生存素基因突变体诱导胃癌细胞凋亡的实验研究

Experimental study on apoptosis induced by pcDNA3-survivin-mutant in gastric cancer cell lines

目的 生存素 (survivin)基因在胃癌组织中高水平表达 ,而在正常胃黏膜中无表达。生存素表达的水平与胃癌的预后密切相关。通过基因重组构建生存素基因突变体 (pcDNA3 survivin mutant,Cys84Ala)质粒 ,并以脂质体转染的方法将质粒DNA转入胃癌细胞株 ,观察其对胃癌细胞的生物学特性及多药耐药性的影响。方法 应用RT PCR、Westernblot、免疫组化等方法检测胃癌细胞中生存素基因mRNA和蛋白的表达 ,流式细胞仪和吖啶橙染色检测胃癌细胞凋亡。结果 通过RT PCR检测 ,在所有的胃癌细胞株中均有不同程度生存素基因表达 ,生存素基因突变体Cys84Ala通过抑制野生型生存素基因的功能诱导胃癌细胞凋亡 ,增加caspase 3活性和促进细胞色素C的释放 ,增加胃癌细胞对化疗药物的敏感性。结论 突变体Cys84Ala能诱导胃癌细胞凋亡和增加胃癌细胞对化疗药物的敏感性。因此 ,生存素基因突变体Cys84Ala有可能成为胃癌治疗的一个候选基因。

Objective Survivin is overexpressed in gastric cancer. However it not expressed in normal gastric mucosa. The expression of survivin is tightly related to the prognosis of gastric cancer.By gene reconstruction we generated pcDNA3 survivin mutant(Cys84Ala) plasmid, and observed its effect on the gastric carcinoma cell lines. Methods The survivin mRNA and protein expression levels were determined by reverse transcription polymerase chain reaction(RT PCR) analysis,Western blot and immunohistochemical staining respectively . Flowcytometry and acridine orange staning were employed to detect apoptosis. Results Overexpression of survivin mRNA and protein were detected in the gastric cancer cell lines. Inhibition of survivin by survivin mutant cDNA induced apoptosis,activated caspase 3 activity,cleaved PARP and promoted cytochrome C releasing in gastric cancer cells,and effectively sensitized gastric cancer cells to chemotherapeutic agents. Conclusion Inhibition of survivin may induce apoptosis in gastic cancer and sensitize gastric cancer cells to chemotherapeutic agents.Survivin targeted therapeutic protocol may potentially benefit gastric cancer therapy.