镉诱导细胞凋亡与Bcl-2抗凋亡机理研究

发布时间：2018-03-22 05:02

本文选题：HEK293细胞　切入点：凋亡　出处：《南京师范大学》2006年硕士论文　论文类型：学位论文

【摘要】：镉可以诱导机体多种组织细胞发生凋亡。研究镉诱导细胞的凋亡对于镉的分子毒理学机制的阐明和镉所致的损伤与致癌机理的研究具有重要意义。本文对镉诱导细胞凋亡发生机制作了如下研究工作： 1 镉诱导HEK293细胞凋亡与DNA损伤在37℃、5％CO_2条件下，HEK 293细胞分别在不同浓度氯化镉和N-乙酰半胱氨酸的10％小牛血清DMEM培养液中孵育6-8小时后，用MTT染色检测细胞生长抑制，台盼蓝染色检测细胞死亡率，丫啶橙／溴乙啶双荧光染色检测细胞凋亡率，以琼脂糖凝胶电泳DNA Ladder检测细胞DNA损伤。结果显示：不同浓度的镉离子能不同程度地诱导HEK 293细胞的凋亡，并呈现剂量效应正相关，其细胞DNA也不同程度的出现DNA ladder现象。提示：镉能引起HEK 293细胞的凋亡并引起DNA损伤。 2 镉诱导HEK293细胞凋亡的线粒体途径通过激光共聚焦技术，以罗丹明123为探针实时监测HEK 293细胞线粒体膜电位水平及其变化规律，通过流式细胞术分别以DCFH-DA和罗丹明123为探针检测镉诱导以及N-乙酰半胱氨酸共处理的镉诱导细胞的胞内ROS水平和线粒体膜电位。结果显示：镉离子可导致HEK 293细胞线粒体膜电位失去节律变化特征并大幅下降从而导致细胞凋亡。 3 镉诱导HEK293细胞凋亡与Bcl-2抗凋亡作用建立以pcDNA3／Bcl-2转染的HEK 293 Bcl-2过表达细胞作为研究对象，，通过激光共聚焦技术，以罗丹明123为探针实时监测HEK 293细胞线粒体膜电位水平及其变化规律。以Western蛋白印迹法检测胞内Caspase-3和Bcl-2的表达水平。以琼脂糖凝胶电泳DNA ladder检测DNA损伤。结果显示：Bcl-2
[Abstract]:Cadmium can induce apoptosis in many tissues and cells. The study of cadmium induced apoptosis is important for elucidating the molecular toxicological mechanism of cadmium and studying the mechanism of damage and carcinogenesis induced by cadmium. The mechanism of apoptosis was studied as follows:. 1 cadmium induced apoptosis and DNA damage in HEK293 cells. After incubating in 10% calf serum DMEM medium with different concentrations of cadmium chloride and N-acetylcysteine for 6-8 hours, the cell growth inhibition was detected by MTT staining, and the cell death rate was detected by trypan blue staining. The apoptosis rate was detected by acridine orange / ethidium bromide double fluorescence staining, and the DNA damage was detected by agarose gel electrophoresis (DNA Ladder). The results showed that different concentrations of cadmium ions could induce apoptosis of HEK 293 cells to different extent. There was a positive dose-effect correlation, and the DNA ladder phenomenon appeared in the DNA of the cells to some extent. It was suggested that cadmium could induce HEK. 293 cells apoptosis and DNA damage. Mitochondrial Pathway of cadmium induced apoptosis in HEK293 cells. The mitochondrial membrane potential of HEK 293 cells was monitored by laser confocal technique with Rhodamine 123 as a probe. The intracellular ROS level and mitochondrial membrane potential of cadmium induced cells and N-acetylcysteine co-treated cells were detected by flow cytometry using DCFH-DA and Rhodamine 123 as probes, respectively. The results showed that cadmium ion could induce HEK 293 fine. Mitochondrial membrane potential (MMP) lost its rhythmic characteristics and decreased significantly, leading to apoptosis. 3 cadmium induced apoptosis of HEK293 cells and Anti-apoptotic effect of Bcl-2. HEK 293 Bcl-2 overexpression cells transfected with pcDNA3/Bcl-2 were established as the research object, and laser confocal technique was used. The mitochondrial membrane potential of HEK 293 cells was monitored by Rhodamine 123 as a probe, the expression of Caspase-3 and Bcl-2 was detected by Western Western blotting, and DNA damage was detected by agarose gel electrophoresis (DNA ladder).
【学位授予单位】：南京师范大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R363

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