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目的构建Survivin-siRNA真核表达载体,并探讨其对胃癌SGC-7901细胞增殖和凋亡的影响。方法化学合成4条能转录出siRNA的模板DNA,各75个碱基,退火形成2条双链DNA,双酶切后插入pSUPER.basic载体。将阳性重组质粒转染SGC-7901细胞,进行细胞计数,并用MTT法检测细胞的增殖活性;半定量RT-PCR检测细胞Survivin基因mRNA的转录水平;流式细胞术检测细胞周期的变化。结果PCR和酶切鉴定表明Survivin-siRNA真核表达载体构建正确,其能下调SGC-7901细胞Survivin基因mRNA的转录水平,抑制SGC-7901细胞生长和增殖,并促进细胞凋亡,使G0/G1和亚G1期细胞增多,S期细胞减少。结论已成功构建了Survivin-siRNA真核表达载体,其能下调SGC-7901细胞中Survivin基因mRNA的转录水平,使细胞增殖减弱,凋亡增加,为RNA干扰技术应用于胃癌的基因治疗提供了一定的实验依据。
Objective To construct the eukaryotic expression vector of Survivin-siRNA and investigate its effect on the proliferation and apoptosis of gastric cancer cell line SGC-7901. METHODS: Four template DNAs capable of transcribing siRNA were chemically synthesized, 75 bases each, annealed to form two double-stranded DNAs, and double-digested to insert the pSUPER.basic vector. The positive recombinant plasmids were transfected into SGC-7901 cells for cell counting. The cell proliferation was detected by MTT assay. The transcription level of Survivin mRNA was detected by semi-quantitative RT-PCR. The cell cycle was detected by flow cytometry. Results PCR and restriction enzyme digestion showed that Survivin-siRNA eukaryotic expression vector was constructed correctly. It could down-regulate the transcription level of Survivin mRNA, inhibit the growth and proliferation of SGC-7901 cells and promote the apoptosis of SGC-7901 cells. And sub-G1 phase cells increased, S phase cells decreased. Conclusion Survivin-siRNA eukaryotic expression vector has been successfully constructed, which can down-regulate Survivin mRNA expression in SGC-7901 cells and attenuate cell proliferation and increase apoptosis, and provide a certain method for the gene therapy of gastric cancer with RNA interference Experimental basis.