RNA干扰抑制FAK基因表达对人胰腺癌细胞凋亡及吉西他滨敏感性的影响

目的 探讨RNA干扰技术抑制黏着斑激酶（FAK）基因表达增强人胰腺癌PANC-1细胞对化疗药物敏感性及凋亡能力的研究.方法 针对FAK mRNA序列设计合成短发夹状干扰RNA（siRNA）的DNA模板,构建pRNAT-FAK重组表达载体,转染人胰腺癌PANC-1细胞;通过RT-PCR分析其对PANC-1细胞内源性FAK表达的影响;激光共聚焦显微镜检测PANC-1细胞凋亡的形态学改变;用四甲基偶氮唑蓝法观察PANC-1细胞对化疗药物吉西他滨敏感性的改变;采用分光光度计检测 Caspase 活性.结果 成功构建 pRNAT-FAK重组质粒,并成功转染PANC-1细胞;RT-PCR证实重组质粒在mRNA显著抑制FAK基因表达（P＜0.01）;吉西他滨组及吉西他滨加pRNAT-FAK组细胞则检测出明显的细胞凋亡;四甲基偶氮唑蓝结果证明在和吉西他滨联合作用下,pRNAT-FAK组PANC-1细胞的生长抑制率明显增高（P＜0.01）;Caspase 3活性明显高于空白对照绀和阴性对照组.结论 pRNAT-FAK可抑制FAK在人胰腺癌PANC-1细胞中的表达,并增强PANC-1细胞对吉西他滨敏感性.

Enhances gemcitabine chemosensitivity and apoptosis of human pancreatic carcinoma cells after decreasing FAK level by RNA interference

Objective To explore the relationship between the expression of FAK and Gemcitabine Chemosensitivity and Apoptosis of human pancreatic carcinoma PANC-1 cells by decreasing FAK gene expression by RNA interference. Methods One pair of DNA template coding siRNA was synthesized against PANC-1 to reconstruct pRNA-FAK,which was transfected into PANC-1 cells. The FAK expression in PANC-1 ceils were transfected with pRNAT-FAK, and it was detected by RT-PCR. MTT was used to observe the growth inhibiting ratio induced by gemcitabine in PANC-1 cells. Cell apoptosis was analyzed by fuorescence microscopy. Caspase-3 activity was measured by spectrofluorometer. Results RT-PCR analyses demonstrated that pRNAT-FAK could significantly inhibit the expression of FAK in PANC-1 cells （P 〈 0. 01 ） ;after PANC-1 cells were exposed to Gemeitabine,the growth inhibiting ratio, Caspase-3 activity and cell apoptosis of pRNAT-FAK transfected cells was significantly increased compared to that of control group and negative group（ P 〈 0.01 ）. Conclusion pRNAT- FAK could significantly inhibit the expression of FAK,increase Chemosensitivity and apoptosis of PANC-1 cells.