摘要目的 探讨低温机器灌注保存对大鼠离体肝脏再生能力的影响.方法 取SD大鼠12只,采用随机数字表法将大鼠分为静态冷保存组(SCS组)和低温机器灌注组(HMP组,每组6只,获取两组大鼠肝脏,SCS组肝脏在获取后置于4℃组氨酸-色氨酸-酮戊二酸溶液(HTK液)中冷保存6h,HMP组肝脏在获取后门静脉置管并连接于低温肝脏灌注系统保存6h.保存6h后,取两组部分肝组织,用100 g/L甲醛溶液固定,用于后续免疫组织化学分析;其余新鲜肝组织置于-80℃保存,采用免疫组织化学法进行检测肝组织中ki67和PCNA蛋白的表达,采用蛋白质印迹法检测肝组织中细胞周期蛋白Cyclin D1和Cyclin E1的表达,采用RT-PCR法检测肝组织中细胞增殖相关基因cdc25a、cdk1和cdk6的表达.结果 HMP组中PNCA表达明显,Ki67可见表达,而SCS组中PNCA和Ki67几乎不表达.与SCS组比较,HMP组ki67、PCNA表达明显较高,两组差异均有统计学意义(P＜0.05).HMP组cdc25a和cdk1基因的表达均显著高于SCS组,两组比较,差异均有统计学意义(P＜0.05);HMP组肝组织cdk6的表达仍高于SCS组,但两组差异无统计学意义(P＞0.05).HMP组大鼠肝组织中细胞周期蛋白Cyclin D1和Cyclin E1的表达量均显著高于SCS组,两组比较,差异均有统计学意义(P＜0.05).结论 低温机器灌注保存较静态冷保存能够更好的维持大鼠肝脏的再生能力,有望成为供肝保存的理想方法.

abstractsObjective To study the effects of different preservation methods on the isolated liver injury and regeneration.Methods Twelve Sprague-Dawley rats were randomly assigned to two groups:static cold storage (SCS) and hypothermic machine perfusion (HMP) (n =6 each).Histidinetryptophan-ketoglutarate solution (HTK) was used as preservation solution.The graft was preserved in HTK (4 C) for 6 h in SCS group,and that was perfused using HTK in HMP group.Liver tissue was obtained and fixed in 10％ neutral formalin for immunohistochemistry.The fresh liver tissue was collected and stored in-80℃ for RT-PCR and Western blotting analyses.Results Compared to SCS,HMP improved liver regeneration ability in terms of PCNA and ki67 detection and promoted cell proliferation in PCR levels (Cdc25A,cdk1,cdk6) and Western blotting levels (Cyelin D1,Cyclin E1).Conclusion HMP is superior to SCS in liver regeneration,and expected to be the ideal method for preservation of donor liver.