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1、腺相关病毒载体转染供肝方法学的实验研究作者：邰升苏志雷王志兵方泰石商文刚吴德全韩德恩【摘要】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
2、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。【关键词】腺相关病毒载体；转染【Abstract】ObjectiveToinvesti
3、gatetheeffectiverouteandpropermethodintransfectinggeneintolivergraftmediatedbyadeno-associatedvirusvector.MethodsThreeroutesincludinghepaticartery,portalveinandhepaticartery+portalvein,and3methods,i.e.routine,circulationandclampingwereemployedforinfusion.Thebestinfusio
4、nrouteandmethodofgenetransfectionintolivergraftweredeterminedbyobservingthecolorchangeofliveranddetectingliverfunctionandtransfectionrateoflivercells.Thesafetyofthesemethodswasevaluated.ResultsInalltheinfusionprocedures,thecolorofthelivergraftsturnedfromredtowhite,noap
5、parentcolordifferenceoftheliversandnoenlargementnormottlingwereobservedundersurgicalmicroscope.Thelivercolorwasbacktonormalimmediatelyafterbloodflowwasrestored.NosignificantlystatisticaldifferencesoftheALTvalueswereobservedamongallthegroups（F=0.343,1.265,0.055,P>0.0
6、5）.Adeno-associatedvirusvectorscodingfortheenhancedgreenfluorescenceprotein（AAV2-EGFP）weresuccessfullytransfectedintolivercellsbythe3infusionroutes1weeklater,andthedifferencesoftransfectionratesviathe3routeshadnostatisticalsignificance（F=0.080,0.091,0.045,P>0.05）.Th
7、etransfectionrateofAAV2-EGFPwasthehighestatanytimepointswhenusingtheclampingmethod,andthenfollowedby5circulationmethodandroutinemethod,withstatisticaldifference（F=3.880,2.976,5.129,P<0.05）.ThetransfectionratesofAAV2-EGFPwereincreasedprogressivelyandpeakedatthe6thwee
8、k,andthentheyweredecreasedgradually.ConclusionsInfusionviahepaticarteryistheeffectiverouteforgenetransfectionandclamp
