慢病毒介导的PLC-γ1小干扰RNA的构建及其对人大肠癌细胞株凋亡的影响
[Construction of lentivirus producing PLC-gamma1 siRNA and its effect on apoptosis of human colorectal carcinomas cell lines].
作者信息
Tan Li, Luo Shen-qiu, Lin Jun
机构信息
Department of Cell Biology, Southern Medical University, Guangzhou 510515, China.
出版信息
Nan Fang Yi Ke Da Xue Xue Bao. 2007 Mar;27(3):255-8.
OBJECTIVE
To construct recombinant lentivirus that stably suppresses phospholipase C (PLC) gamma1 expression in human colorectal carcinoma LoVo cells to obtain LoVo cell lines deficient in PLC gamma1 for investigation of the role of PLC gamma1 gene.
METHODS
Recombinant lentivirus producing PLC gamma1 siRNA were constructed to infect LoVo cells, and the stably transduced cells were selected with blasticidin. The protein and mRNA expression of PLC gamma1 was examined by Western blotting and RT-PCR, and the effect of the lentivirus on cell apoptosis was analyzed by flow cytometry.
RESULTS AND CONCLUSION
PLC gamma1 siRNA significantly suppressed PLC gamma1 expression in LoVo cells, suggesting high efficiency of gene silencing induced by the siRNA produced by the recombinant lentivirus. Concomitantly, cell apoptosis induced by 5-FU was significantly increased.
目的
构建能稳定抑制人结直肠癌LoVo细胞中磷脂酶C（PLC）γ1表达的重组慢病毒，以获得PLCγ1基因缺陷的LoVo细胞系，用于研究PLCγ1基因的作用。
方法
构建产生PLCγ1小干扰RNA（siRNA）的重组慢病毒并感染LoVo细胞，用杀稻瘟菌素筛选稳定转导的细胞。通过蛋白质免疫印迹法和逆转录-聚合酶链反应检测PLCγ1的蛋白质和mRNA表达，采用流式细胞术分析慢病毒对细胞凋亡的影响。
结果与结论
PLCγ1 siRNA显著抑制了LoVo细胞中PLCγ1的表达，提示重组慢病毒产生的siRNA诱导基因沉默的效率较高。同时，5-氟尿嘧啶诱导的细胞凋亡显著增加。
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