NOS反义RNA重组腺相关病毒载体体内提高脑细胞耐缺血能力的实验研究

Recombinant AAV mediated transfection of me-AsnNOS or AsiNOS into rat brain resists against ischemic neuronal injury and inhibits neuronal apoptosis

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）提高脑细胞耐受缺血能力的作用机制。方法应用立体定位技术将预处理好的病毒载体转染至将要梗死侧的基底节区，转染病毒滴度为2×10^9mL～，并将SD大鼠分为4组：即rAAV-AsnNOS组，rAAV-AsiNOS组，rAAV—LacZ组和对照组；处理后运用MACO建立缺血模型，每组分为缺血早期和缺血晚期，流式细胞术（FCM）检测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。结果一定剂量的重组病毒载体转染到大鼠海马区域，无神经损伤症状；转染rAAV-AsnNOS病毒载体的脑神经细胞在缺血早期（缺血1～6h），NT阳性细胞百分比、细胞凋亡率以及nNOS、p38MAPK和Caspase-3 mRNA表达量均较对照组、rAAV-LacZ组和rAAV—AsiNOS组降低；转染rAAV—AsiNOS病毒载体的脑神经细胞在缺血晚期（缺血24—72h），NT阳性细胞百分比、细胞凋亡率以及nNOS、p38MAPK和Caspase-3 mRNA表达量均较对照组、rAAV-LacZ组和rAAV-AsnNOS组降低，差异有统计学意义。结论转染重组病毒载体后动物模型脑神经细胞能够耐受缺血损伤，转染rAAV-AsnNOS病毒载体的脑神经细胞能够在缺血早期抑制nNOS、p38MAPK和Caspase-3的表达，转染rAAV—AsiNOS病毒载体的脑神经细胞能够在缺血晚期抑制iNOS、038MAPK和Caspase-3的表达，从而在缺血后抑制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 Recombinant AAV （ rAAV）-AsnNOS or rAAV-AsiNOS transfection in vitro. Methods Recombi- nantAAV viral particles containing either AsnNOS or AsiNOS were transfected into the right basal ganglia areas of SD rats using stereotactic technology with infectious titer of 2 × 10^9/ml. Rats were divided into 4 groups： rAAV-AsnNOS, rAAV-AsiNOS, rAAV-LacZ, and control groups. FCM was applied to analyze the percentages of NT positive as well as the apoptotic cells. Semiquantitiative RT-PCR was conducted to determine the expression of nNOS, iNOS, p38 MAPK and Caspase-3 mRNA. Results There was no neuronal injury in the rats transfected with rAAV vectors. The percentages of NT positive neurons and apoptotic ceils of rAAV-AsnNOS group were lower than those of controls. Similarly, the percentages of NT positive neurons and apoptotic ceils of rAAV-AsiNOS group were lower than those of controls. RT-PCR showed that mRNA expressions of nNOS, iNOS, P38MAPK and Caspase-3 were significantly decreased in the rAAV-As nNOS group after 1 -6h of ischemia and in the rAAV-As iNOS group after 24 - 72 h. Conclusions Recombinant AAV mediated transfection of meAsnNOS or AsiNOS into rat brain can resist against ischemic injury, rAAV-AsnNOS and rAAV-AsiNOS inhibit expressions of nNOS, p＇38MAPK and Caspase-3 at the early and late stage of ischemia, respectively.