逆转录病毒载体介导针对RANKL的重组蛋白对小鼠肝缺血再灌注损伤的保护作用  

Protective functions of recombinant protein targeted at RANKL against hepatic ischemia/reperfusion injury transfected by retrovirus in mice

作　　者：时建[1] 李晓花[1] 孙倍成[1] 林绪涛[1] 张兴元[1] 张帆[1] 欧琨[1] 陈强谱[1] 

机构地区：[1]山东省滨州医学院附属医院肝胆外科,256603

出　　处：《中华医学杂志》2011年第38期2719-2724,共6页National Medical Journal of China

摘　　要：目的探讨重组蛋白核因子-κB（NF—κB）受体活化因子（RANK）-Fc对小鼠肝缺血再灌注损伤保护作用及可能的机制。方法据不同处理将小鼠随机分为无血清培养基对照组（Sham组）；目地基因病毒组（RANK—Fc组）；空载体病毒组（eGFP组），各组均经尾静脉将2．5ml溶液（含或不含病毒）6s内注入。3d后RANK—Fc及eGFP组建立70％肝缺血再灌注损伤模型，热缺血90min后再灌注不同时间，Sham组行假手术。各组均在1、3、6及24h剖杀小鼠5只，留取血清及肝组织标本。RT—PCR法检测eGFPmRNA表达，Western印迹检测RANK—Fc蛋白表达。HE染色组织学变化及肝脏酶学水平用于肝损伤判断，Western印迹及免疫组织化学方法测定NF-κB的活化程度。c-Jun-氨基末端激酶（JNK）活化水平亦经Western印迹检测。酶联免疫吸附测定法定量检测组织匀浆肿瘤坏死因子α（TNF-α）、白细胞介素6（IL-6）水平。细胞凋亡情况由TUNEL法判断。相同时间点各组间比较采用单因素方差分析，两两比较用t检验。结果应用流体力学体内转染方法，RANK-Fc及eGFP在肝脏成功表达。相同时间点RANK—Fc组[ALT（U／L）：1149±109、1574±258、2778±217、461±65；AST（U／L）：1027±276、1467±236、2208±378、506±160]肝脏酶学水平均较eGFP组[ALT（U／L）：2828±345、5194±944、8403±806、969±147；AST（U／L）：2770±343、4778±684、8050±1160、977＋115]改善（P〈0．01）。与eGFP组相比，RANK—Fc组小鼠肝细胞NF-κBp65（t=6．726）及JNK（t=6．713）表达水平降低，促炎症介质TNF-α[[（235±56）比（527±109）pg／ml，t=4．779]、IL-6[（303±73）比（702±126）pg／ml，t=5．482]水平降低，差异均有统计学意义（均P〈0．01），免疫组织化学检查显示细胞核NF-κB阳性染色细胞数减少，HE染色显示肝组织学病变减轻，TUNEL染色阳性细胞显著减少。结论RANK—Fc对小鼠缺血再灌注�Objective To explore the protective functions of recombinant protein RANK-Fc against hepatic ischemia/reperfusion injury and clarify its possible mechanism. Methods Sixty male Balb/e mice were randomly divided into 3 groups according to different treatments： serum-free medium control （Sham） group, target gene retrovirus （RANK-Fc） group and empty vector retrovirus （eGFP） group. All mice were injected with 2. 5 ml solution（with or without retrovirus）within 6 seconds via tail vein. After 3 days, the mode/ of 70% hepatic ischemia/reperfusion was induced under warm conditions for 90 minutes after different periods of reperfusion in RANK-Fc and eGFP groups ; Sham group underwent the same procedure without the occlusion of blood supply. Blood and liver samples were obtained at different time points （ 1, 3, 6 and 24 h ; n =5 in each）. Reverse transcription-polymerase chain reaction （RT-PCR） was used for the evaluation of eGFP mRNA expression. RANK-Fc was assessed by Western blot. Liver transaminases and histopathological changes were used for the evaluation of hepatic injury. The activity of NF-κB in liver nucleus was analyzed by Western blot and immunohistochemistry. The activation level of ,INK was also assessed by Western blot. Liver homogenate levels of tumor necrosis factor （TNF）-α and interleukin （IL）-6 were detected by enzymelinked immunosorbent assay （ELISA）. Apoptosis was identified by the terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） analysis. The differences between three treatment groups at each time point were detected by the one-way ANOVA. Statistical analysis for inter-comparison was performed by Student＇s t test. Results RANK-Fc and eGFP were successfully expressed in liver after hydrodynamicsbased transfecfion. Compared with eGFP group, RANK-Fc significantly improved liver functions at the same time point （P 〈 0. 01 ）, decreased NF-κB nuclear translocation （ t = 6. 726, P 〈 0. 01 ） and JNK phosphorylation（t = 6. 713, P �

关 键 词：基因疗法 再灌注损伤 核因子-κB受体活化因子 核因子-κB受体活化因子 配基 

分 类 号：R575[医药卫生—消化系统]