一氧化氮合酶反义RNA重组腺相关病毒载体体内抑制脑缺血神经细胞凋亡的研究  

To explore the mechanisms of resistance to ischemic injury of neurons and inhibition for neuronal apoptosis after rAAV-AsnNOS or rAAV-AsiNOS transfection in vitro

作　　者：石松生[1] 杨卫忠[1] 陈春美[1] 王春华[1] 易海波[1] 陈晓斌[1] 蔡冬生[1] 杨意堃[1] 

机构地区：[1]福建医科大学附属协和医院神经外科 福建省神经外科研究所,福州350001

出　　处：《中华实验外科杂志》2008年第4期471-473,共3页Chinese Journal of Experimental Surgery

基　　金：福建省教育厅科技计划资助项目（JA04200）

摘　　要：目的探讨二种分别携带神经元型一氧化氮合酶（nNOS）、诱导型一氧化氮合酶（iNOS）反义RNA重组腺相关病毒载体（rAAV-AsnNOS和rAAV-AsiNOS）在脑缺血时抑制神经细胞凋亡的作用机制。方法运用MACO法建立脑缺血模型，闭塞1h后分别经右侧颈内动脉缓慢注入重组病毒载体（rAAV-AsnNOS、rAAV-AsiNOS和rAAV-LaeZ），继续闭塞大脑中动脉，每只转染病毒滴度为1×10^10个病毒颗粒／ml，并于分别缺血早期（缺血5h）和缺血晚期（缺血25h）时处死模型，流式细胞术（FCM）检测硝基酪氨酸（NT）阳性细胞百分比和细胞凋亡率，逆转录反应系统（RT-PCR）分析nNOS、iNOS，p38MAPK，Caspase-3 mRNA的表达。结果在脑缺血早期，rAAV-AsnNOS组的NT阳性百分比、凋亡率以及nNOS、p38MAPK、Caspase-3 mRNA表达量较对照组、rAAV-LacZ组和rAAV-AsiNOS组的低均降低，差异有统计学意义；在脑缺血晚期，rAAV-AsiNOS组的NT阳性百分率、凋亡率以及nNOS、p38MAPK、Caspase-3 mRNA表达量较对照组、rAAV-LacZ组和rAAV-AsiNOS组低，差异有统计学意义。结论在体内缺血动物模型中，rAAV-AsnNOS和rAAV-AsiNOS重组病毒载体能够分别在缺血早期和晚期通过抑制NOS表达，从而抑制p38MAPK和Caspase-3基因的表达，抑制缺血后神经细胞凋亡的发生。Objective To explore the mechanisms of resistance to ischemic injury of neurons and inhibition for neuronal apoptosis after rAAV-AsNOS transfecfion in vivo. Methods Cerebral ischemia was induced by MCAO for 60 minutes followed by slow dextrocarotid injection of vector solutions （ rAAV- AsnNOS ,rAAV-AsiNOS and rAAV-laeZ） with infectious titer of 1 × 10^10/ml. FCM was used to determine the percentage of NT positive cells and the apoptosis rate. The expression of nNOS,iNOS, p38 MAPK and Caspase-3 mRNA was semi-quantified by RT-PCR. Results At the early phase of focal ischemia,percentage of NT positive calls, apoptosis rate and mRNA expression of iNOS, iNOS, p38 MAPK and Caspase-3 in the rAAV-AsnNOS group were the lowest among 4 groups with statistical significance. At the late phase, percentage of NT positive cells, apoptosis rate and mRNA expression of nNOS, iNOS, p38MAPK and Caspase-3 in the rAAV-AsiNOS group were also the lowest with statistical significance. Conclusion In vivo ischemia models, rAAV vectors transfected neurons could resist the following ischemic injury, rAAV- AsnNOS and rAAV-AsiNOS could inhibit the NOS-induced neurotoxicity respectively in the early and late phase after cerebral ischemia onset.

关 键 词：脑缺血 一氧化氮合酶 脱噬作用 重组腺相关病毒载体 反义寡核苷酸类 

分 类 号：R74[医药卫生—神经病学与精神病学]