靶向Hiwi基因的shRNA表达载体的干扰效果评价  

The evaluation of the interference effect of shRNA expression vectors targeting Hiwi gene

作　　者：陈岐辉[1] 卢绩[1] 郑学清[1] 荆明远 王春喜[1] 

机构地区：[1]吉林大学第一医院,吉林长春130021 [2]抚松县人民医院

出　　处：《中国实验诊断学》2010年第3期380-382,共3页Chinese Journal of Laboratory Diagnosis

摘　　要：目的评价靶向Hiwi基因的shRNA表达载体的干扰效果。方法通过构建靶向Hiwi基因的短发夹RNA(shout-hairpin RNA,shRNA)真核表达载体pGenesil-2-Hiwi1和pGenesil-2-Hiwi2263,并以pGenesil-2-control为阴性对照,转染膀胱癌T24细胞,从mRNA和蛋白水平观察3种干扰载体的干扰效率。结果pGenesil-2-Hiwi1和pGenesil-2-Hiwi2263组膀胱癌T24细胞Hiwi mRNA与GAPDH灰度比值较Normal control组Hiwi mRNA与GAPDH灰度比值显著降低(P<0.01),pGenesil-2-control、pGenesil-2-Hiwi1和pGenesil-2-Hiwi2263的干扰效率分别为5.7%、100%和100%。pGenesil-2-Hi-wi1和pGenesil-2-Hiwi2263组膀胱癌T24细胞Hwi蛋白与GAPDH灰度比值较Normal control组Hiwi蛋白与GAPDH灰度比值明显著降低(P<0.01),pGenesil-2-control、pGenesil-2-Hiwi1和pGenesil-2-Hiwi2263的干扰效率分别为3.2%、74.2%和64.5%。结论pGenesil-2-Hiwi1和pGenesil-2-Hiwi2263表达载体沉默膀胱癌T24细胞中Hiwi基因表达,干扰效率较高,为今后通过RNAi技术抑制Hiwi基因表达,从基因与蛋白质水平探索Hiwi基因在干细胞及肿瘤中的作用打下基础。Objective To evaluate the interference effect of shRNA expression vectors targeting Hiwi gene.Methods We contructed shRNA expression vectors targeting HIWI gene, pGenesil-2-Hiwil and pGenesil-2-Hiwi2263. And we used pGenesil-2-control as a negative control. Then these expression vectors are transfected into bladder carcinoma cell line 324, and the interference efficiency was observed by the mRNA and protein level. Results The grey scale ratio of HIWI/GAPDH mRNA in pGenesil-2-Hiwil group and p Genesil-2-Hiwi2263 group is significantly lower than in Normal control group. （P 〈 0.01 ）. The interference efficiency in pGenesil-2- control group, pGenesil-2-Hiwil group and pGenesil-2-Hiwi2263 group are 5.7%, 100% and 100% respectively. The grey scale ratio of HIWI/GAPDH protein in pGenesil-2-Hiwil group and pGenesil-2-Hiwi2263 group is also significantly lower than in Normal control group （P 〈 0.01 ）. The interference efficiency in pGenesil-2-control group, pGenesil-2-Hiwil group and pGenesil-2-Hiwi2263 group are 5.7% ,74.2% and 64.5% respectively. Conclusion pGenesil-2-Hiwil and pGenesil-2-Hiwi2263 expression vector can silence HIWI expression in T24 cell line and the efficiencies are high. The results demonstrate that we can inhibit HIWI expression by RNAi and those lay the foundation of exploring roles of HIWI gene in stem cell and tumors on the level of gene and protein.

关 键 词：Hiwi基因 膀胱癌组织 表达 

分 类 号：R346[医药卫生—基础医学] R73-3