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目的通过构建维生素D受体(VDR)基因特异性siRNA(small interfering RNA)真核表达载体,检测其对血管内皮细胞(EC)中VDR基因的沉默作用,为进一步探讨VDR与心血管疾病发生发展关系的研究奠定基础。方法构建小鼠VDR特异性siRNA表达载体并测序,进行人EC/鼠EC的培养,进行siRNA转染试验,倒置荧光显微镜下观察细胞转染效率,Western blot进行鉴定。结果针对VDR基因的4个不同位点,设计、构建了4个siRNA干扰载体,经测序,序列全部正确。成功完成了人EC/鼠EC的培养及转染,荧光显微镜观察发现转染的重组质粒在EC内出现不同的转染效率,1号质粒的转染效率约为65%,2号质粒的转染效率约为50%,3、4号质粒的转染效率比较低。取转染效率较高的1、2号质粒做进一步转染干预,转染72h后,采用Western blot方法检测人EC/鼠EC内VDR蛋白质表达,与空白对照组比较1,、2号质粒在人EC中对VDR的表达无明显抑制作用,而在鼠EC中则有明显抑制作用,其中以1号质粒抑制效果最为明显,目的蛋白IOD/内参蛋白IOD比值发现两干扰组在人EC中对VDR的表达无明显抑制作用,而在鼠EC中则有明显抑制作用,其中以1号干扰质粒抑制效果最明显,抑制率达到了70%。结论成功完成了人EC/鼠EC的培养、转染及转染干预,并行Western blot鉴定,发现抑制效率明显的为鼠EC 1号质粒组。表明针对鼠VDR基因的siRNA表达载体有很强的特异性,并且对VDR基因干扰的位点不同表现的抑制效应不一。
Objective To construct a eukaryotic expression vector of small interfering RNA (VDR) gene to detect the silencing effect of VDR gene in vascular endothelial cells (ECs). To further explore the development of VDR and cardiovascular diseases, The relationship between the research laid the foundation. Methods The mouse VDR-specific siRNA expression vector was constructed and sequenced. The human EC / mouse EC was cultured and transfected with siRNA. The transfection efficiency was observed under inverted fluorescence microscope and identified by Western blot. Results Four siRNA interference vectors were designed and constructed according to four different sites of VDR gene. All the sequences were correctly sequenced. The human EC / mouse EC was successfully cultured and transfected. Fluorescence microscopy showed that the transfection efficiency of recombinant plasmids was different in EC. The transfection efficiency of plasmid # 1 was about 65% The dyeing efficiency is about 50%, and the transfection efficiency of 3,4 and 4 plasmids is relatively low. 1 and 2 plasmids with higher transfection efficiency were used for further transfection. After transfection for 72h, VDR protein expression in human EC / EC was detected by Western blot. Compared with the blank control group, plasmid 1 and 2 There was no significant inhibitory effect on the expression of VDR in human EC, but significant inhibition in murine EC. The inhibition effect was most obvious on plasmid No. 1, and the IOD / IOD ratio of the target protein found that the two interfering groups were in human EC The expression of VDR had no obvious inhibitory effect, but the inhibitory effect was obvious in murine EC. The inhibitory rate of VDR was the most obvious, and the inhibition rate was 70%. Conclusion The culture, transfection and transfection of human EC / murine EC were successfully completed. Western blot analysis showed that the inhibitory effect was obvious. The results showed that the siRNA expression vector targeting murine VDR gene was highly specific and had different inhibitory effects on the different sites of VDR gene interference.