维生素D受体特异性siRNA表达载体的构建  

Identification of human vascular endothelial cells/rat vascular endothelial cells transfected with vitamin D receptor gene in vitro

作　　者：向伟[1] 何小解[2] 马燕琳[3] 易著文[2] 郑维[4] 曹艳[2] 赵水平[2] 马志超[1] 吴明[1] 符生苗[1] 马建林[1] 王洁[5] 康红[4] 

机构地区：[1]海南省人民医院(海南医学院附属人民医院)儿科,海南海口570311 [2]中南大学湘雅二医院,湖南长沙410008 [3]海南医学院,海南海口570311 [4]湖南省人民医院,湖南长沙410008 [5]海南省妇幼保健院(海南省妇儿医院),海南海口570006

出　　处：《中国热带医学》2011年第5期521-524,共4页China Tropical Medicine

基　　金：国家自然科学基金(30560161);国家自然科学基金(81060073);海南省自然科学基金(805106);国家人事部留学人员科技活动课题项目基金(2006年)

摘　　要：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。Aim To Identify of human/rat vascular endothelial cells transfected with vitamin D receptor gene in vitro.Methods The plasmid expression vector with human pcDNATM6.2-GW/EmGFPmiR was amplified in E.coli.The plasmid was extracted,purified by EndoFree Plasmid Maxi Kit.The plasmid of pcDNATM6.2-GW/EmGFPmiR was analyzed and identified by enzymes digestion and sequencing analysis.human vascular endothelial cells /rat vascular endothelial cells was cultured and transfected VDR gene were selected with G418.The expression of bFGF mRNA and its products in the transfected BMSCs were detected by real time PCR,immunohistochemistry,immunofluorescence and Western-blot.The proliferative cycle of transfected BMSCs were examined by flow cytometry analysis.Results BMSCs expressing bFGF gene were obtained by transfection with pcDNA3.1-bFGF gene via Lipofectamine.The cell lineage was confirmed that the expressed position of bFGF was mainly in cytoplasm after transfection with bFGF gene.Higher proportion of cells in proliferation cycle was shown in transfected BMSCs compared with non-transfected BMSCs（P0.05）.Concluson pcDNA3.1-bFGF can be transfected into BMSCs cultured in vitro via Lipofectamine.Genetic modification of BMSCs with bFGF may promote the proliferation of BMSCs.

关 键 词：维生素D受体 血管内皮细胞 转染 质粒 

分 类 号：Q565[生物学—生物化学]