腺相关病毒载体转染供肝方法学的实验研究

Methodology of transfecting gene into liver graft mediated by adeno-associated virus vector

目的探讨腺相关病毒载体基因转染的有效途径和方法。方法我们设计肝动脉、门静脉、双重灌注3种途径和传统、循环、夹闭3种转染方法。通过观察肝脏灌注后颜色、检测肝脏功能和肝细胞转染率，确定灌注途径和转染方法，并探讨其安全性。结果肝脏灌注后颜色无明显差别，无肿胀或花斑出现，血流开放以后肝脏颜色迅速恢复正常，3种灌注途径ALT比较差异无统计学意义（F=0．343，1．265，0．055，P〉0．05）;1周后肝脏组织均有免疫荧光染色，并且携带增强型绿色荧光蛋白基因的腺相关病毒载体，转染率比较差异无统计学意义（F=0．080，0．091，0．045，P〉0．05）。传统法的转染率略低于循环法，而夹闭法的转染率在各时间段均高于前两种方法，差异有统计学意义（F=3．880，2．976，5．129，P〈0．05）。3种方法转染率随时间逐渐升高，6周左右达到高峰，以后缓慢下降。结论经肝动脉灌注是基因转染的有效途径，夹闭法可以提高目的基因的转染率，两者均无肝脏功能损害，腺相关病毒载体的转染呈缓慢、持续的过程。

Objective To investigate the effective route and proper method in transfecting gene into liver graft mediated by adeno-associated virus vector. Methods Three routes including hepatic artery, portal vein and hepatic artery ＋ portal vein, and 3 methods, i.e. routine, circulation and clamping were employed for infusion. The best infusion route and method of gene transfection into liver graft were determined by observing the color change of liver and detecting liver function and transfeetion rate of liver cells. The safety of these methods was evaluated. Results In all the infusion procedures, the color of the liver grafts turned from red to white, no apparent color difference of the livers and no enlargement nor mottling were observed under surgical microscope. The liver color was back to normal immediately after blood flow was restored. No significantly statistical differences of the ALT values were observed among all the groups （ F =0.343, 1. 265, 0. 055, P 〉 0.05 ）. Adeno-assoeiated virus vectors coding for the enhanced green fluorescence protein （AAV2-EGFP） were successfully transfeeted into liver cells by the 3 infusion routes 1 week later, and the differences of transfection rates via the 3 routes had no statistical significance （ F =0.080, 0. 091, 0.045, P 〉 0.05）. The transfection rate of AAV2-EGFP was the highest at any time points when using the clamping method, and then followed by circulation method and routine method, with statistical difference （ F = 3. 880, 2. 976, 5. 129, P 〈 0. 05 ）. The transfeetion rates of AAV2-EGFP were increased progressively and peaked at the 6th week, and then they were decreased gradually. Conclusions Infusion via hepatic artery is the effective route for gene transfection and clamping the vessels can elevate the transfection rate of AAV2-EGFP. All procedures were performed without detectable liver injury. The transfection of gene into liver graft mediated by adeno-assoeiated virus vector is a slow and persistent process.