| 张松陈敏黄淑玲许春红王军徐桂芳邹晓平△.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[J].,2012,12(10):1812-1816 |
| 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 andtheir Expression in Human Gemcitabine-Resistant Pancreatic Cell Line |
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| DOI： |
| 中文关键词: HSP27 shRNA SW1990/Gem 胰腺癌 载体构建 |
| 英文关键词: HSP27 RNAi shRNA SW1990/Gem cell line Pancreatic cancer Vector construction |
| 基金项目:南京市青年科技人才启动项目（QYK11166），中央高校基本科研业务费（1117021408），
南京市卫生青年人才工程第三层次（13） |
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| 中文摘要: |
| 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、Western
Blot 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。 |
| 英文摘要: |
| 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. |
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