基础医学与临床 ›› 2008, Vol. 28 ›› Issue (6): 588-594.

• 研究论文 • 上一篇    下一篇

人内皮型一氧化氮合酶基因在小鼠肺内的表达

赵祝香 李冰 冉丕鑫   

  1. 广州医学院附属市一人民医院 广州医学院附属一院广州呼吸疾病研究所 广州呼吸疾病研究所
  • 收稿日期:2006-09-12 修回日期:2007-08-22 出版日期:2008-06-25 发布日期:2008-06-25
  • 通讯作者: 赵祝香

Expression of Recombinant Human Endothelial Nitric Oxide Synthase gene in Lung tissue of mouse

Zhu-xiang ZHAO, Bing LI, Pi-xin RAN   

  1. the First Affiliated People's hospital of Guangzhou municikpality,Guangzhou Mediacl College
  • Received:2006-09-12 Revised:2007-08-22 Online:2008-06-25 Published:2008-06-25
  • Contact: Zhu-xiang ZHAO,

摘要: 目的 观察heNOS重组腺病毒感染小鼠肺组织后HENOS基因在小鼠肺组织内的表达。方法 采用反转录PCR(RT-PCR)法克隆HENOS基因,利用体外连接法构建复制缺陷型heNOS重组腺病毒表达载体;免疫组织化学分析heNOS在小鼠肺组织内的表达情况。结果 (1)序列分析表明,克隆所得HENOS基因片段含有完整开放阅读框架,与GenBank中HENOS cDNA序列(*163729)同源性达99.93%。(2)该基因在转染细胞中能表达具有生物活性功能的NOS蛋白。(3)体外连接法成功构建出复制缺陷型heNOS重组腺病毒转移载体。(4)滴入heNOS重组腺病毒后,小鼠肺血管内皮细胞、平滑肌细胞、细支气管上皮细胞内HENOS阳性表达明显增加。结论 本实验成功构建的复制缺陷型heNOS重组腺病毒转移载体,能经呼吸道转移至肺组织,并在小鼠肺血管、支气管和肺泡上皮细胞表达所需的目的基因HENOS。

Abstract: Objective:To detect whether HENOS we cloned could be expressed in mouse lung after intratracheal administration of recombinant adenovirus vector. Methods:HENOS cDNA were obtained by RT-PCR from total RNA which extracted from human HUVEC. The replication-deficient heNOS recombinant adenovirus vector was constructed with a ligation method in vitro. The high titer of recombinant adenovirus was obtained by chromatographic methods. The expression of HENOS protein was determined by immunohistochemistry staining after intratracheal administration of recombinant adenovirus. Results:Sequence confirmed the cloned cDNA containing the whole ORF and the HENOS cDNA was about 3731bp in size.It showed 99.93% identity with that of HENOS cDNA in GenBank(*163729).The biological activity of HENOS protein was examined in transfected cos7 cell line with HENOS cDNA in eukaryotic expression vector of pcDNA3.0. The replication-deficient recombinant adenovirus vector containing HENOS cDNA was constructed successfully and the purified viral titer was 2.0×1010pfu/ml. After intratracheal administration of the recombinant adenovirus, HENOS expression was detected in the majority of bronchial epithelium, alveolar lining cells, endothelial cells and smooth muscle cells of pulmonary vessels. In control, little endogenous eNOS immunoreactivity was detected in pulmonary vessels and it was no eNOS immunoreactivity in bronchial and alveolar epithelial cells. Conclusion:The replication-deficient recombinant human endothelial nitric oxide synthase mediated by adenovirus vector constructed could be delivered into the lung tissue of mouse by intratracheal administration of recombinant adenovirus and can be expressed in lung tissue in high-efficiency.