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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 and screen RNAi vector of heme oxygenase-2 (HO-2) gene and observe its expression in mouse cerebrovascular endothelial cells. METHODS: Four HO-2 gene interference sequences were designed according to the c DNA sequence of mouse HO-2 gene and cloned into the interference vector p GPU6-GFP-Neo. The interference vectors were used to detect the expression of HO-2 gene in mouse cerebrovascular endothelial cells Transfection. Real-time PCR and Western Blot were used to detect HO-2 expression in mouse cerebrovascular endothelial cells. Results: The interfering vector significantly inhibited the expression of HO-2 in mouse cerebrovascular endothelial cells, and the expression of HO-2 mRNA was significantly inhibited by the interference vector p GPU6-GFP-Neo-HO-2-mus-768 (P < 0.01). The protein expression was inhibited to a significant level (P <0.05). Conclusion: The HO-2 expression vector was successfully constructed and screened, which laid the foundation for the functional identification of HO-2 gene in the next step.