针对HO-2基因的四种RNA干扰序列构建和效应观察

Construction and identification of four RNA interference vectors targeting on HO-2 gene

目的:构建和筛选出血红素氧合酶-2(HO-2)基因的RNA干扰载体,并观察其在小鼠脑血管内皮细胞中的表达情况。方法:根据小鼠HO-2基因的c DNA序列设计了4个HO-2基因干扰序列,克隆到干扰载体p GPU6-GFP-Neo上,利用电穿孔法将干扰载体对小鼠脑血管内皮细胞进行转染。Real-time PCR和Western Blot检测小鼠脑血管内皮细胞中HO-2的表达。结果:干扰载体显著抑制小鼠脑血管内皮细胞中HO-2的表达,其中干扰载体p GPU6-GFP-Neo-HO-2-mus-768对HO-2 mRNA的表达抑制达显著水平(P<0.01),蛋白的表达抑制达显著水平(P<0.05)。结论:成功构建并筛选了HO-2表达干扰载体,为下一步的HO-2基因的功能鉴定奠定了基础。

Objective： To construct heme oxygenase-2 interference vectors,and observe their expression in cerebrovascular endothelial cells of mice. Methods： Four interference sequences were designed according to the the c DNA sequence of HO-2 gene,which were cloned into interference vector p GPU6-GFP-Neo. Then cerebrovascular endothelial cells were transfected with the interference vector using electroporation,the Real-time PCR and Western Blot was used to detect HO-2 mRNA and protein levels in cerebrovascular endothelial cells. Results： The interference vectors highly suppressed the HO-2 gene expression of cerebrovascular endothelial cells,and the interference vector p GPU6-GFP-Neo-HO-2-mus-768 extremely significant suppressed the mRNA expression of HO-2（ P 〈 0. 01）,and the protein expression inhibition also reached significant level（ P 〈 0. 05）. Conclusion： The interference vector p GPU6-GFP-Neo-HO-2 was successfully constructed and selected,which may be benefit for further study the mechanisms and function of HO-2 gene.