HIV-1反式激活蛋白-血红素氧合酶-1融合蛋白对大鼠供肝冷保存期肝细胞凋亡的影响

摘要

目的 探讨HIV-1反式激活蛋白(transactiviting protein,TAT)与血红素氧合酶-1(heme oxygenase,HO-1)融合蛋白(TAT-HO-1)对大鼠供肝冷保存期肝细胞凋亡的影响.方法 选择雄性SD大鼠48只,体重250 g～300 g.根据所用保存液的不同随机分为两组(n=24):C组,肝脏应用4℃ HTK液进行灌注和冷保存;P组,肝脏应用4℃含TAT-HO-150 mg/L的HTK液进行灌注和冷保存.参照Kamada等的方法切取肝脏,分别于冷保存0、6、12、18 h后,收集保存液,切取肝脏组织.免疫组化染色法分析TAT-HO-1转导入肝脏的情况,全自动生化分析仪测定保存液中谷丙转氨酶(alanine aminotransferase,ALT)水平,放射免疫分析法检测肝组织中透明质酸(hyaluronic acid,HA)水平,TUNEL法检测肝实质细胞和肝窦内皮细胞(sinusoidal endothelial cell,SECs)凋亡指数,免疫组化染色法检测肝组织Caspase-3表达情况.结果 免疫组化染色结果显示P组肝组织中HO-1含量在冷保存6、12、18 h时均高于C组(P＜0.05).两组保存液中ALT水平,肝组织中HA含量,肝实质细胞和SECs凋亡率以及肝组织Caspase-3表达水平均随保存时间延长而升高(P＜0.05).冷保存6、12、18 h后保存液中ALT水平、肝组织HA含量、肝实质细胞、SECs凋亡指数以及Caspase-3阳性表达水平P组均低于C组(P＜0.05).结论 TAT-HO-1可以在冷保存期间转导进入大鼠肝脏并减轻肝实质细胞和SECs凋亡,对肝脏功能具有一定保护作用.蛋白转导技术将成为减轻肝移植冷保存期损伤的新措施.%Objective To study the effects of TAT-HO-1 fusion protein,HIV-1 transactiviting protein (TAT) and heme oxygenase-1 (HO-1),on hepatic cell apoptosis of rat donors in cold storage stage.Methods Forty-eight male SD rats were randomly divided into two groups.Rat livers were flushed and preserved with 4℃ HTK solution containing(group P) or uncontaining(group C) 50 mg/L of TAT-HO-1.The preserved solution and hepatic tissue were collected at 0,6,12,18 h of cold storage stage.TAT-HO-1 transducing into liver,alanine aminotransferase(ALT) level in preserved solution,hyaluronic acid(HA) level and the expression of caspase-3 in hepatic tissue,and the apoptotic index (AI) of hepatocytes and sinusoidal endothelial cells (SECs) were measured or detected.Results ALT level in preserved solution,HA level and the expression of caspase-3 in hepatic tissue,and the AI of hepatocytes and SECs increased time-dependently in cold storage stage in both groups (P＜0.05),with lower increasing extent in group P than that in group C (P＜0.05) at 6h,12h and 18h of cold storage stage.A stronger accumulation of HO-1 staining was also detected at the same time-points in group P than that in group C(P＜0.05).Conclusion TAT-HO-1 may transduce efficiently into rat livers,exerting protective effects on both hepatocytes and SECs during cold storage stage.Protein transduction technology may be a novel therapeutic means to reduce donor liver injury in preservation period for transplantion.