复制缺陷的重组腺病毒载体直接转染猪慢性缺血心肌的研究

Direct in Vivo Gene Transfer Into Porcine Chronic Ischemic Myocardium Using Replication Deficient Recombination Adenovirus Vector

目的 建立猪慢性心肌缺血模型 ,评价腺病毒载体的转染效率和持续时间。 方法 应用磷酸钙沉淀法制备携带大肠杆菌 L ac Z基因复制缺陷的重组腺病毒 ( Ad.L ac Z) ,将健康家猪 8条随机分为实验组和对照组 ,每组 4条。两组猪均经左前外侧开胸 ,于冠状动脉左回旋支 ( L CX)放置 Ameroid环 ,2 8天后二次开胸 ,实验组 :在缺血心肌部位每点直接注射 Ad.L ac Z 10 0μl,10 1 0 噬斑形成单位 ,共 10点 ;对照组 :在缺血心肌部位每点注射磷酸盐缓冲液 ( PBS) 10 0μl,共10点。于注射后 3天、7天和 2 8天对缺血心肌进行染色和病理观察。 结果 冠状动脉造影证实 L CX完全闭塞 ,心肌有缺血和小面积心肌梗死 ;实验组注射 Ad.L ac Z后第 3天、7天和 2 8天 X- gal染色有阳性细胞 ,以 7天时明显 ,对照组无阳性细胞。 结论 应用 Ameroid环可成功建立猪慢性心肌缺血模型 ,腺病毒载体转染缺血心肌基因表达可持续 4周。

Objective To establish a model of chronic myocardial ischemia with Ameroid constrictor and evaluate the expression efficacy and time of adenovirus vector. Methods Replication deficient recombinant adenoviral vector carrying LacZ reporter genes (Ad.LacZ) was constructed by the calcium phosphate method. Eight health swine were divided into two groups, experimental group( n =4) and control group ( n =4). Four weeks after Ameroid constritor placing around left circumflex (LCX) artery, the left chest was reopened for myocardial administration. In experimental group, 100μl of Ad.LacZ(10 10 pfu) was injected directly into ten separate sites in the ischemic myocardium, and with phosphate buffered saline (PBS) in control group . The histological changes and beta galactosidase activity were observed at the 3rd, 7th, 28th day after injection. Results The LCX was occluded completely and minimal infarction and ischemia were detected by histologic assessment.In experimental group ,X gal staining positive cells were detected at the 3rd day after injection, increased markedly at the 7th day, and then declined at the 28th day. In control group, no positive cells were observed. Conclusion Direct intramyocardial injection of adenovirus vector can succeed in expressing target gene in the model of chronic ischemic myocardium for 4 weeks.