携带一氧化氮合酶反义基因重组腺相关病毒载体抑制大鼠脑胶质瘤生长增殖的研究

Inhibition of rat brain glioma proliferation resulting from recombinant adeno-associated virus with nitricoxide synthaser of antisense gene

目的 建立大鼠脑胶质瘤模型,诱导型一氧化氮合酶反义RNA重组腺相关病毒载体（rAAV-AsiNOS）在体内抑制大鼠脑C6胶质瘤生长增殖以及对诱导型一氧化氮合酶（iNOS）和血管内皮生长因子（VEGF）基因表达的影响.方法 采用立体定向技术将C6胶质瘤细胞接种于SD大鼠的右侧尾状核区,建立大鼠脑胶质瘤模型,造模7d后根据注入肿瘤内物质不同分为对照组（生理盐水）、rAAV-LacZ组（重组腺相关病毒载体rAAV-LacZ）和rAAV-AsiNOS组,设立空白组（只进行类似立体定向开颅手术但未接种C6细胞）.各组于造模前（第1天）、造模后7d（第8天,转染前）和转染7d（第15天）分别进行神经功能缺失评分;于转染前和转染7d接受MR平扫及增强检查;于转染7d测定大鼠脑胶质瘤重量和体积,半定量逆转录-聚合酶链反应（RT-PCR）分析大鼠脑胶质瘤中iNOS和VEGF mRNA表达水平.结果 对照组和rAAV-LacZ组转染后神经功能缺失均加重,rAAV-iNOS组转染后神经功能缺失改善.转染后7d MR结果示对照组和rAAV-LacZ组脑肿瘤大于转染前,rAAV-iNOS组病灶缩小,部分坏死.RT-PCR结果示：对照组、rAAV-LacZ组及rAAV-iNOS组中iNOS mRNA表达分别为：0.67 ±0.04、0.72±0.07、0.21±0.02;对照组、rAAV-LacZ组及rAAV-iNOS组中VEGF mRNA表达分别为：0.71±0.08、0.76±0.07、0.29±0.02; iNOS和VEGF mRNA在rAAV-iNOS组表达均比对照组和rAAV-LacZ组明显下降,差异均有统计学意义（P＜0.05）.结论 携带iNOS反义RNA腺相关病毒载体rAAV-iNOS,能够抑制大鼠脑胶质瘤中iNOS表达,调控VEGF合成,减少胶质瘤血管形成,发挥体内抗胶质瘤机制.

Objective To establish the rat brain glioma model and explore the proliferation of brain glioma and the expression of inducible nitric oxide synthase （iNOS） and vascular endothelial growth factor （VEGF） gene influenced by the recombinant adeno-associated virus （rAAV-AsiNOS） in vivo.Methods C6 cells were injected into the right caudate nucleus areas of SD rats by stereotactic technology.After 7 days,rats were divided into 4 groups：control,rAAV-LacZ,rAAV-AsiNOS and blank.The neurological deficit score of all rats was separately acquired before the establishment of rat model,7 days after the establishment of rat model and 7 days after transfection.Seven days after transfection,the weight and volume of brain glioma were calculated by plain and enhanced MR scan.The expression of iNOS and VEGF was detected by using semi-quantified analysis of reverse transcriptase-polymerase chain reaction （RT-PCR）.Resuits The neurological deficit score in rAAV-iNOS group was increased,but that in control and rAAVLacZ groups was reduced.MR images showed that brain tumors in control and rAAV-LacZ groups at 7th day after transfection were larger than before transfection,but the brain tumors in rAAV-iNOS group were obviously reduced.RT-PCR showed that mean expression levels of iNOS mRNA in control,rAAV-LacZ and rAAV-iNOS groups were separately 0.67 ±0.04,0.72 ±0.07 and 0.21 ±0.02,and mean expression levels of VEGF mRNA in control,rAAV-LacZ and rAAV-iNOS groups were separately 0.71 ± 0.08,0.76 ± 0.07 and 0.29 ± 0.02.The expression of iNOS and VEGF mRNA in rAAV-iNOS group was lower than that in control and rAAV-LacZ groups （P ＜0.05）.Conclusion rAAV-iNOS can inhibit the expression of iNOS,regulate the synthesis of VEGF,reduce glioma angiogenesis and perform the mechanism of anti-glioma in vivo.