摘要
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 a eukaryotic expression vector for Survivin-siRNA and investigate its effect on proliferation and apoptosis of gastric cancer SGC-7901 cells.Methods Four single-stranded template DNAs encoding siRNA against Survivin,each consisting of 75 bp,were synthesized chemically,based on which two double-stranded DNAs were formed by annealing,then identified by restriction analysis and inserted into vector pSUPER.basic.SGC-7901 cells were transfected with positive recombinants,then counted and determined for proliferation activity by MTT method,and for cell cycle by flow cytometry.The transcription level of Survivin mRNA was determined by semi-quantitative RT-PCR.Results Both PCR and restriction analysis proved that the eukaryotic expression vector for Survivin-siRNA was constructed correctly.The constructed expression vector down-regulated the transcription level of Survivin mRNA,inhibited the growth and proliferation and promoted the apoptosis of SGC-7901 cells.The number of transfected SGC-7901 cells at G0 /G1 and sub-G1 stages increased,while that at S stage decreased.Conclusion The eukaryotic ex-pression vector for Survivin-siRNA was successfully constructed,which down-regulated the transcription of Survivin mRNA in SGC-7901 cells,and inhibited the proliferation and promoted the apoptosis of the cells.It provided a certain experimental basis for gene therapy of gastric cancer by RNAi technique.
出处
《中国生物制品学杂志》
CAS
CSCD
2009年第10期975-978,982,共5页
Chinese Journal of Biologicals
基金
甘肃省自然科学基金项目资助(3ZS051-A25-079)
