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目的:分离,克隆编码β-Smac的基因,观察胃癌细胞SGC7901中β-Smac基因过表达对顺铂敏感性的影响。方法:从胃癌细胞株SGC7901扩增β-Smac的cDNA。构建含β-Smac基因cDNA的表达载体pcDNA3.1-β-Smac。将pcDNA3.1-β-Smac质粒转入SGC7901细胞;用RT-PCR、Western blot分别从核酸和蛋白水平检测外源基因的表达情况。选用顺铂(0、2、8、12μg/ml)处理转染pcDNA3.1(对照组)或pcDNA3.1-β-Smac(实验组)48h后的SGC7901细胞24h,流式细胞仪分析细胞凋亡百分率。结果:成功构建了含β-Smac基因的表达质粒,核酸序列分析显示克隆的β-Smac基因序列与GeneBank中登记的β-Smac基因序列100%同源,引入的Flag序列完全正确。同对照组比较,实验组转染24h后,SGC7901细胞β-Smac mRNA水平明显增高,48h后Western blot在β-Smac预期位置检测到Flag蛋白特异性条带。顺铂处理24h后,仅2μg/ml组显示出实验组和对照组细胞凋亡差异有显著性(P=0.009),而其余各组间均未显示有统计学差异。结论:成功构建含β-Smac基因序列的重组质粒并转染靶细胞,外源性β-Smac基因过表达后,未显示能显著提高人胃癌细胞株SGC7901对顺铂的敏感性。
OBJECTIVE: To isolate and clone the gene encoding β-Smac and observe the effect of β-Smac gene overexpression on the sensitivity of cisplatin in gastric cancer cell line SGC7901. Methods: cDNA of β-Smac was amplified from gastric cancer cell line SGC7901. Construction of expression vector pcDNA3.1-β-Smac containing β-Smac cDNA. The pcDNA3.1-β-Smac plasmid was transfected into SGC7901 cells. The expression of foreign genes was detected by RT-PCR and Western blot respectively. SGC7901 cells transfected with pcDNA3.1 (control group) or pcDNA3.1-β-Smac (experimental group) 48h were treated with cisplatin (0, 2, 8, 12μg / ml) for 24 hours. Flow cytometry Dead percentage. Results: The expression plasmid containing β-Smac gene was successfully constructed. Nucleotide sequence analysis showed that the cloned β-Smac gene sequence was 100% homologous with the β-Smac gene sequence registered in GeneBank. The introduced Flag sequence was completely correct. Compared with the control group, the expression ofβ-Smac mRNA in SGC7901 cells was significantly increased 24 h after transfection in experimental group, and Flag protein specific bands were detected by Western blot in the expected position of β-Smac 48 h after transfection. Cisplatin treatment 24h, only 2μg / ml group showed that the experimental group and the control group apoptosis was significantly different (P = 0.009), while the rest did not show any significant difference between groups. Conclusion: The recombinant plasmid containing β-Smac gene was successfully constructed and transfected into target cells. The overexpression of exogenous β-Smac gene did not show a significant increase in the sensitivity of human gastric cancer cell line SGC7901 to cisplatin.