F10下调通过细胞色素C促进KLE细胞凋亡

[摘要] 目的:建立F10基因表达稳定下调的KLE细胞系,探讨F10基因沉默对KLE细胞凋亡的影响及机制。方法:将两条合成的寡核苷酸退火后形成的双链DNA连接到逆转录病毒载体pRetroQ上构建表达质粒pRetroQ-i F10，脂质体转染KLE细胞，嘌呤霉素筛选，获得单细胞克隆；荧光定量RT-PCR鉴定阳性克隆。流式细胞仪检测细胞凋亡，荧光定量RT-PCR和western blot检测细胞色素 C和Bcl-2 表达.结果: 两个抗性克隆呈现F10 mRNA表达显著下调。F10基因沉默的KLE细胞克隆凋亡率较对照组增加近9倍，细胞色素C mRNA表达上调、胞浆细胞色素C表达增加，BCL-2mRNA和蛋白表达下调。结果：成功构建F10基因表达稳定下调的KLE细胞系，F10基因表达下调导致细胞凋亡；细胞色素C的释放可能是F10沉默诱导KLE细胞凋亡的途径之一。关键词：F10基因 KLE 细胞 RNA干扰

KLE cell apoptosis is Promoted By F10 RNAI Through Cytochrome C

Objective To establish a cell line in which F10 gene is stablely knocking-down,and to explore the effect of F10 silence on apoptosis of KLE.Methods The recombinant plasmid pRetroQ-i F10 producing short hairpin RNA of F10 gene was constructed by ligating the double DNA annealed with two synthesizing oligonucleotides to pRetroQ.pRetroQ-i F10 plasmid was transfected into KLE cells,and the cells were screened by puromycine.When single clone was established,the expression level of F10 gene in stable cell clones was detected by quantitative RT-PCR and positive clones were identified.Apoptosis was detected by FCM(flow cytometry).The expression of cytochrome and Bcl-2 mRNA and protein were further examined by quantitative RT-PCR and western blot.Results Two clones showed significant down-regulation of F10 gene.The apoptosis rate of positive clone cells increased by about 15% in contrast to control cells.Quantitative RT-PCR and western blotting showed that the level of cytochrome C in cytoplasm of positive clone was significantly up-regulated,but the expression of bcl-2 was significantly down-regulated.Conclusion The KLE cell lines with stablely down-regulated F10 is successfully established.The down regulation of F10 gene induces apoptosis of KLE cells.Release of cytochrome C from mitochondria to cytoplasm is involved in the apoptosis.