腺相关病毒介导的PD-L1基因转染对大鼠肝移植术后肝脏的保护作用  

Gene transfer of programmed death ligand-1 prolongs liver allograft survival in rat

作　　者：邵永[1] 王槐志[2] 董家鸿[2] 王曙光[2] 别平[2] 李晓武[2] 张玉君[2] 陈永文[3] 倪斌[3] 

机构地区：[1]徐州医学院附属医院普外科,江苏徐州221002 [2]第三军医大学西南医院肝胆外科研究所,重庆400038 [3]第三军医大学免疫研究所,重庆400037

出　　处：《中国现代普通外科进展》2013年第12期925-930,共6页Chinese Journal of Current Advances in General Surgery

基　　金：国家自然科学基金资助(No.30400418)

摘　　要：目的：探讨腺相关病毒（AAV）介导的PD—L1基因转染对大鼠移植肝的保护作用及其可能机制。方法：96只近交系大鼠随机分为等基因肝移植组（G1）、同种异体肝移植组（G2）、AAV空载体组（G3）和PD—L1转染组（G4），每组12对。通过大鼠肝移植急性排斥模型，将PD—L1-AAV2-RFP（RFP）经门静脉灌注夹闭法转染供肝，观察移植后大鼠中位生存时间（MST），全自动生化分析仪测定肝功能，在荧光显微镜、光镜同视野下观察RFP在移植肝的表达和病理变化。采用免疫组织化学染色法评估PD—L1蛋白的表达及炎性细胞浸润情况。结果：G1在观察期内全部存活；G2与G3MST分别为18（17-20）d和18（17-19）d；G4显著延长了移植肝的存活（MST29d，P〈0．05）。术后14dG4的肝功能及病理变化与术后7dG2和G3相似。G3、G4术后7d中观察到明显的红色荧光，随时间逐渐增强。移植肝PD—L1蛋白的变化趋势与RFP的表达相似。G1移植肝T细胞含量极少；术后14dG2、G3移植肝CD4^＋、CD8^＋、CD25^＋不同程度淋巴细胞浸润，G4移植肝T淋巴细胞浸润明显减少，尤以CD8^＋细胞为著，与各组相比差异显著（P〈0．05）。结论：腺相关病毒介导的PD—L1基因转染可延长移植肝的存活，其机制可能是PD—L1基因转染增强了PD—L1／PD-1信号通路，通过抑制移植肝CD4^＋、CD8^＋、CD25^＋T细胞浸润及其功能，从而发挥对移植肝的保护作用。Objective： To investigate the protective effect of PD-L1 on rat by infecting liver allograft with PD-L1-AAV2-RFP. Methods： All rats were divided into isogenic liver transplantation group（G1）, allogeneic liver transplantation group（G2）, allogeneic liver transplantation group infected by AAV2-RFP （G3） and PD-L1-AAV2-RFP （G4）. AAV mediated PD-L1 gene transfer was per- formed in LEW rat donor livers placed in BN recipients. Median survival time （MST） of grafts were investigated. The function of liver was measured with automatic biochemical analyser. The expressions of RFP in liver and pathological changes were observed under fluorescent microscope and light microscope. The expression of PD-L1 protein and inflammatory cell infiltrating in grafts were assessed by immunohistochemical staining. Results： Liver allograft survival was significantly prolonged by PD-L1-AAV2-RFP treatment （MST, 29 （range： 28-31） days） compared with either AAV2-RFP treated group or virus dilution buffer alone： 18（17-20） and 18（17-19） days respectively （P〈0.05）. The pathological changes of liver and liver function in G4 on POD 14 were similar to those of G2 and G3 on POD 7. Evident red fluorescence was observed in sections of allograft on POD 7 and ever increased in the later. The trend of PD-L1 protein changes in graft was similar to that of RFP. The amount of CD4^＋, CD8^＋ and CD25^＋ lymphocytes infiltrating the graft increased in G2 and G3, accompanying with greatly decreased lymphocytes in G4 （P〈0.05）. Conclusion： AAV-mediated gene transfer of PD-L1 into cold preserved fiver allograft before transplantation results in long term survival of liver allograft. PD-L1 gene may take the effect via suppression of INF-y and IL-17 production as well as stimulation of IL-10 and TGF- β1 production.

关 键 词：肝移植 PD—L1 腺相关病毒 基因转染 近交系大鼠 

分 类 号：R657.3[医药卫生—外科学] R617[医药卫生—临床医学]