HSP27特异性shRNA表达载体的构建及在耐吉西他滨人胰腺癌细胞株SW1990/Gem中的表达

Construction of shRNA Expression Vector Targeting the HSP27 Gene and their Expression in Human Gemcitabine-Resistant Pancreatic Cell Line

目的:构建HSP27基因的短发夹RNA(short hairpin RNA,shRNA)真核表达载体及观察其在耐吉西他滨人胰腺癌细胞株SW1990/Gem中的表达,为进一步探索肿瘤的基因治疗打下前期基础。方法:参考文献及shRNA设计原则,设计并合成2条能转录shRNA的DNA序列,退火连接后,插入含绿色荧光蛋白(green fluorescence protein,GFP)基因和U6启动子的真核表达载体pRNAT-U6.3中,构建重组载体pRNAT-shHSP27。重组载体经鉴定后转染SW1990/Gem,倒置荧光显微镜观察转染情况,RT-PCR、Western Blot从mRNA及蛋白水平探讨转染对耐吉西他滨人胰腺癌细胞株SW1990/Gem的影响。结果:成功构建了针对HSP27基因的shRNA表达载体。倒置荧光显微镜下显示转染48h后SW1990/Gem细胞内存在GFP表达。RT-PCR、WesternBlot结果提示转染后HSP27的mRNA及蛋白表达水平较对照组有明显抑制(P<0.05)。结论:成功构建针对HSP27基因的特异性shRNA真核表达载体,转染细胞后可抑制HSP27表达,为进一步研究HSP27与胰腺癌生物学行为及化疗耐药等相关性奠定了基础。

Objective： To construct eukaryotic expression vectors of small hairpin RNA（shRNA） for HSP27 gene and investigate their expression in human gemcitabine-resistant pancreatic cells（SW1990/Gem）.Methods： One pair of HSP27 shRNA sequence was selected and ligated to the pRNAT-U6.3 vector contained GFP gene and U6 promoter.pRNAT-shHSP27 or empty vector was introduced into SW1990/Gem cells by liposome-mediated transfection respectively.The transfected SW1990/Gem cells were then confirmed by fluoroscopy.The expression of HSP27 mRNA and protein was detected by reverse transcription polymerase chain reaction（RT-PCR） and Western blot,respectively.Results： Restriction enzyme digestion and sequence analysis showed that recombinant pRNAT-shHSP27 shRNA vector was successfully constructed.The expression of GFP was observed in transfected SW1990/Gem Cells by fluorescent microscopy.The expression levels of HSP27 mRNA and protein in SW1990/Gem cells transfected with pRNAT-shHSP27 shRNA vector were significantly lower than those in SW1990/Gem cells transfected with empty or negative vector.Conclusion： Recombinant pRNAT-shHSP27 shRNA vector was successfully constructed.Its transfection can silence the expression of HSP27 gene in SW1990/Gem cells.The pRNAT-shHSP27 shRNA vector lay a foundation for further study of the role of the HSP27 gene in the pathogenesis of pancreatic cancer.