银杏黄酮预防早产、保护胎儿神经系统作用及作用机制的研究

The project intends to take advantage of aglient microarray and transcription factor activity chip, RT-PCR, Western blot and other technologies to screen and verify the differentially expressed GPCR (G protein-coupled receptor) and transcription factors in preterm uterine smooth muscle cells pretreated by Ginkgetin. It can be validated that the role of Ginkgetin in the prevention of preterm birth through observing the pregnant duration of preterm models pretreated by different levels of doses of Ginkgetin. To illustrate the mechanism of ginkgetin to prevent preterm birth ,we detect the transcription factor activity ,IL-6 expression and [Ca2 +] levels in vitro and vivo cell whose GPCR gene was silenced or overexpressed. Fetuses neuro protective effects can be validated by measuring biological characteristics of neural stem cells in premature rats’ brain pretreated by different levels of Ginkgetin. To further reveal Ginkgetin’s protection mechanism of premature rats’ nervous system, we measure the resisting damage ability, levels of MDA, SOD , NO and expression levels of Bax and Bcl-2 in vitro and in vivo of brain tissue and neural stem cells, combined with observing the neurological status of animal models in short and long term. It can provide experimental evidence for effective Chinese medicine monomer containing our own intellectual property rights, which can effectively prevent preterm birth and protect fetal nervous system.

本项目拟利用GPCR和转录因子活性芯片、RT-PCR、Western blot等技术，筛选、验证银杏黄酮作用下早产子宫平滑肌中的差异表达的GPCR和转录因子；通过观察不同等级剂量的银杏黄酮对早产模型动物妊娠时限的影响，确定其预防早产的作用；通过对其在体及离体细胞GPCR基因沉默、过表达情况下，转录因子活性和下游基因IL-6表达及［Ca2+］i水平的影响，揭示银杏黄酮预防早产作用的机制。通过测定不同等级剂量的银杏黄酮对早产鼠仔脑组织中神经干细胞生物学性状的影响、近远期神经系统功能的变化，确定其对胎仔神经保护作用，进一步对在体及离体胎脑组织和神经干细胞抗损伤能力、NO、 MDA、SOD和凋亡基因（Bax）和抑制凋亡基因(Bcl-2)表达等指标的变化，揭示银杏黄酮对鼠仔神经系统保护作用的机制。为寻找我国拥有自主知识产权、有效的预防早产、保护胎儿神经系统的有效中药单体提供实验依据。

本课题首先使用EGB761干预NMB诱发的小鼠早产模型，确定银杏黄酮具有预防早产和保护胎儿神经系统的作用；进而运用Western Blotting、qPCR，联合基因沉默等技术检测EGB761和其单体槲皮素干预后小鼠子宫平滑肌组织和培养的人原代子宫平滑肌细胞中的p65、p-p65 (Ser536)、C-JUN、p-C-JUN (Ser73)、COX-2及IL-6的表达，确定银杏黄酮预防早产的作用机制。同时，检测EGB761干预后小鼠脑组织和培养的小鼠神经干细胞中HSP-70、MMP-9、MDA、NO、T-SOD、Bax 、Bcl-2、Caspase-3的表达，确定银杏黄酮保护胎儿神经系统的作用机制。为阐明银杏黄酮防治早产，保护胎儿神经系统的作用及机制，NMBR作为宫缩抑制剂靶点，银杏黄酮作为宫缩抑制剂和胎儿神经保护剂提供实验依据，为以后早产的预防和早产儿的脑保护提供思路。