内质网钙稳态在铅镉联合诱导大鼠肾小管上皮细胞凋亡中的作用

项目名称： 内质网钙稳态在铅镉联合诱导大鼠肾小管上皮细胞凋亡中的作用

项目编号： No.31472251

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 农业科学

项目作者： 王林

作者单位： 山东农业大学

项目金额： 86万元

中文摘要： 内质网是重要的胞内钙库，且内质网钙稳态在调控细胞凋亡过程中发挥重要作用。前期研究表明铅镉联合可诱导大鼠肾小管上皮细胞凋亡并伴随胞浆钙超载，但内质网钙稳态在此过程中的调控机制尚不明确。本项目在应用RNA干扰与基因过表达技术对原代大鼠肾小管上皮细胞进行特定基因沉默与过表达处理的基础上，结合特异性信号转导抑制剂处理，分别对模型细胞进行铅、镉单独与联合暴露。染毒后，采用免疫印迹技术、荧光定量PCR技术、流式细胞术及激光共聚焦显微技术检测Calpain/caspase-12活化通路、IP3R信号通路、钙缓冲蛋白及Bcl-2家族蛋白参与调控内质网钙稳态过程中关键信号分子的表达、细胞凋亡及胞内不同区域钙分布。本项目将阐明内质网钙稳态在铅镉联合诱导大鼠肾小管上皮细胞凋亡中的作用，进而从细胞和分子水平探讨铅镉联合所致肾毒性损伤的信号转导机制。

中文关键词： 铅镉联合；肾小管上皮细胞；细胞凋亡；内质网钙稳态；信号转导

英文摘要： Endoplasmic reticulum (ER) is an important intracellular calcium store and ER calcium homeostasis plays a key part in the regulation of apoptosis. Cytoplasm calcium overload occurred in the apoptosis of primary cultures of rat proximal tubular (rPT) cells induced by co-exposure to lead and cadmium in previous study. However, the role of ER calcium homeostasis in this process has not yet been defined explicitly. In this project, the primary cultures of rPT cells will be treated with RNA inference method and gene overexpression technology to make some genes silenced and overexpressed. In addition, some rPT cells will be incubated with specific inhibitors. Then all of the model cells will be exposed to different doses of lead and/or cadmium, respectively. After the exposure, immunoblot assays and real-time quantitative PCR analyses will be performed on rPT cells to assess key signaling molecules expression levels in regulation of ER calcium homeostasis mediated by Calpain/caspase-12 activated pathways, IP3R signaling pathways, calcium buffer proteins and Bcl-2 family proteins, respectively. Likewise, apoptosis and calcium distribution in different domains in rPT cells will be identified by flow cytometry and confocal fluorescent microscopy, respectively. Therefore, the role of ER calcium homeostasis in rPT cells exposed to lead and cadmium will be clarified. Furthermore, this project will contribute significantly to signal transduction mechanism implicated in combined toxic effects of lead and cadmium on rat kidney from the cellular and molecular level.

英文关键词： Combination of lead and cadmium;Renal tubular epithelial cells;Apoptosis;Endoplasmic reticulum calcium homeostasis;Signal transduction