 acetylated estrogen receptor α, added 17β-E2 in the culture medium, the amount of the spinous of transfected cells would increase 3-folds than the control ones; the hormone level of GnRH increased for about 6.2-folds. In this proposal, we will use the codon expanding technique, RT-PCR and Western-blotting, luciferase tracking tool, focus on the molecular pathways to the development and function mature of K266 site acetylated estrogen receptor GnRH neurons, it included the estrogen receptor-NDR1/2 pathway; estrogen receptor α- extracellular regulated protein (ERK) pathway; and the switch behaviors of the acetylated estrogen receptor itself from cell cytoplasm to the nucleus, and then activate the regulatory factor genes of cell development and GnRH hormone. The research on GnRH neurons biological functions which express K266 acetylated estrogen receptor α and the studies on the mechanisms that cause physiological changes will offer efficient experimental data, and deep our understandings on the molecule mechanisms of human puberty onset.

中枢神经系统中GnRH神经元的正常发育及激素分泌功能的成熟，与青春期启动有密切的关系。在前期工作中，我们对分泌GnRH激素的GT1-7细胞的研究发现，GT1-7细胞中雌激素受体α乙酰化水平的升高可以增强其对雌激素的敏感性。转染可表达K266位点乙酰化雌激素受体α的质粒至GT1-7细胞，饲喂雌二醇后，细胞棘突生长的长度是对照组的3倍，GnRH激素分泌量是对照组的6.2倍。我们拟使用密码子扩展技术、RT-PCR及Western-blotting、荧光素追踪等技术手段，重点研究转染K266位点乙酰化雌激素受体的GT1-7细胞棘突发育及激素分泌所涉及的分子途径，明确GT1-7细胞内NDR1/2分子介导的棘突发育调控途径；雌激素受体-细胞外调节蛋白（ERK）通路与激素分泌的关系；乙酰化的雌激素受体是否在细胞内发生细胞质到细胞核转移，进而调控棘突发育和激素分泌相关基因转录表达的机制。

中枢神经系统中GnRH神经元的正常发育及激素分泌功能的成熟，与青春期启动有密切的关系。我们对分泌GnRH激素的GT1-7细胞的研究发现，GT1-7细胞中雌激素受体α乙酰化水平的升高可以增强其对雌激素的敏感性。转染可表达K266位点乙酰化雌激素受体α的质粒至GT1-7细胞，饲喂雌二醇后，细胞棘突生长的长度是对照组的3倍，GnRH激素分泌量是对照组的6.2倍。.同时，我们利用本课题已经建立好的密码子扩展技术平台，选取了异柠檬酸脱氢酶1（isocitrate dehydrogenase,IDH1）为研究对象。IDH1是胞质中重要的代谢酶，除催化异柠檬酸氧化脱羧生成α-KG、产生NADPH外，还可间接调节氨基酸和脂类代谢，参与氧化应激反应等。现有研究发现，IDH1在肺癌组织和血浆中有高表达，抑制IDH1可以抑制肺癌细胞增殖，促进癌细胞凋亡。我们将利用已经建立完善的码子扩展技术平台研究乙酰化修饰对IDH1酶活力的影响及其在肿瘤细胞增殖的影响，进而为肺癌的诊断和治疗提供潜在靶点。为了探索乙酰化对IDH1的调控机制，构建了活性位点的赖氨酸-谷氨酰胺(KQ)和赖氨酸-精氨酸(KR)突变体，分别模拟赖氨酸乙酰化和脱乙酰化状态。动力学实验表明，K93Q突变体的活性比野生型降低了8倍，K224Q突变体的活性比野生型高3倍。利用分子动力学模拟，推断K224Q突变可能破坏S94和D279之间的关键氢键，导致更开放的构象和增加的催化活性。K93Q突变可能会破坏酶的整体稳定性，从而降低活性。我们进一步验证了活性降低的突变体抑制肺癌细胞的增殖。基于我们的研究结果，希望能找到IDH1的对肺癌细胞的调节机制。