9型重组腺相关病毒介导 R65核酶基因体外转染人血管不同细胞的对比研究  

Comparative study of in vitro transfection of R65 ribozyme gene to different human vascular cells mediated by recombinant adeno-associated virus 9

作　　者：翟慧[1] 杨毅宁[1] 马依彤[1] 李晓梅[1] 向阳[1] 陈邦党[1] 刘芬[1] 谢佳[1] 李东泽[1] 

机构地区：[1]新疆医科大学第一附属医院冠心病实验室,乌鲁木齐830054

出　　处：《中华微生物学和免疫学杂志》2013年第12期938-942,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金项目（81160042）;教育部新世纪优秀人才计划（NCET-11-1074）

摘　　要：目的：研究9型重组腺相关病毒（recombinant adeno-associated virus 9，rAAV9）载体介导R65核酶基因及增强型绿色荧光蛋白（ enhanced green fluorescent protein ，EGFP）对体外培养人脐静脉内皮细胞（ human umbilical vein endothelial cells ， HUVECs ）、人主动脉平滑肌细胞（ human aortic smooth muscle cells ， HASMCs）的转染及病毒载体对两种不同细胞的增殖情况和对NF-κB通道P65表达的影响。方法 rAAV9-EGFP-R65按转染复数（multiplicity of infection，MOI）为1×105、1×106、1×107转染HUVECS、HASMCS细胞，倒置荧光显微镜下观察 HUVECs、HASMCs 细胞中EGFP 的表达，流式细胞仪检测rAAV9-EGFP-R65对HUVECs、HASMCs细胞的转染效率，Alamar Blue 检测rAAV9-EGFP-R65载体对HUVECS、HASMCs细胞增殖的影响。 Western blot法检测rAAV9-EGFP-R65对HUVECs和HASMCs两种细胞中NF-κB通道P65表达的影响。结果 rAAV9-EGFP-R65转染的荧光强度随着MOI的增加和转染时间的延长而增加。 HUVECs和HASMCs在rAAV9-EGFP-R65转染24 h后EGFP 开始表达。 HUVECs 在转染后第6天达到高峰，而HASMCs在转染后第5天达高峰。高峰表达时流式细胞术检测rAAV9-EGFP-R65对HUVECs的转染率分别为MOI＝1×105组：（1．40±1．20）％，MOI＝1×106组：（12．30±1．35）％，MOI＝1×107组：（52．80±2．05）％。rAAV9-EGFP-R65对HASMCs的转染率分别为MOI＝1×105组：（5．30±1．04）％，MOI＝1×106组：（18．30±2．24）％，MOI＝1×107组：（52．40±3．21）％。 rAAV9-EGFP-R65转染HUVECs和HASMCs细胞后，细胞生长及形态均正常，Alamar Blue法进一步检测表明HUVECs和HASMCs各细胞内转染组与未转染组之间A值差异均无统计学意义。 Western blot 法检测显示rAAV9-EGFP-R65转染HUVECs 和HASMCs 两种细胞后NF-κB通道P65的表达减弱。结论 rAAV9-EGFP-R65能够有效转染人脐静脉内皮细胞和人主动脉平滑肌细胞，对两种细胞增殖均无抑制作用，均能有效抑制�Objective To evaluate in vitro transfection of anti-nuclear factor-κB （ NF-κB） ribozyme gene to human umbilical vein endothelial cells （HUVECs） and human aortic smooth muscle cells （HASMCs） mediated by recombinant adeno-associated virus 9 carrying enhanced green fluorescent protein gene （ rAAV9-EGFP-R65 ） and to study their effects on cell proliferation and NF-κB P65 expression.Methods HUVECs and HASMCs were respectively transfected with rAAV9-EGFP-R65 at different multiplicity of infection （ MOI=1 ×105 , 1 ×106 and 1×107）.The expression of EGFP was observed with fluorescence microscopy .Flow cytometry was performed to evaluate the transfection efficiency .Alamar Blue assay was used to measure the proliferation of the transfected cells.Western blot was used to detect NF-κB P65 expression .Results The fluorescence intensity was enhanced along with an increased MOI and an extended time of transfection .HUVECs and HASMCs transfected with rAAV 9-EGFP-R65 began to express EGFP at 24 h after transfection .The expression peak appeared on the sixth day in HUVECs, and the fifth day in HASMCs.The efficiencies of transfection in HUVECs at MOI of 1×105, 1×106 and 1×107 on the sixth day were （1.40±1.20）%, （12.30±1.35）%and （52.80±2.05）%, respectively.The trans-fection efficiencies of HASMCs on the fifth day were （5.30±1.04）%, （18.30±2.24）% and （52.40±3.21）%at MOI of 1×105 , 1×106 and 1×107 .Cell growth and morphology were not affected by transfection .Alamar Blue assay confirmed that there was no significant difference in the absorbance value between the transfected cells and two types of control cells .Western blot assay showed that the expression of NF-κB P65 was decreased by the trans-fection of rAAV9-EGFP-R65 in HUVECs and HASMCs .Conclusion rAAV9-EGFP-R65 can be efficiently trans-fected into two types of human vascular cells .It shows no inhibitory effects on cell proliferation , but can repress NF-κB P65 expression.

关 键 词：腺相关病毒 核酶基因 转染 NF-ΚB 动脉粥样硬化 

分 类 号：R543.5[医药卫生—心血管疾病]