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目的构建pDsRED2-HMGB1重组质粒,探讨转染高迁移率族蛋白1(HMGB1)基因对奥沙利铂诱导的SGC-7901细胞凋亡的影响。方法用逆转录聚合酶链反应(RT-PCR)扩增HMGB1 mRNA全编码序列,将PCR产物插入到pD-sRED2-N1载体的Xho I和EcoR I位点,构建pDsRED2-HMGB1重组质粒;通过脂质体法转染SGC-7901胃癌细胞,荧光显微镜检测HMGB1红色荧光融合蛋白表达。Westernblot鉴定HMGB1蛋白表达。应用流式细胞仪(Annexin-V/PI标记)分析HMGB1过表达对奥沙利铂诱导胃癌细胞凋亡的影响。结果成功构建真核表达质粒pDsRED2-HMGB1,转染SGC-7901细胞后,荧光显微镜下可见细胞内有HMGB1红色荧光融合蛋白的表达;流式细胞术检测发现转染pD-sRED2-HMGB1后SGC-7901细胞凋亡水平较转染pDsRED2-N1组下降22.4%。结论 HMGB1过表达可抑制奥沙利铂诱导的SGC-7901胃癌细胞凋亡。
Objective To construct recombinant plasmid pDsRED2-HMGB1 and investigate the effect of transfection of HMGB1 gene on the apoptosis of SGC-7901 cells induced by oxaliplatin. Methods The complete coding sequence of HMGB1 mRNA was amplified by reverse transcription-polymerase chain reaction (RT-PCR). The PCR product was inserted into Xho I and EcoR I sites of pD-sRED2-N1 vector to construct pDsRED2-HMGB1 recombinant plasmid. SGC-7901 gastric cancer cells were transfected by plastid method, and the expression of HMGB1 red fluorescence fusion protein was detected by fluorescence microscopy. Western blot was used to identify HMGB1 protein expression. The effect of HMGB1 overexpression on the apoptosis of gastric cancer cells induced by oxaliplatin was analyzed by flow cytometry (Annexin-V / PI). Results The eukaryotic expression plasmid pDsRED2-HMGB1 was successfully constructed and transfected into SGC-7901 cells. The expression of HMGB1 red fluorescent fusion protein was observed under fluorescence microscope. The results of flow cytometry showed that SGC- Compared with pDsRED2-N1 group, the apoptosis of 7901 cells decreased by 22.4%. Conclusion Overexpression of HMGB1 can inhibit the apoptosis of SGC-7901 gastric cancer cells induced by oxaliplatin.