镉诱导HEK293细胞凋亡及其线粒体凋亡途径

CADMIUM INDUCED APOPTOSIS OF HEK293 CELLS AND ITS MITOCHONDRIAL APOPTOSIS PATHWAY

本课题研究了氯化镉(CdCl_2)诱导HEK293细胞(人胚胎肾细胞系)的凋亡,初步探讨了凋亡过程中Caspase-3、Bcl-2的变化和凋亡诱导因子(AIF)的转移以及它们的意义。MTT法检测CdCl_2对HEK293细胞增殖的抑制作用;通过倒置显微镜、电镜、琼脂糖凝胶电泳、流式细胞术、激光共聚焦观察细胞凋亡;应用Western blot法和荧光免疫法测定Caspase-3酶原、Bcl-2蛋白的变化以及检测AIF蛋白在细胞中的定位。结果显示:CdCl_2对HEK293细胞具有显著的生长抑制作用,并呈明显的剂量和时间依赖性。在琼脂糖凝胶电泳中,显示有凋亡细胞特有的DNA梯状条带,其中30μmol/L作用6-9h梯状条带最为清晰,时间过长或浓度过高则梯状条带逐渐模糊,表明镉浓度过高或处理时间过长,细胞有坏死。流式细胞仪检测也印证了这一结果。形态学观察可见明显的细胞凋亡特征。同时线粒体膜电位明显下降,发现Caspase-3酶原蛋白、Bcl-2蛋白含量减少,并具有时间依赖性;另外检测到线粒体AIF向细胞核转移。而Bcl-2转染后有一定的抑制凋亡作用。实验结果提示,CdCl_2能够诱导HEK293细胞凋亡,线粒体损伤导致AIF转移与细胞色素c释放,从而引发的非Caspases与Caspases凋亡途径可能在镉引发的细胞凋亡过程中起重要作用,而Caspase-3, Bcl-2起着重要的调控作用。

HEK293 cell was chose to study the kidney damage of cadmium and to explore the significance of caspase 3,Bcl-2 and AIF （apoptosis inducing factor） in the apoptosis of cells induced by cadmium. Inhibition of the cell proliferation was measured by MTT assay. The structure of apoptotic cells was observed by light microscopy and electron microscopy; moreover, apoptotic cells were detected by DNA electrophoresis, flow cytometry and confocal laser microscopy. Furthermore,the expressions of Pro-caspase-3,Bcl-2 and the location of AIF in cells （mitochondria,cytoplasm or nuclei） were tested by western blot and immunofluorescence assay. CdCl2 exhibited antiproliferative activity in dosage and time-dependent manner. DNA ladders of HEK293 cells were showed on agarose gel electrophoresis and the fragments of DNA were integral of 180-200 bp. 6- 9 hours after 30μmol/L CdCl2 treatment,DNA ladders were distinct. However,mistiness DNA ladder or smear was found when HEK293 cells were treated with CdCl2 on higher concentration or treated longer. It suggests that necrosis may happen, and flow cytometry results confirmed it. Morphological examination showed cell shrinkage,chromosomal condensation, karyotheca margination, nucleus cracking, vacuoles formed in cytoplasm and the presence of apoptotic bodies. At the same time,mitochondrial membrane potential （MMP） decreased, and the expression of Pro-caspase-3, Bcl-2 were decreased in time-dependent manner. Furthermore, AIF was released from mitochondria,and then traveled to nuclei. It suggests that CdCl2 may induce the apoptosis of HEK293 cells involving mitochondrial disruption including AIF migration and Cyt c release through both caspaseindependent and -dependent pathways, and Bcl-2 and Caspase-3 are important factors which participate in the processes.