重组9型腺相关病毒载体转染动脉粥样硬化模型小鼠主动脉:最佳在体表达时间点的确定  

Recombinant adeno-associated virus serotype 9 transfection of atherosclerosis mice:determination of the optimal expression time in vivo

作　　者：谢佳[1] 杨毅宁[1] 马依彤[1] 李晓梅[1] 陈邦党[1] 刘芬[1] 陈清杰[1] 翟慧[1] 

机构地区：[1]新疆医科大学第一附属医院心脏中心,新疆维吾尔自治区乌鲁木齐市830054

出　　处：《中国组织工程研究》2015年第5期733-738,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金项目(81160042)~~

摘　　要：背景:重组9型腺相关病毒载体对心肌组织具有较高的亲和力,关于携带增强型绿色荧光蛋白报告基因的重组9型腺相关病毒(recombinant adeno-associated virus serotype 9-enhanced green fluorescent protein,r AAV9-e GFP)在动脉粥样硬化模型小鼠主动脉中的表达情况还不确定。目的:探索携带r AAV9-e GFP转染动脉粥样硬化模型小鼠主动脉最佳的在体表达时间点。方法:给予30只Apo E-/-小鼠连续高脂饮食饲养16周建立为动脉粥样硬化模型,然后随机取其中25只通过尾静脉注射转染5.0×1011 vg(virus genomes)r AAV9-e GFP,另5只作为对照组给予生理盐水。转染病毒的动脉粥样硬化模型小鼠分别于转染后14,21,28,35,60 d处死,留取主动脉组织,应用激光共聚焦显微镜观察增强型绿色荧光蛋白荧光表达情况,Western blot法检测主动脉增强型绿色荧光蛋白的蛋白表达,观察增强型绿色荧光蛋白体内表达情况,确定病毒在体表达最佳时间点。结果与结论:r AAV9-e GFP可有效转染动脉粥样硬化模型小鼠主动脉血管,增强型绿色荧光蛋白可在主动脉组织中表达,其表达强度随着转染时间的增加而逐渐增强,于转染35 d时表达最强,60 d趋于稳定,各时间点主动脉斑块中增强型绿色荧光蛋白表达的水平差异有显著意义(P<0.001)。表明r AAV9-e GFP可有效在动脉粥样硬化模型Apo E-/-小鼠主动脉中表达,r AAV9-e GFP可成为动脉粥样硬化治疗的最佳载体。BACKGROUND：Recombinant adeno-associated virus serotype 9 has a high affinity in myocardial tissue, and the expression of recombinant adeno-associated virus serotype 9-enhanced green fluorescent protein （rAAV9-eGFP） in the aorta of atherosclerosis mice is not clear. OBJECTIVE：To explore the optimal time point of rAAV9-eGFP expression in the aorta of atherosclerosis mice. METHODS：Atherosclerosis model was established with high-fat diet in 30 ApoE-/-mice for 16 weeks. Among them, 25 mice were injected with 5.0＆#215;1011 vg （virus genomes） rAAV9-eGFP through the tail vein, while the remaining 5 mice were injected with saline, serving as the control group. The virus-transfected mice were kil ed at 14, 21, 28, 35 and 60 days after transfection, and aortic tissue was harvested. The expression of enhanced green＆amp;nbsp;fluorescent protein was detected with laser scanning confocal microscope. Western blot assays were used to detect the expression of enhanced green fluorescent protein in aorta. The expression of enhanced green fluorescent protein in vivo was observed and the optimal expression time point was determined. RESULTS AND CONCLUSION：rAAV9-eGFP effectively transfected the aorta of atherosclerosis mice, enhanced green fluorescent protein was expressed in aortic tissue, and the expression intensity increased gradual y with the increasing transfection time. The highest expression level was found at 35 days after transfection and then maintained stable at 60 days. There were significant differences at different time points after transfection （P〈0.001）. These data indicate that rAAV9-eGFP can be effectively expressed in the aorta of atherosclerosis ApoE-/-mice and rAAV9-eGFP can be regarded as the optimal vector in the treatment of atherosclerosis.

关 键 词：实验动物 心脏及血管损伤模型 动脉粥样硬化 组织构建 9型腺相关病毒 主动脉 转染 APOE-/-小鼠 国家自然科学基金 

分 类 号：R318[医药卫生—生物医学工程]