镉诱导人肝肾细胞自噬及相关蛋白作用初步研究
发布时间：2019-05-17 19:16
【摘要】：已有研究表明镉(20μM)可诱导人肝肾细胞凋亡,但是,镉能否诱导人肝肾细胞自噬目前还不是很清楚。本研究旨在分析镉诱导HEK293和WRL68细胞的自噬,及自噬与凋亡的关系。本研究分为三部分：1.构建pcDNA3.1-GFP-LC3B重组质粒,为建立快速检测细胞自噬的方法打下基础；2.探究镉(0～10μM)能否引起HEK293和WRL68胞自噬；3.探究HEK293细胞自噬与凋亡的关系。 本实验运用细胞培养、基因重组及基因转染、Western blot、流式细胞术、显微及超微结构观察等细胞分子生物学技术,构建了pcDNA3.1-GFP-LC3B真核表达载体。检测了镉(0～10μM)诱导人肝肾细胞的自噬标志基因LC3B-Ⅱ/Ⅰ的表达；初步探究了自噬过程中ERK1/2和AKT的作用。用流式细胞术检测了镉(0～-10μM)诱导HEK293细胞的凋亡。探究了自噬抑制剂3-MA对自噬与凋亡的影响,并分析了自噬与凋亡的关系；观察了极低浓度镉(0～2.0μM)长时间40d处理HEK293后细胞形态变化情况,以分析自噬与和转化的关系。实验结果如下： 1.通过PCR扩增出LC3B基因,与pcDNA3.1-GFP载体相连,成功构建了pcDNA3.1-GFP-LC3B真核表达载体,且能在HEK293和WRL68细胞中成功表达。 2.低浓度镉(0～10μM)处理HEK293和WRL68细胞,均可引起细胞自噬,表现为LC3B-Ⅱ表达量增加、荧光显微镜下转染细胞出现绿色荧光点状聚集、透射电镜下观察到自噬泡。 3.低浓度镉(0～10μM)处理HEK293细胞后,ERK1/2和AKT被激活。流式细胞仪也检测出自噬镉浓度下同时发生细胞凋亡；加入自噬抑制剂3-MA后,Cleaved Caspase-3的表达量增加。 本研究表明了,低浓度镉(0-10gM)能诱导HEK293细胞自噬,且自噬与凋亡相伴发生；加入自噬抑制剂后,自噬减弱,凋亡增强；ERK1/2和AKT可能介导细胞自噬。
[Abstract]:It has been shown that cadmium (20 渭 M) can induce apoptosis of human hepatorenal cells, but it is not clear whether cadmium can induce autophagy of human hepatorenal cells. The purpose of this study was to analyze the autophagy of HEK293 and WRL68 cells induced by cadmium and the relationship between autophagy and apoptosis. This study is divided into three parts: 1. The recombinant plasmid pcDNA3.1-GFP-LC3B was constructed, which laid the foundation for the establishment of a rapid method for the detection of autophagy. To investigate whether cadmium (0 ~ 10 渭 M) can cause HEK293 and WRL68 cell autophagy. To explore the relationship between autophagy and apoptosis in HEK293 cells. In this study, pcDNA3.1-GFP-LC3B eukaryotic expression vector was constructed by cell culture, gene recombination and, Western blot, flow cytometry, microscopic and ultrastructure observation. The expression of autophagy marker gene LC3B- 鈪，
本文编号：2479343
[Abstract]:It has been shown that cadmium (20 渭 M) can induce apoptosis of human hepatorenal cells, but it is not clear whether cadmium can induce autophagy of human hepatorenal cells. The purpose of this study was to analyze the autophagy of HEK293 and WRL68 cells induced by cadmium and the relationship between autophagy and apoptosis. This study is divided into three parts: 1. The recombinant plasmid pcDNA3.1-GFP-LC3B was constructed, which laid the foundation for the establishment of a rapid method for the detection of autophagy. To investigate whether cadmium (0 ~ 10 渭 M) can cause HEK293 and WRL68 cell autophagy. To explore the relationship between autophagy and apoptosis in HEK293 cells. In this study, pcDNA3.1-GFP-LC3B eukaryotic expression vector was constructed by cell culture, gene recombination and, Western blot, flow cytometry, microscopic and ultrastructure observation. The expression of autophagy marker gene LC3B- 鈪，
本文编号：2479343
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