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pEGFP-C1-STAT3真核表达载体的构建及其在SGC7901细胞株中对Snail表达的影响

Construction of pEGFP-C1-STAT3 Recombinant Eukaryotic Expression Vector and Its Effect on Expression of Snail in SGC7901 Cell Line

  • 摘要: 目的 研究信号转导与转录激活因子3(STAT3)基因转染人胃癌细胞株SGC7901后,对锌指转录因子Snail1(Snail)及切除修复交叉互补基因1(excision repair cross complementation group 1,ERCC1)表达的影响。 方法 利用基因重组技术构建重组pEGFP-C1-STAT3真核表达载体;脂质体介导转染技术转染胃癌细胞株SGC7901,荧光显微镜、Western blot 检测转染后SGC7901细胞株中STAT3、pSTAT3、Snail及ERCC1的表达,流式细胞仪检测转染后顺铂处理下细胞凋亡率。 结果 重组质粒经Hind Ⅲ、SacⅡ双酶切测序与STAT3基因序列一致;利用脂质体介导将pEGFP-C1-STAT3重组质粒转染SGC7901细胞株,获得了STAT3基因表达,Western blot 检测pSTAT3、Snail、ERCC1表达增高,具有统计学意义,流式细胞术分析显示转染重组质粒pEGFP-C1-STAT3后,细胞在顺铂作用下的早期凋亡率减低。 结论 成功构建STAT3重组质粒并转入中分化胃腺癌细胞株SGC7901后,细胞Snail及ERCC1显著增高,细胞对顺铂敏感度下降。

     

    Abstract: Objective To investigate the effect on the expression of zinc finger transcript factor Snail1(Snail) and excision repair cross complementation group 1 (ERCC1) after transfected with the signal transducer and activator of transcription factor(STAT3) in SGC7901 cell line. Methods We used recombinant DNA technology to construct the pEGFP-C1-STAT3 recombinant eukaryotic expression vector and transfected by liposome 2000 in SGC7901.The expression of EGFP was observed in transfection group by fluorescent microscopy.We detected the expression of STAT3,pSTAT3,Snail, ERCC1 and the apoptosis rate using Western blot and flow cytometry after cisplatin (DDP) treated. Results The recombinant plasmid was confirmed by double restriction enzyme digestion and the sequence was consensus with STAT3 gene sequencing.SGC7901 cells were transfected with recombinant plasmid pEGFP-C1-STAT3 and the product of recombinant plasmid was obtained.Western blot detection of the pSTAT3,Snail and ERCC1 expression showed significantly increased protein levels.Flow cytometry analysis revealed obviously decreasing of cell early apoptosis after the DDP treatment in pEGFP-C1-STAT3 transfection group. Conclusion We constructed the pEGFP-C1-STAT3 recombinant eukaryotic expression vector successfully and enhanced the expression of Snail and ERCC1 after transfection in SGC7901 cell line,which decreased its sensitivity to cisplatin.

     

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