Fas/FasL途径介导的线粒体通路在镉致神经细胞凋亡中的调控机制

Cadmium-induced apoptosis in neurons is the major reason in brain comprised of abnormal form of neurons and low intelligence. However, the mechanism of cadmium-induced apoptosis in neurons remains to be further elucidated. Our previous study have demonstrated that the mitochondrial pathway involved in Cd-induced apoptosis in neurons, however, the role of Fas/FasL signaling pathway played in Cd-induced mitochondrial pathway is not clear. In this project, we are planning to use lentivirus-mediated shRNA to knock down the expression of Fas/FasL in rat, primary rat cerebral cortical neurons and PC12 cells, following exposure to Cd and specific inhibitors, using real-time PCR, Western-blot, FACS, immunohistochemistry and the techniques of Confocal Laser Scanning Microscopy to study the role of of Fas/FasL pathway in Cd-induced apoptosis by quantifying the levels of the key proteins and apoptosis. Therefore, the mechanism of Fas/FasL pathway mediated mitochondrial pathway in neuroal cell apoptosis caused by cadmium will be clarified. The results of this study have potential benefits on the mechanism of neurotoxicology caused by cadmium, regulatory network of signaling pathway and novel drug targets for neurorestoration.

镉致神经细胞凋亡是神经细胞形态异常和智力发育低下的主要原因，但镉致神经细胞凋亡的信号通路尚不明确。前期研究表明，镉可经线粒体通路引起神经细胞凋亡，但Fas/FasL途径在镉致神经细胞凋亡线粒体通路中的调控机制尚未阐明。本研究拟利用慢病毒介导的shRNA 沉默大鼠、原代大鼠大脑皮质神经元和PC12细胞中Fas/FasL的表达，结合特异性抑制剂，然后进行镉染毒，制备镉中毒模型，采用荧光定量PCR、western-blot、流式细胞术、免疫组化和激光共聚焦显微技术，检测Fas/FasL和线粒体信号通路中关键蛋白及细胞凋亡率的量化指标，从体内、外两个角度明确Fas/FasL途径介导的线粒体通路在镉致神经细胞凋亡中的调控机制。研究结果将对于进一步深入了解镉的神经毒性机制、信号通路的调控网络及神经修复药物新标靶点研究具有指导价值。

镉是有害金属，可严重损害包括脑部在内的多种器官。镉致神经细胞凋亡是神经细胞形态异常和智力发育低下的主要原因，但镉致神经细胞凋亡的信号通路尚不明确。前期研究表明，镉可经线粒体通路引起神经细胞凋亡，但Fas/FasL途径在镉激活神经细胞凋亡线粒体通路中的作用机制尚未阐明。本研究应用RNA干扰技术沉默大鼠、原代大鼠大脑皮质神经元和PC12细胞中Fas的表达，结合特异性抑制剂，然后进行镉染毒，制备镉中毒模型，采用荧光定量PCR、western-blot、流式细胞术、免疫组化和激光共聚焦显微技术，检测Fas/FasL途径和线粒体信号通路中关键蛋白及细胞凋亡的量化指标，从体外和体内阐明Fas/FasL途径介导的线粒体通路在镉致神经细胞凋亡中的调控机制。体外试验结果显示，Cd可激活神经细胞Fas/FasL途径和线粒体通路；Fas shRNA、Fas siRNA显著或极显著抑制Cd致神经细胞Fas mRNA 转录水平和Fas/FasL途径相关蛋白表达量升高；Fas shRNA、Fas siRNA、Anti-FasL或Z-IETD-FMK显著或极显著抑制Cd致神经细胞凋亡Fas/FasL途径和线粒体通路的激活。体内试验结果显示，Fas shRNA能够缓解Cd致大鼠大脑皮质病理组织损伤和超微结构损伤，抑制Cd诱导的大鼠大脑皮质细胞凋亡，极显著抑制Cd致大鼠大脑皮质Fas mRNA转录水平升高，显著或极显著抑制Cd致大鼠大脑皮质Fas/FasL途径和线粒体通路的激活。说明Fas/FasL途径在Cd致神经细胞凋亡过程中具有重要作用，Fas/FasL途径通过调控线粒体通路参与Cd致神经细胞凋亡。研究结果对于进一步深入了解镉的神经毒性机制、信号通路的调控网络及神经修复药物新标靶点研究具有指导价值。