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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 the eukaryotic expression vector of short hairpin RNA (shRNA) of HSP27 gene and observe its expression in the gemcitabine human pancreatic cancer cell line SW1990 / Gem to lay a foundation for further exploration of gene therapy of tumors. Methods: Two DNA sequences that can transcribe shRNA were designed and synthesized according to the references and shRNA design principles. After annealed, the eukaryotic expression vector pRNAT-U6 containing green fluorescent protein (GFP) gene and U6 promoter .3, a recombinant vector pRNAT-shHSP27 was constructed. The recombinant vector was transfected into SW1990 / Gem cells and transfected into SW1990 / Gem cells by inverted fluorescence microscope. The effects of transfection on the growth of human gemcitabine-resistant human pancreatic cancer cell line SW1990 / Gem were investigated by RT-PCR and Western Blot. Results: shRNA expression vector targeting HSP27 gene was successfully constructed. Inverted fluorescence microscopy showed GFP expression in SW1990 / Gem cells 48h after transfection. The results of RT-PCR and Western Blot showed that the mRNA and protein expression of HSP27 were significantly inhibited (P <0.05) compared with the control group after transfection. CONCLUSION: The specific shRNA eukaryotic expression vector targeting HSP27 gene is successfully constructed, and the expression of HSP27 can be inhibited after transfection of cells, which lays the foundation for further study on the correlation between HSP27 and biological behavior of pancreatic cancer and chemoresistance.