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 |
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引用本文: | 石松生,杨卫忠,陈春美,王春华,易海波,陈晓斌,蔡冬生,杨意堃. 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[J]. 中华实验外科杂志, 2008, 25(4) |
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作者姓名: | 石松生 杨卫忠 陈春美 王春华 易海波 陈晓斌 蔡冬生 杨意堃 |
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作者单位: | 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 |
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基金项目: | 福建省教育厅资助项目 |
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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1 h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-LacZ),继续闭塞大脑中动脉,每只转染病毒滴度为1×1010个病毒颗粒/ml,并于分别缺血早期(缺血5 h)和缺血晚期(缺血25 h)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(RTPCR)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-LaeZ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.
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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 |
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 |
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Abstract: | Objective To explore the mechanisms of resistance to ischemic injury of neurons and inhibition for neuronal apoptosis after rAAV-AsNOS transfection in vivo.Methods Cerebral ischemia was induced by MCAO for 60 minutes foilowed by slow dextrocarotid injection of vector solutions (rAAV-AsnNOS,rAAV-AsiNOS and rAAV-lacg)with infectious titer of 1×1010/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 cels,apoptosis rate and mRNA expression of iNOS,iNOS,p38MAPK 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 vlvo ischemia models,rAAV vectors transfected neurons could resist the following ischemic iniury,rAAV-AsnNOS and rAAV-AsiNOS could inhibit the NOS-induced neurotoxicity respectively in the early and late phase after cerebral ischemia onset. |
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Keywords: | Ischemia of neurons NOS Apoptotis rAAV Antiserse oligonucleoticles |
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