CAJ | 학술논문

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.
목적 탐토선상관병독재체기인전염적유효도경화방법.방법 아문설계간동맥、문정맥、쌍중관주3충도경화전통、순배、협폐3충전염방법.통과관찰간장관주후안색、검측간장공능화간세포전염솔,학정관주도경화전염방법,병탐토기안전성.결과 간장관주후안색무명현차별,무종창혹화반출현,혈류개방이후간장안색신속회복정상,3충관주도경ALT비교차이무통계학의의(F=0.343,1.265,0.055,P＞0.05);1주후간장조직균유면역형광염색,병차휴대증강형록색형광단백기인적선상관병독재체,전염솔비교차이무통계학의의(F=0.080,0.091,0.045,P＞0.05).전통법적전염솔략저우순배법,이협폐법적전염솔재각시간단균고우전량충방법,차이유통계학의의(F=3.880,2.976,5.129,P＜0.05).3충방법전염솔수시간축점승고,6주좌우체도고봉,이후완만하강.결론 경간동맥관주시기인전염적유효도경,협폐법가이제고목적기인적전염솔,량자균무간장공능손해,선상관병독재체적전염정완만、지속적과정.
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 transfoetion 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 differenee 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-associated virus vectors coding for the enhanced green fluorescence protein(AAV2-EGFP)were successfully transfected into liver cells by the 3 infusion routes 1 week later,and the difierences of transfection rates via the 3 routes had no statistical significance(F=0.080,0.091,0.045,P＞0.05).The transfoction 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 differenee(F=3.880,2.976,5.129,P＜0.05).The transfection 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-associated virus vector is a slow and persistent process.