【摘要】：已有研究表明鎘(20μM)可誘導(dǎo)人肝腎細(xì)胞凋亡,但是,鎘能否誘導(dǎo)人肝腎細(xì)胞自噬目前還不是很清楚。本研究旨在分析鎘誘導(dǎo)HEK293和WRL68細(xì)胞的自噬,及自噬與凋亡的關(guān)系。本研究分為三部分：1.構(gòu)建pcDNA3.1-GFP-LC3B重組質(zhì)粒,為建立快速檢測細(xì)胞自噬的方法打下基礎(chǔ)；2.探究鎘(0～10μM)能否引起HEK293和WRL68胞自噬；3.探究HEK293細(xì)胞自噬與凋亡的關(guān)系。 本實驗運用細(xì)胞培養(yǎng)、基因重組及基因轉(zhuǎn)染、Western blot、流式細(xì)胞術(shù)、顯微及超微結(jié)構(gòu)觀察等細(xì)胞分子生物學(xué)技術(shù),構(gòu)建了pcDNA3.1-GFP-LC3B真核表達(dá)載體。檢測了鎘(0～10μM)誘導(dǎo)人肝腎細(xì)胞的自噬標(biāo)志基因LC3B-Ⅱ/Ⅰ的表達(dá)；初步探究了自噬過程中ERK1/2和AKT的作用。用流式細(xì)胞術(shù)檢測了鎘(0～-10μM)誘導(dǎo)HEK293細(xì)胞的凋亡。探究了自噬抑制劑3-MA對自噬與凋亡的影響,并分析了自噬與凋亡的關(guān)系；觀察了極低濃度鎘(0～2.0μM)長時間40d處理HEK293后細(xì)胞形態(tài)變化情況,以分析自噬與和轉(zhuǎn)化的關(guān)系。實驗結(jié)果如下： 1.通過PCR擴增出LC3B基因,與pcDNA3.1-GFP載體相連,成功構(gòu)建了pcDNA3.1-GFP-LC3B真核表達(dá)載體,且能在HEK293和WRL68細(xì)胞中成功表達(dá)。 2.低濃度鎘(0～10μM)處理HEK293和WRL68細(xì)胞,均可引起細(xì)胞自噬,表現(xiàn)為LC3B-Ⅱ表達(dá)量增加、熒光顯微鏡下轉(zhuǎn)染細(xì)胞出現(xiàn)綠色熒光點狀聚集、透射電鏡下觀察到自噬泡。 3.低濃度鎘(0～10μM)處理HEK293細(xì)胞后,ERK1/2和AKT被激活。流式細(xì)胞儀也檢測出自噬鎘濃度下同時發(fā)生細(xì)胞凋亡；加入自噬抑制劑3-MA后,Cleaved Caspase-3的表達(dá)量增加。 本研究表明了,低濃度鎘(0-10gM)能誘導(dǎo)HEK293細(xì)胞自噬,且自噬與凋亡相伴發(fā)生；加入自噬抑制劑后,自噬減弱,凋亡增強；ERK1/2和AKT可能介導(dǎo)細(xì)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，

本文編號：2479343