重组腺相关病毒介导的血红素加氧酶1基因和绿色荧光蛋白基因在肝移植大鼠肝脏中的表达  

Expression of heme oxygenase-1 and GFP gene mediated by recombinant adeno-associated-virus in transplanted liver in rats

作　　者：孙亮[1] 乔海泉[1] 石铁锋[1] 姜宪[1] 唐波[1] 姜洪池[1] 孙学英[1] 

机构地区：[1]哈尔滨医科大学第一临床医学院普外科,150001

出　　处：《中华外科杂志》2008年第11期851-853,共3页Chinese Journal of Surgery

基　　金：国家自然科学基金资助项目（30571753）

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2h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。Objective 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 （ 3-5 μm） 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. Conclusions High 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.

关 键 词：肝移植 模型 动物 血红素加氧酶-1 基因治疗 

分 类 号：R686[医药卫生—骨科学]