【摘要】 目的构建C35的小干扰RNA(siRNA),观察其在乳腺癌细胞株T47D中对C35基因的表达抑制作用。方法设计两对针对C35编码区的siRNA并合成,构建到转录载体pTZU6+1上,形成重组质粒siRNA-C35,在脂质体介导下转染乳腺癌细胞株T47D,RT-PCR分析RNA干扰后mRNA的变化,Western blot检测表达蛋白的变化。结果DNA测序和双酶切证明重组表达载体构建成功。RT-PCR结果显示,质粒pTZU6+1、pshRNA-C35-1和pshRNA-C35-2组的相对表达率分别是脂质体对照组的95.3%、40.0%和28.4%。Western blot结果显示,以转染脂质体组的蛋白带为标准,其他组与之比较,转染质粒pTZU6+1、psiRNA-C35-1和psiRNA-C35-2组的相对表达率分别是转染对照组(脂质体组)的94.9%、29.6%和32.2%。结论通过构建siRNA转录载体转染乳腺癌细胞株能有效抑制乳腺癌细胞中C35基因的表达。更多还原

【Abstract】 Objective To construct the plasmid containing small interfering RNA (siRNA) to suppress the expression of the C35 gene in T47D cells. Methods siRNA was designed according to the C35 coding region, synthesized and inserted into vector pTZU6+1 to generate a new plasmid named siRNA-C35. The plasmid was transfected using lipofectamine into breast cancer cells T47D to detect the effect of C35 expression, including gene and protein by RT-PCR and western blot, respectively. Results The recombinant plasmids were successfully constructed by sequencing and enzyme digestion. RT-PCR indicates the expression rate of vector pTZU6+1, psiRNA-C35-1 and psiRNA-C35-2 was 95.3%, 40.0% and 28.4% compared with the lipofectamine control group. Western blot showed that the expression rate of vector pTZU6+1, psiRNA-C35-1 and psiRNA-C35-2 was 94.9%, 29.6% and 32.2% according to the band of the lipofectamine group’s protein. Conclusion The constructed siRNA plasmid could efficiently suppress the expression of C35 in breast cancer cells.更多还原