心包穿刺简易装置在基因转染中的应用

Application of Gene Transfer by a Simple Intrapericardial Puncture Device

目的探讨自制心包穿刺装置转染心脏的安全性、可行性。方法应用磷酸钙沉淀方法制备携带大肠杆菌LacZ基因复制缺陷的重组腺病毒(Ad-LacZ),将12头中国小型猪分为实验组和对照组,采用球囊堵塞前降支第一对角支远端,心肌梗死模型建立后即刻,采用自制简易心包腔穿刺装置经皮剑突下穿刺,成功后置中心静脉导管于心包腔内并行转染,28 d后处死。实验组:胶原酶1200 U及透明质酸酶3000 U预处理心包后,在心包腔内注射Ad-LacZ基因2.0×109p.f.u;对照组:同样方法预处理心包后,在心包腔内注射生理盐水1 mL。于注射后3、7及28 d分别对缺血心肌进行染色及病理观察。结果冠状动脉造影证实前降支远端完全闭塞,病理显示心肌有缺血和梗死;实验组注射Ad-LacZ基因后第3、第7天及28d后X-gal染色有阳性细胞,以第7天最明显,对照组无阳性细胞。结论自制的心包腔简易穿刺装置将腺病毒载体转染至缺血心肌是安全的,可行的,并且腺病毒可持续表达4周。

Objective To explore the feasibility and safety of gene transfer via the pericardial cavity by a simple puncture device, made by ourself. Methods Replication-deficient recombinant adenoviral vector carrying LacZ reporter genes （Ad-LacZ） was constructed by calcium phosphate precipitation method. Twelve healthy pigs were randomly divided into two groups： experimental group （n = 6） and control group （n= 6）. AMI model was constructed by balloon occlusion of the distal part of D1 branch of LAD at the same time the intra-pericardial cavity injections were performed through a small incision of the abdominal wall below the xyphoid appendix using a device made by ourself. After succeeding, gene transfer was performed by central venous catheter. In both the experimental and control groups, the pericardium was pre-treated by injection of a mixture of collagenase and hyaluronidase. Then 2.0 × 10^9 p.f. u Ad-LacZ was injected into the pericardium. The histological changes and beta-galactosidase activity of the isehemic myocardium were assessed at the 3rd, 7th and 28th day after injection. Results The LAD was occluded completely and infarction and isehemia were detected by histological examination. In the 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 the control group, no positive cells were observed. Conclusion Adenovirus vector can be transfered into isehemic myocardium and succeed in expressing target gene in the model for 4 weeks via pericardial cavity pre-treated by injecting a mixture of collagenase and hyaluronidase by a simple puncture device made by ourself.