摘要目的 构建提纯重组腺相关病毒介导的血红素加氧酶1基因(HO-1)和绿色荧光蛋白基因(GFP),并探讨其在肝移植大鼠肝脏中的表达情况.方法 克隆大鼠HO-1,构建重组腺相关病毒-HO-1(rAAV-HO-1)载体,酶切鉴定并进行测序,氯化钙共沉淀法与辅助质粒Virus helper、AAV-cap-rep转染包装细胞,应用肝素层析柱法纯化浓缩病毒,实时荧光定量PCR测定病毒滴度.应用二套管法建立Wistar→Wistar大鼠原位肝移植模型.将提纯的重组腺相关病毒-GFP(rAAV-GFP)在供肝冷保存阶段经门静脉靶向转染并孵育2 h后行大鼠原位肝移植,分别于术后1、3个月处死大鼠取材,冰冻切片荧光显微镜下观察不同组织GFP的表达情况及转染效率.结果 rAAV-HO-1重组子经酶切电泳检测表明插入片段大小正确,测序结果与Genbank一致.rAAV-GFP靶向转染供肝1、3个月冰冻切片荧光显微镜下观察,移植肝GFP表达效率均＞80%,且在心、肺、脾、肾、肠等组织中未见报告基因的表达.结论 成功构建并提纯了携带HO-1、GFP基因的高滴度的腺相关病毒,验证了腺相关病毒介导的GFP在肝移植大鼠肝脏中稳定高效的表达.

abstractsobjective To construct and purify heme oxygenase-1,GFP gene mediated by recombinant adeno-associated-virus and identify expression rate of GFP in transplanted liver in rats.Methods Heme oxygenase-1 gene of rat was cloned and subcloned to rAAV vector,the gene sequence was confirmed correct by restriction enzyme and DNA sequencing.The rAAV-HO-1 was then cotransfected into 293 cell line with accessory plasmid virus helper and AAV-cap-rep through CaCl2 coprecipition.Virus particles were purified by heparin column chromatography and titre were detected by Real-time PCR.An orthotopic liver transplantation model by Wistar to Wistar was set up using Kamada's two cuff technique.Purified rAAV-GFP was injected into portal vein and incubated for 2 hours at the donor liver cold preserved stage,and then performed OLT.Recipients were killed and visceral organs were sampled at 1 and 3 months after operation respectively,frozen section(3-5μm)were prepared and gene expression rate in different tissues Was examined under fluorescence microscope.Results The inserted segment of HO-1 was identified through restriction enzyme cutting followed with electrophoresis,the result of DNA sequencing was in accordance with which found in Genbank.The GFP expression rate was over 80%in allograft at 1 and 3 month after transfection whereas there was no GFP expression in heart,lung,spleen,kidney and small bowel.Condusions Hish titre rAAV carried HO-1 and GFP were constructed successfully.Steady and effective expression of GFP mediated by rAAV was demonstrated in liver allograft in rats.