重组腺相关病毒介导的PD-L1基因在大鼠移植肝中的表达

Expression of PD-L1 gene mediated by recombinant adeno-associated virus 2 in rat liver allograft

目的探讨供肝转染PD-L1-AAV2-RFP后基因的表达情况。方法近交系BN大鼠随机分3组:同基因肝移植组(ISO组)、腺相关病毒空载体组(AAV组)、基因转染组(PD-L1组),每组6对。ISO组供肝冷保存期经门静脉注射生理盐水2mL,保存2h后采用改良"二袖套法"行原位肝移植;AAV、PD-L1组所用灌注液分别为AAV2-RFP空病毒颗粒和PD-L1-AAV2-RFP(1×1011v.g./只)。术后7、14d各取3只大鼠采血测定肝功能,取肝组织即刻行冰冻切片在荧光显微镜下观察RFP的表达,免疫组化染色法检测PD-L1蛋白的表达。结果3组术后7d谷丙转氨酶(ALT)、谷草转氨酶(AST)均明显增高,14d后基本恢复正常,各组间相比均无显著性差异(P>0.05);术后7d在荧光显微镜下AAV、PD-L1组移植肝可见少量而微弱的红色荧光,14d可见高亮度的红色荧光,而各组肝外组织及ISO组各时段均未见红色荧光;免疫组化染色结果示未转染组PD-L1蛋白均微弱表达,而PD-L1转染7d后随时间延长靶基因表达逐渐增强(P<0.05)。结论重组腺相关病毒介导,体外冷保存期经门静脉途径供肝转染PD-L1,可以使PD-L1基因在肝内获得安全、有效的表达。

Objective To investigate the transfection and expression of PD-L1-AAV2-RFP after rat liver transplantation. Methods All rats were divided into isogenie liver transplantation group （ISO group）, liver transplantation group infected by AAV2-RFP （AAV group） and PD-L1-AAV2-RFP （PD-L1 group）. Recombinant virus were respectively injected into portal vein and kept for 2 hours to transfect grafts during cold preservation in vitro. Three rats were killed on postoperative day 7 and 14 respectively. Serum samples were collected for decting liver function. Transgene expression of PD-L1 was assessed by means of fluorescent microscope and immunohistochemistry. Results Serum levels of ALT and AST increased obviously on the 7th day after transplantation, then gradually returned back to normal with time. The liver function of PD-L1 group was similar to those of ISO and AAV groups. Otherwise, the expression of RFP in PD-L1 group was significant higher than those of ISO and AAV groups observed by fluorescent microscope. The expression of PD-L1 protein was up-regulated in the liver of donor after transfection in PD-L1 group, but still lower in other groups. Conclusion During cold preservation in vitro through portal vein injection to donor liver, AAV mediated gene transfection can successfully achieve local gene expression.