维生素D受体特异性shRNA慢病毒载体及ASMCs稳转株的构建

Construction of vitamin D response-shRNA lentivirus vector and establishment of its stably transfected ASMC cells line

目的构建特异性靶向大鼠维生素D受体(VDR)基因的RNA干扰慢病毒载体,并筛选构建VDR基因稳定沉默的ASMCs细胞株。方法设计大鼠VDR的4组短发夹状RNA(shRNA)靶序列,合成的DNA双链与线性化的慢病毒LV3载体相连,构建LV3-sh-VDR重组慢病毒质粒。经酶切及测序鉴定后,进行病毒包装和病毒滴度检测,而后转染大鼠ASMCs,分组为干扰组(转染VDR shRNA)、阴性对照组(仅转染慢病毒载体)和空白对照组(未转染载体)。经嘌呤霉素抗性筛选后,建立ASMCs的VDR-shRNA稳定细胞株,用逆转录聚合酶链反应和Western Blotting法分别检测各组ASMC细胞中VDR mRNA及蛋白的表达水平。结果经酶切和测序鉴定,成功构建4个VDR重组慢病毒载体。慢病毒转染ASMCs后,经嘌呤霉素抗性成功筛选到ASMCs的VDR-shRNA稳定细胞株,干扰组ASMCs的VDR mRNA和蛋白的表达量与阴性对照组及空白对照组相比均明显下降。结论成功构建VDR稳定沉默表达的大鼠ASMCs细胞株,为研究VDR对哮喘生物学行为的影响提供细胞模型。

Objective To construct a recombinant lentiviral vector specificly targeting rat vitamin D receptor(VDR)gene by RNA interference and select airway smooth muscle cell(ASMC)line model stably silencing the VDR gene expressing.Methods Four short hairpin RNA(shRNA)sequences targeting VDR gene were designated and the synthetic double-strained DNA chains were connected with linear LV3 carriers,then the LV3-sh-VDR lentivirus plasmid was constructed.Being indentified by restriction enzyme digestion and sequencing,the recombinant lentiviral vector VDR was packaged and the virus titer was measured.Then the recombinant lentiviral vector was transfected into rat ASMCs.And these transfected cells were divided into three groups:interference group(infected with the VDR shRNA),negative control group(infected only with control-shRNA)and blank control group(without transfection).ASMCs stably silencing VDR expressing were selected by puromycin.Then real-time polymerase chain reaction and Western Blotting were used to examine the expression of VDR mRNA and protein in them.Results DNA restriction enzyme digestion and sequencing demonstrated that four LV3-VDR-shRNA recombinant lentivirals were constructed successfully.And ASMCs were successfully transfected with LV3-VDR-shRNA recombinant lentivirus vector by puromycin.Compared with the negative control group and blank control group,the expression of VDR mRNA and protein were significantly down-regulated in interference groups.Conclusion A stable ASMC cells line with VDR gene silencing are successfully constructed,which provided a cellular model to study the role of VDR in the biological behavior of asthma.