大鼠叉头蛋白转录因子O1基因的shRNA慢病毒载体构建与RNA干扰效率的鉴定

目的 构建针对大鼠又头蛋白转录因子哦O1（FoxO1）基因的shRNA慢病毒载体，并在大鼠系膜细胞（RMCs）上鉴定其沉默效率。方法利用四质粒合成法合成慢病毒载体。分别用阴性对照慢病毒载体（LV-shNC-GFP组）、shRNA慢病毒载体（LV-shRNA-FoxO1组）和感染增强剂（NC组）处理RMCs。72h后，采用流式细胞仪检测绿色荧光蛋白（GFP）阳性表达率。采用荧光定量PCR检测Fox01mRNA表达情况。15d后，采用蛋白免疫印迹法检测Fox01蛋白的表达情况。结果Lv_shNc_GFP、LV-shRNA-Fox01组GFP阳性表达率分别为87．4％、95．8％；Lv-shRNA-FoxO1组FoxO1的mRNA与蛋白表达量较NC、LV-shNC-GFP组均降低（P〈0.05）。其mRNA及蛋白抑制率分别达到86．2％和77．3％，而Lv_shNC-GFP、Lv_shRNA-FoxO1组FoxO1的mRNA及蛋白表达量差异无统计学意义（P〉0．05）。结论成功构建大鼠FoxO1基因的shRNA慢病毒载体能高效、稳定地抑制FoxO1的表达，为进一步研究FoxO1的功能和作用机制奠定研究基础。

Construction of shRNA lentiviral vectors targeting rat FoxO1 gene and identification of RNAi efficiency

Objective To construct the shRNA lentiviral vectors targeting rat FoxO1 gene and to detect its efficiency of gene silence in rat mesangial cells （RMCs）. Methods The FoxO1 shRNA lentiviral vectors were constructed with four-plasmids packaging approach which encoding rat FoxO1 gene. RMCs were transfected with transfection negative control lentiviral vectors （LV-shNC-GFP group）, shRNA lentiviral vectors （LV-shRNA-FoxO1 group）, and infection enhancer （NC group）. After 72 hours, the positive expression rate of GFP was determined by Fluorescence Activated Cell Sorting （FACS）, the mRNA expression of FoxO1 was detected by qRT-PCR. After 15 days, Western blotting was performed to detect the protein expression of FoxO1. Results The positive expression rates of GFP in the LV-shNC-GFP group and LV-shRNA-FoxO1 group were 87.4% and 95.8% respectively. The expressions of FoxO1 mRNA and FoxO1 protein were lower in the LV-shRNA-FoxO1 group than in the NC group and LV-shNC-GFP group （all P〈0. 05）, and the inhibition efficiency of FoxO1 mRNA and FoxO1 protein were 86. 2% and 77.3% respectively, but there was no statistical difference in the expressions of FoxO1 mRNA and FoxO1 protein between the LV-shNC-GFP group and LV-shRNA- FoxO1 group （P〉0.05）. Conclusion The shRNA lentiviral vectors targeting rat FoxO1 gene are constructed successfully, which can significantly inhibit the expression of FoxO1 stably and efficiently. These findings lie the research foundation for the further study of the function and mechanism of FoxO1.