NO介导甲状腺素调节大鼠卵泡发育机制的研究

NO (Nitric Oxide) regulates cell development through different signaling pathways in the different cell types. Although NO plays an important role in the normal reproduction function, the mechanisms involved have not been elucidated. Our preliminary studies indicate that the combination of T3 and FSH increase NOS3 protein content and induce preantral follicle growth in vitro. Meanwhile, T3 and FSH induced granulosa cell development via Src/PI3K/Ak pathway. Dysregulation of TH [TH (T3 and T4)] support induces disorder of reproductive endocrinology, which is associated with impaired follicular development. In addition, hypo- and hyper-thyroidism modified NOS (nitric oxide synthase) content in rat ovary. However, whether TH promotes ovarian follicle growth through regulating ovarian cells proliferation and apoptosis, which is mediated by NO, is unclear. The overall objective of this program is to improve our understanding on the cellular and molecular mechanisms by which TH and NO regulate ovarian follicle growth, and specifically on the expression of NOS intermediates in the regulation of follicle destiny during follicular development base on the previous study. The present study will use follicle and granulosa cell culture system in vitro. These findings will advance the current understanding on the role of TH and NO in the regulation of reproductive function and provide important insight into improving and curing the ovarian dysfunction in the condition of TH dysregulation.

一氧化氮(NO)可调节多种细胞的发育，且对生殖功能有重要作用，然而其调节卵巢细胞发育的机制并不清楚。前期结果表明，T3联合FSH可提高颗粒细胞NOS3的蛋白含量并促进腔前卵泡生长，且通过Src/PI3K/Akt通路调节细胞发育。另外，甲状腺素[TH (T3和T4)]分泌失调会导致机体生殖内分泌紊乱，从而使卵泡发育受阻，还可影响卵巢细胞NOS(nitric oxide synthase)表达。然而NO是否介导TH调节卵巢细胞增殖及凋亡进而影响卵泡发育尚不清楚。在前期研究基础上，本课题利用体外卵泡、颗粒细胞培养模型，以NOS为切入点，研究卵泡发育过程中TH对卵巢细胞NOS表达及下游信号通路的调节，从而进一步探明NO在激素调节卵泡发育过程中的作用机理。通过这些研究最终阐明NO和TH调节卵泡发育的初步分子机制，研究结果对改善、治疗甲状腺机能紊乱引起的卵巢功能障碍具有一定的意义。

TH对于雌性生殖非常重要，TH紊乱后会引起卵泡发育受阻。尽管在体外T3能促进FSH诱导的腔前卵泡发育，但是其具体机制还不清楚。葡萄糖是卵泡发育过程中不可或缺的营养物质， GLUT介导它的吸收，然而该过程是否受TH调节并不清楚。本研究通过大鼠颗粒细胞和卵泡培养模型，旨在确定T3联合FSH如何调节GLUT的表达并影响细胞对葡萄糖的吸收。. 结果表明，T3和FSH共同处理颗粒细胞后，显著增加了细胞GLUT1、4的表达、转位以及对葡萄糖的吸收，而这些过程由p-mTOR和PI3K/Akt信号通路所介导。另外，我们还发现L-arg上调GLUT1、4的表达和转位，进而增加葡萄糖的吸收。然而，这些反应被L-NAME阻断。¬同时，激素处理颗粒细胞后，细胞中NOS3的表达量增加、总NOS和NOS3的活性增高，以及NO的含量也相应增加。而且，抑制NO的合成可降低激素诱导的GLUT的表达、转位以及细胞对葡萄糖的吸收作用。以上结果表明，T3和FSH共同处理颗粒细胞后，可以通过上调GLUT1、4的表达和转位来促进细胞摄取葡萄糖，这一过程是通过激活mTOR/PI3K/Akt信号通路来实现的。同时，NOS3/NO也参与了这个调节系统。.为了进一步揭示NO如何参与调节颗粒细胞对葡萄糖的吸收，用SNAP共培养细胞，结果表明SNAP上调了细胞GLUT1、4的表达和转位及葡萄糖的摄取，并伴随着cGMP水平的上升。用sGC抑制剂ODQ和PKG抑制剂KT5823预处理细胞后，我们发现NO的上述调节作用被抑制了。另外用siRNA敲降PKG-I表达后，显著减弱了颗粒细胞中GLUT1/4表达，转位和葡萄糖摄取。另外，PKG-I的siRNA也降低了SNAP诱导的CREB磷酸化。此外，CREB siRNA减弱了颗粒细胞中NO诱导的GLUT表达，转位和葡萄糖吸收效应。这些研究表明，NO通过激活cGMP/ PKG-I信号通路上调颗粒细胞GLUT表达、转位及增加细胞对葡萄糖的摄取。同时，激活的CREB也参与了这一过程。. 综上所述，T3联合FSH通过活化mTOR/PI3K/Akt信号通路，调节细胞中GLUT1/4的表达、转位和葡萄糖的吸收，这一过程由NO介导；而NO则是通过激活cGMP/ PKG信号通路实现上述作用的。