一氧化氮合酶反义RNA重组腺相关病毒载体体外提高PC12细胞耐缺氧的能力  

Transfection of rAAV-AsnNOS or rAAV-AsiNOS in vitro reinforcing tolerance of PC12 cells to hypoxic injury and inhibiting neuronal apoptosis

作　　者：杨卫忠[1] 陈春美[1] 王春华[1] 石松生[1] 黄勇[1] 宋施委[1] 王锐[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》2007年第6期731-733,共3页Chinese Journal of Experimental Surgery

基　　金：福建省教育厅科技计划项目(JA04200)

摘　　要：目的探讨2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(iN- OS)反义RNA重组腺相关病毒载体(rAAV-/mnNOS和rAAV-AsiNOS)提高PC12细胞耐氧能力和抑制PC12细胞凋亡的作用机制。方法将PC12细胞培养基换成分别含有rAAV-AsnNOS、rAAV-Asi- NOS和rAAV-LacZ(携带β-半乳糖苷酶的结构基因重组腺相关病毒载体)的10%胎牛血清DMEM,设立对照组,测定不同rAAV转染后PC12细胞在不同缺氧时间下细胞生长趋势和乳酸脱氢酶(LDH)活性改变,流式细胞仪(FCM)测定细胞凋亡率,FCM和半定量RT-PCR分别分析nNOS、iN- OS、p38MAPK和Caspase-3阳性细胞百分比和mRNA的表达。结果一定感染复数的rAAV对PC12细胞生长趋势无明显影响;转染rAAV-AsnNOS的PC12细胞在缺氧1～6 h时LDH活性、细胞凋亡率以及nNOS、p38MAPK和Caspase-3阳性细胞百分比和mRNA表达量均较对照组、rAAV-LacZ组和rAAV-AsiNOS组均降低;转染rAAV-AsiNOS的PC12细胞在缺氧6～24h时LDH活性、细胞凋亡率以及iNOS、p38MAPK和Caspase-3阳性细胞百分比和mRNA表达量也均较对照组、rAAV-LacZ组和rAAV-AsnNOS组降低。结论在体外神经细胞缺氧模型中,转染后PC12细胞能够耐受缺氧损伤;转染rAAV-AsnNOS病毒载体的PC12细胞能够在缺氧早期抑制nNOS、p38MAPK和Caspase-3的表达,转染rAAV-AsiNOS病毒载体的PC12细胞能够在缺氧晚期抑制iNOS、p38MAPK和Caspase- 3的表达。Objective To explore the mechanisms of rAAV-AsnNOS or rAAV-AsiNOS transfection in vitro reinforcing the tolerance of PC12 ceils to hypoxic injury and inhibiting neuronal apoptosis. Methods PC12 ceils were divided into 4 groups：rAAV-AsnNOS,rAAV-AsiNOS,rAAV-LacZ and blank control groups. Ceils to be infected were incubated in DMEM medium supplemented with 10% FBS and infectious rAAV was added at a multiplicity of infection （MOI） of 100. The influence of rAAV vectors on PC12 ceil growth was determined by cytometry. LDH activity was measured respectively after different hypoxic periods. FCM was used to determine the apoptosis rate. Both the percentage of positive ceils and mRNA expression of nNOS, iNOS, p38 MAPK and Caspase-3 mRNA was determine by FCM and RT-PCR. Results rAAV vectors had no distinct effect on PC12 ceil growth and there was no statistically significant difference （ P 〉 0. 05 ） in comparison to controls. LDH activity, apoptosis rate ,positive ceil percentage and mRNA expression of nNOS,P38MAPK and Caspase-3 were decreased in the PC12 ceils of rAAV-As nNOS group as compared with rAAV-AsiNOS group,rAAV-LacZ group and controls at the early stage （1-6 h）. LDH activity, apoptosis rate, positive ceil percentage and mRNA expression of iNOS, P38MAPK and Caspase-3 were decreased in the PC12 ceils of rAAV-AsiNOS group as compared with rAAV-AsnNOS group,rAAV-LacZ group and controls at the early stage （6-24 h）. Conclusion In the in vitro hypoxia models,transfected PC12 neurons could tolerate the hypoxic insult. PC12 ceils transfected with rAAV-As nNOS displayed early inhibition of nNOS,p38MAPK and Caspase-3 while those transfected with rAAV-As iNOS presented late suppression of iNOS,p38MAPK and Caspase-3.

关 键 词：缺氧 一氧化氮合酶 脱噬作用 重组腺相关病毒载体 

分 类 号：R651[医药卫生—外科学]