PPARγ基因靶向shRNA真核表达载体的构建及表达

Construction and expression of eukaryotic expression plasmids of shRNA targeting the PPARγ gene

目的研究短发夹RNA(shorthairpin RNA,shRNA)真核表达载体在人脐静脉内皮细胞(human umbilical veinendo-thelial cell,HUVECs)中对人过氧化物酶体增殖物激活受体γ(PPARγ)基因表达的抑制作用。方法设计3条靶向PPARγ的shRNA,经退火成互补双链,克隆到质粒pGPU6/GFP/Neo中构建重组载体,经酶切鉴定和测序确认后,将3个重组表达载体转染到HUVECs,利用Western blot检测并筛选出抑制效果最好的重组表达载体。结果通过酶切鉴定和测序分析,靶向PPARγ的3个pGPU6/GFP/Neo-shR-NA重组载体构建成功,Western blot结果显示pGPU6/GFP/NeoshRNAPPARγ3可有效抑制高糖诱导HUVECs中PPARγ基因的表达,抑制率为63.2%。结论 PPARγ基因靶向shRNA真核表达载体构建成功,且能有效抑制HUVECs中PPARγ基因的表达。

Aim To construct eukaryotic expressing plasmids of short hairpin RNA（shRNA） targeting the PPARγ gene and to evaluate their inhibitory effect on PPARγ expression in human umbilical veinendothelial cells（HUVECs）.Methods Three pairs of complementary shRNA oligonucleotides targeting the PPARγ gene were designed,synthesized,annealed and inserted into the pGPU6/GFP/Neo plasmid.The recombinant plasmids were identified by restriction enzyme analysis and sequence analysis.The inhibitory effect of recombinant plasmids on PPARγ expression in HUVECs was detected by Western blot.Results After restriction enzyme analysis and sequence analysis,three eukaryotic expression plasmids of shRNA targeting the PPARγ gene were successfully constructed.Western blot analysis showed that pGPU6/GFP/NeoshRNAPPARγ3 reduced PPARγ expression by 63.2% in HUVECs induced with high glucose.Conclusion Three eukaryotic expression plasmids of shRNA targeting the PPARγ gene are successfully constructed.These recombinant plasmids can efficiently inhibit PPARγ expression in HUVECs.