重组腺相关病毒对内皮细胞增殖和凋亡的影响  

Effects of recombinant adeno-associated virus on proliferation and apoptosis of endothelial cells

作　　者：翟慧[1] 向阳[1] 杨毅宁[1] 马依彤[1] 李晓梅[1] 陈邦党[1] 刘芬[1] 张金宇[1] 

机构地区：[1]新疆医科大学第一附属医院心脏中心,乌鲁木齐830054

出　　处：《中华实验外科杂志》2014年第8期1690-1693,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（81160042、81060021）

摘　　要：目的 通过重组9型腺相关病毒（rAAV9）载体介导R65核酶基因对体外培养人脐静脉内皮细胞（HUVECs）进行转染,观察R65核酶基因表达对高脂血清刺激后HUVECs增殖和凋亡的影响.方法 体外培养HUVECs,高脂血清诱导建立动脉粥样硬化内皮损伤模型,重组rAAV9介导抗核因子（NF）-κB核酶基因（rAAV9-R65）腺相关病毒载体转染HUVECs.实验分为3组：（1）正常对照组;（2）高脂血清诱导组;（3）高脂血清诱导＋病毒转染组.通过Western blot检测HUVECs细胞中p65核蛋白的表达;酶联免疫吸附试验（ELISA）检测NF-κB信号通路下游肿瘤坏死因子（TNF）-α和白细胞介素（IL）-6的变化;Alamar Blue法检测rAAV9-R65转染后HUVECs的体外增殖;rAAV9-R65转染细胞96 h后流式细胞仪检测高脂血清刺激后HUVECs凋亡率的变化.结果 高脂血清诱导HUVECs 24 h后,细胞由卵圆形铺路石样逐渐变成类圆形,Western blot检测显示p65的表达水平分别为：空白组：0.0229 ±0.0024;高脂血清诱导组：0.224 3±0.008 6;rAAV9-R65转染＋高脂血清诱导组：0.071 4 ±0.007 0.HUVECs细胞中高脂血清诱导后p65蛋白表达明显增高,而病毒转染后p65水平降低.ELISA检测结果显示HUVEC分泌的TNF-α水平3组分别为：15.98 ±0.43、27.94±0.83、19.73±0.43.而3组IL-6水平分别为：3.94 ±0.12、6.32 ±0.21、5.08 ±0.12.高脂血清诱导后TNF-α和IL-6的分泌显著高于空白组,而rAAV9-R65干预＋高脂血清诱导后TNF-α和IL-6的浓度明显降低.Alamar blue法检测显示R65基因表达能明显减轻高脂血清刺激后HUVECs的增殖抑制.流式细胞术检测显示与空白组比较,高脂血清诱导后细胞早期凋亡比率为20.4％,晚期凋亡比率为4.7％;而rAAV9-R65转染＋高脂血清诱导组早期凋亡比率降至7.2％,晚期凋亡比率降为2.9％,证明R65表达可明显减轻高脂血清刺激后HUVECs的凋亡.结论 R65基因表达能有效减轻高脂血清刺激后HUVECs的增殖抑制,并Objective To investigate the effects of type 9 recombinant adeno-associated virus （rAAV9） mediated anti-nuclear factor-κB （NF-κB） ribozyme on the proliferation and apoptosis of high lipid serum-induced human umbilical vein endothelial cells （HUVECs） in vitro,and to provide an important support that R65 nuclear enzyme gene would be an optimal gene for gene therapy in AS.Methods HUVECs were cultured in vitro,endothelial damage model of AS was established by using high lipid serum,and the HUVECs were transfected by the rAAV9-R65 recombinant adenovirus vector.Cells were divided into three experimental groups：control group,high lipid serum induced group and high lipid serum induced ＋ rAAV9-R65 transfected group.The expression of p65 protein was detected by using Western blotting.The cell supernatant fluid was collected for determination of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） by using enzyme linked immunosorbent assay （ELISA）.Proliferation of HUVECs transfected by rAAV9-R65 was detected by using Alamar blue.The apoptosis rate was detected by using flow cytometry 96 h after transfection by rAAV9-R65.Results The cells were changed from long spindle into class circle after 24 h induced by high lipid serum.Western blotting showed that the expression of p65 in control group was 0.022 9 ± 0.002 4 ; In high lipid serum induced group was 0.224 3 ± 0.008 6 ; In rAAV-R65 transfected and high lipid serum induced group was 0.071 4 ±0.007 0.The expression of p65 in control group was increased after high lipid serum stimulating HUVECs,and decreased statistically significant in rAAV-R65 transfected and high lipid serum induced group.ELISA showed that the concentrations of TNF-α in the supernatant among three groups were：15.98 ±0.43,27.94 ± 0.83 and 19.73 ± 0.43,respectively.And the concentrations of IL-6 among three groups were：3.94 ±0.12,6.32 ± 0.21 and 5.08 ± 0.12,respectively.The levels of TNF-α and IL-6 were significantly higher than control group after 24 h indu

关 键 词：R65核酶基因 腺相关病毒 高脂血清 增殖 脱噬作用 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]