双球囊导管法基因导入血管的实验研究

　　【摘要】　目的　探讨双球囊导管法腺病毒介导性基因导入犬股动脉的效率及时间曲线。方法　选成年杂交犬20只，经股动脉分支逆行插入7F双球囊导管至髂动脉，采用气囊法剥脱血管内皮后，充盈双球囊，经球囊间侧孔注入0.3 mL约含3.0×109　pfu的含有细菌lacZ基因的重组腺病毒入间腔，培养30分钟。术后1、3、7、14、21、28及35天处死动物，组织化学分析法检测lacZ基因在股动脉的表达。结果　导入基因后24小时即见lacZ基因在血管内膜表达，7天达高峰，持续4周左右。高峰时，培养间腔血管内侧近100%表达了外源基因，深度达血管中膜2/3。结论　双球囊导管法腺病毒介导性基因导入犬股动脉呈高效及一过性特点。

Experimental study on gene transfer into vessel using double-balloon catheter

Li Jianjun, Huang Congxin, Li Gengshan

　　（Department of Cardiology, First Affiliated Hospital, Hubei Medical University, Wuhan 430060）

　　【Abstract】　Objective　To evaluate the efficiency and time curve of gene expression following adenovirus-mediated gene transfer into canine injured femoral arteries through double-balloon catheter. Methods　Femoral arteries were first subjected to the balloon injury and 3.0×109 pfu of adenovirus expressing bacterial lacZ gene was filled into a closed space created by inflated double-balloon catheter, subsequently incubated for 30 min. The vessel were harvested at 1,3,7,14,21,28 and 35 days after gene transfer.Results　The lacZ expression was found in 100% of arterial inner surface and in two-third of arterial media, which detected at 1 day, peaked at 7 days and lasted up to 28 days.Conclusion　This study indicated that adenovirus-mediated gene transfer into arteries was transient but high-efficient.

　　【Key words】　Adenovirus Gene transfer Gene therapy Vessel

　　心血管疾病的基因治疗引人注目，其实验研究报道已迭见文献［1～3］，但高效率地导基因入血管之研究报道尚少。本文选用含细菌lacZ基因的重组腺病毒载体，经双球囊导管法导入犬球囊剥脱内皮后股动脉，获得了十分满意的效果，现报道如下。

　　材料与方法

　　1.重组腺病毒的制作　重组腺病毒的制作方法详见文献［2，3］。简言之，将细菌lacZ基因插入粘性末端质粒载体的SRa启动子与SV40多聚腺苷酸［poly(A)］序列之间。其质粒载体中含有整个人类5型腺病毒的基因组，但已预先去掉其E1a、E1b和E3区使之形成复制缺陷。含有目的基因lacZ的重组腺病毒命名为Adex1SR-lacZ且经293细胞培养同源重组获得，并经常规酶消化法和测定受其感染后细胞中β-半乳糖苷酶的活性而确认。确认后的病毒株经293细胞扩增，尔后经氯化铯密度梯度离心法回收与纯化。病毒滴度采用293细胞受其感染后斑块形成单位(pfu: plaque formation unit)计算。经该法提取，通常可获得1×1010pfu的高滴度病毒。与此同时，制作不含任何外源基因但结构上与重组腺病毒完全相同的腺病毒命名为Adex1w作为对照。

　　2.活体基因导入犬股动脉　成年杂交犬20只，雌雄不拘，体重13～27.5千克。经静脉注

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