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为了深入研究诱导型一氧化氮合酶（ｉｎｄｕｃｉｂｌｅｎｉｔｒｉｃｏｘｉｄｅｓｙｎｔｈａｓｅ，ｉＮＯＳ）基因表达产物在阿片耐受和依赖中作用，采用脂质体介导基因转染技术，将ｉＮＯＳｃＤＮＡ重组逆转录病毒载体导入ＮＧ１０８１５神经细胞，获得Ｇ４１８抗性克隆，命名为ＮＧＬＮＣＸｉＮＯＳ细胞。ＤＮＡ印迹杂交、ＰＣＲ扩增及ＲＴＰＣＲ和蛋白质免疫印迹杂交分析，证实ＮＧＬＮＣＸｉＮＯＳ细胞有外源ｉＮＯＳ基因整合、转录和表达；ＮＡＤＰＨ黄递酶（ＮＡＤＰＨｄｉａｐｈｏｒａｓｅ，ＮＡＤＰＨｄ）组化和免疫组化染色表明，胞质中有ｉＮＯＳ基因功能表达；酶催化活性和ＮＯ－２含量均明显升高；重组酶具有天然酶生化和药理特性，即ＮＡＤＰＨ依赖性，Ｃａ２＋非依赖性和Ｃａ２＋螯合剂抗性以及ＮＯＳ抑制剂使酶活性降低，呈剂量相关性；ｉＮＯＳ基因表达使ＮＯｃＧＭＰ系统上调。成功地建立ｉＮＯＳ基因修饰神经细胞株，为寻找治疗阿片耐受和依赖的新药创造了条件。
In order to further study the effect of inducible nitric oxide synthase (iNOS) gene expression products on the tolerance and dependence of opioid, liposome-mediated gene transfection technique was used to transduce iNOS cDNA recombinant retroviral vector into NG108-15 nerve Cells were obtained G418 resistant clones, named NG NGXiNOS cells. DNA blot hybridization, PCR amplification and RT PCR and Western blot hybridization analysis confirmed NG LNCXiNOS cells have exogenous iNOS gene integration, transcription and expression; NADPH diaphorase (NADPHdiaphorase, NADPH d) and immunohistochemistry The staining of iNOS gene in the cytoplasm showed that the expression of iNOS gene was enhanced in the cytoplasm. The activity of enzyme and the content of NO-2 in the cytoplasm were significantly increased. The recombinant enzyme had biochemical and pharmacological properties of natural enzyme, namely, NADPH-dependent, Ca 2+ -dependent and Ca 2+ chelator anti- Sex and NOS inhibitors decreased the enzyme activity in a dose-dependent manner; iNOS gene expression up-regulated NO-cGMP system. The successful establishment of iNOS genetically modified neural cell lines has created the conditions for finding new drugs for the treatment of opiate tolerance and dependence.