叉头状转录因子O1基因过表达慢病毒载体构建及其大鼠系膜细胞株的建立

Construction of lentiviral vectors targeting Fox O1 gene and overexpression of Fox O1 in rat mesangial cells

目的构建含组成性激活突变型Fox O(CA-Fox O1 1)基因的过表达慢病毒载体,并建立其过表达的大鼠肾小球系膜细胞株。方法通过四质粒合成法构建包含CA-Fox O1编码序列的过表达慢病毒载体(LV-CA-Fox O1)。实验分为3组:空白对照组(NC组)、空慢病毒载体阴性对照组(LV-empty-Fox O1组)和过表达慢病毒载体组(LV-CA-Fox O1组)。培养系膜细胞(RMCs)72 h后,用流式细胞仪检测RMCs中绿色荧光蛋白(GFP)阳性率,并采用实时荧光定量PCR及蛋白免疫印迹法检测Fox O1的m RNA及蛋白表达情况。结果成功构建LV-CA-Fox O1,其病毒滴度为1×108TU/ml。LV-empty-Fox O1组和LV-CA-Fox O1组GFP阳性率分别为84.5%和81.4%;与NC组相比,LV-CA-Fox O1组Fox O1的m RNA与蛋白表达量相当于NC组的27.92倍与5.41倍(均P<0.05);而NC组与LV-empty-Fox O1组Fox O1的m RNA及蛋白表达量差异均无统计学意义。结论成功构建了大鼠Fox O1基因的过表达慢病毒载体,并建立了稳定过表达Fox O1的大鼠肾小球系膜细胞株,为进一步研究Fox O1的功能和作用机制奠定了研究基础。

[ Objective ] To construct the lentiviral vectors （LV） expressing recombinant plasmid which contains rat constitutively active forkhead box transcription factor O1 （CA-FoxO1） gene, and build the rat mesangial cells （RMCs） of overexpressing CA-FoxO1. [ Methods ] The lentiviral vectors were constructed with four-plasmids pack- aging approach which encoding rat CA-FoxO1 gene. RMCs were divided into three groups： the normal control group （NC group）, the empty lentiviral vector group（LV-empty-FoxO1 group） and the overexpression lentiviral vector group （LV-CA-FoxO1 group）. After 72 hours, the positive ratio of green fluorescent protein （GFP） was determined by fluo- rescence-activated cell sorting （FACS）, real-time fluorescence quantification PCR （qRT-PCR） and western blotting were used to detect the mRNA and protein expression of FoxO1 respectively. [Results] LV-CA-FoxOI was suc- cessfully constructed with virus titer of 1∽10s TU/mL. The positive ratio of GFP in LV-empty-FoxO1 group and LV- CA-FoxO1 group were 84.5% and 81.4% ,respectively. The expressions of FoxO1 mRNA and FoxO1 protein in group LV-CA-FoxOI were 27.92 times and 5.41 times of the NC group （t =439.1, t =45.91, all P 〈0.05）, but the differences were not statistically significant between NC group and LV-empty-FoxO1 group. [ Conclusions ] The lentiviral vectors expressing rat constitutively active FoxO1 gene were successfully constructed, and the RMCs of the over-expressing CA-FoxO1 were build. These findings provide a research foundation for further study of the function and mechanism of FoxO1.