靶向LOX-1基因RNAi慢病毒载体的构建及鉴定

发布时间：2018-01-14 13:29

本文关键词：靶向LOX-1基因RNAi慢病毒载体的构建及鉴定　出处：《中国细胞生物学学报》2013年07期 　论文类型：期刊论文

【摘要】：该研究针对LOX-1基因(OLR1)设计并合成3条siRNA干扰序列,与双酶切的U6-vshR-NA-CMV-GFP慢病毒载体连接得到重组载体,包装慢病毒并测定滴度。采用最佳MOI值及转染条件,转染人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC),转染96 h后通过RT-PCR和Western blot检测LOX-1 mRNA及蛋白的表达,流式细胞仪检测转染后ox-LDL诱导HUVEC的凋亡情况。测序证实靶向LOX-1 RNAi慢病毒载体构建成功;RT-PCR和Western blot结果显示转染后LOX-1 mRNA及蛋白均受到不同程度抑制(P0.05);流式检测结果显示抑制LOX-1表达后ox-LDL诱导HUVEC凋亡显著减少(P0.05)。该研究应用RNAi技术成功构建LOX-1 RNAi慢病毒载体,有效抑制了LOX-1的表达,显著减少了ox-LDL诱导的凋亡,为进一步研究LOX-1基因沉默减轻内皮细胞损伤及相应保护机制奠定了基础。
[Abstract]:In this study, three siRNA interference sequences were designed and synthesized for LOX-1 gene. The recombinant vector was obtained by ligating with the double digested U6-vshR-NA-CMV-GFP lentivirus vector to package the lentivirus and determine the titer. The optimal MOI value and transfection conditions were used. Human umbilical vein endothelial cells were transfected into human umbilical vein endothelial cells (HUVECs). After transfection for 96 h, the expression of LOX-1 mRNA and protein was detected by RT-PCR and Western blot. Flow cytometry was used to detect the apoptosis of HUVEC induced by ox-LDL. Sequencing confirmed that the lentivirus vector targeting LOX-1 RNAi was successfully constructed. The results of RT-PCR and Western blot showed that LOX-1 mRNA and protein were inhibited in different degree after transfection. Flow cytometry showed that HUVEC apoptosis induced by ox-LDL was significantly reduced by inhibiting LOX-1 expression (P 0.05). In this study, LOX-1 RNAi lentivirus vector was successfully constructed by using RNAi technique. It effectively inhibited the expression of LOX-1 and significantly reduced the apoptosis induced by ox-LDL, which laid a foundation for the further study of LOX-1 gene silencing to alleviate the injury of endothelial cells and the corresponding protective mechanism.
【作者单位】：辽宁医学院附属第一医院心内科;辽宁医学院附属第一医院组织工程重点实验室;
【基金】：辽宁省科技厅社会发展攻关计划(批准号:2011225015) 辽宁医学院校长基金—奥鸿博泽研究生科研创新基金资助的课题~~
【分类号】：R341;Q78
【正文快照】： 正常情况下,凝集素样氧化性低密度脂蛋白受体-1(lectin-like oxidized low density lipoprotein receptor-1,LOX-1)在内皮细胞、平滑肌细胞、巨噬细胞等有少量表达,可被氧化性低密度脂蛋白(oxi-dized low density lipoprotein,ox-LDL)、血管紧张素II(angiotensin II,Ang II)

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