重组腺病毒气管途径反复转染大鼠肺组织人类eNOS基因的转导效果

Efficiency of transduction of recombinant adenovirus-mediated human endothelial nitric oxide synthase gene into lung tissue by repeated intratracheal transfection in rats

目的重组腺病毒气管途径反复转染大鼠肺组织人类内皮型一氧化氮合酶（eNOS）基因的转导效果。方法雄性Wistar大鼠60只，3～4月龄，体重220—280g，采用随机数字表法，将其随机分为2组：对照组（C组，n=10）和重组eNOS基因转染组（T组，n=50）。麻醉下，气管插管，行机械通气，T组气管内注射滴度为5×109PFU／ml的携带人类eNOS基因的重组腺病毒载体质粒DNA50μl，c组注射等容量病毒保存液，每隔5min重复经气管内注射一次，共注射12次。T组分别于转染后2、5、7、14、21d时取10只大鼠，采集肺动脉血样，采用硝酸还原酶法测定血清NO浓度，分别采用WesternNot法及荧光定量PCR技术检测肺组织eNOS其及RNA表达，采用免疫组化法测定气管、支气管、肺、肝、脾、肾组织eNOS表达。结果与c组比较，T组各时点肺动脉血清NO浓度、肺组织eNOS及其RNA表达升高（P〈0．05）；转染后7、14和21d时明显高于转染后2d（P〈0．05）。T组气管、支气管上皮细胞、肺泡囊内细胞和肺内小血管的内皮细胞均有eNOS表达，而c组未见eNOS表达。转染后7d时肝、脾、肾组织未见eNOS表达。结论重组腺病毒气管途径反复转染大鼠肺组织人类eNOS基因后，可实现外源基因在肺内表达，且具有正常的酶活性，无远隔器官表达。

Objective To investigate the efficiency of transduction of recombinant adenovirus-mediated human endothelial nitric oxide synthase （eNOS） into lung tissue by repeated intratracheal transfection in rats. Methods Sixty 3-4 month old male Wistar rats weighing 220-280 g were randomly divided into 2 groups： control group （group C, n -- 10） and eNOS gene transduction group （group T, n = 50）. The animals were anesthetized with intraperitoneal 10% chloral hydrate 35 mg/kg, traeheally intuhated and mechanically ventilated （VT 2.5 ml, RR 60 bpm, FiO2 1.0）. Recombinant adenovirus carrying human eNOS gene was given as gift by Professor Gerard from Texas University, Southwest Medical Center. In group T 50 μl of the recombinant adenovirus in concentration of.5 × 109 PFU/ml was instilled into trachea every 5 minutes for 12 times, while in group C equal volume of vector conservation solution was instilled instead. Pulmonary arterial blood samples were obtained at 2, 5, 7, 14 and 21 d after intratracheal transfection （ n = 10 at each time point） for determination of serum NO concentration. The ani- mals were immediately sacrificed after blood sample collection for determination of expression of eNOS protein in the lung tissue and RNA. The eNOS expression in the trachea, bronchus, lung, liver, spleen and kidney was detected by immuno-histochemistry.Results The serum NO concentrations were significantly higher at all time points in group T than in group C. The eNOS expression was detected in the epithelial cells of trachea and bronchi, and en- dothelial cells of alveoli and pulmonary blood vessels in group T but not in group C. eNOS expression was not deteeted in liver, spleen and kidney at 7 d after intratracheal transfection in group T. Conclusion Human eNOS gene mediated by recombinant adenovirus was transdueted into rat lung tissue with normal enzyme activity by re- peated intratracheal administration without being detected in distant organs.