大鼠供肝冷保存时间延长损伤移植肝肝细胞和肝窦内皮细胞

目的 探讨供肝冷保存时间与肝移植后肝细胞和肝窦内皮细胞（SEC）损伤的关系。方法 选取健康雄性SD大鼠作为供、受者，建立原位肝移植（OLT）模型。随机分为3组，冷保存1h组（H=48）：供肝获取后，置于4C的冷保存液中保存1h，再行OLT。冷保存12h组（n=48）：供肝获取后，置于4℃的UW液中保存12h，再行OLT。对照组（H=6）：大鼠只打开腹腔，不进行移植。前2组分别于术后1、6、12、24、48、72、96和168h采取血液及组织标本，对照组仅在开腹时取血液及组织标本，检测各组、各时点血清丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）及透明质酸（HA）的水平；观察移植肝的病理形态学变化，透射电镜观察其超微结构改变；原位末端脱氧核糖核酸转移酶标记法（TUNEL）检测移植肝细胞的凋亡情况；观察术后168h时的大鼠存活率。结果 冷保存1h和冷保存12h组肝移植后各时点血清ALT、AST及HA均较对照组明显升高（P〈0．05），并且冷保存12h组又明显高于冷保存1h组（P〈0．05）。冷保存12h组术后24h移植肝组织出现片状坏死，而冷保存1h组病理学改变不明显。冷保存12h组肝窦内皮细胞凋亡指数（AI）明显高于冷保存1h组（F=63．58，P〈0．01），两组大鼠移植肝组织均于术后6h出现凋亡高峰，且肝窦内皮细胞的凋亡指数明显高于肝细胞。冷保存1h组和冷保存12h组大鼠肝移植后168h时的存活率分别为100％和50％，两组比较，差异有统计学意义（F=6．39，P〈0．05）。结论 肝移植后肝细胞和肝窦内皮细胞的损伤程度与冷保存时间密切相关。肝窦内皮细胞对冷保存及再灌注损伤的敏感性高于肝细胞，其损伤方式以细胞凋亡为主。

Injury to hepatocytes and sinusoidal endothelial cells caused by cold preservation of donor liver after transplantation in rats

Objective To investigate the primary target of injury resulting from cold preservation/reperfusion after liver transplantation in rats. Methods Male SD rats were divided into three groups randomly: sham group,UW 1-h group and UW 12-h group. Orthotopic liver transplantation was performed by using the technique described by Kamada with a modification. Survival rate of rats within 168 h postoperation was observed. Liver tissue specimens and blood samples were collected at 8 time points predetermined as 1,6,12,24,48,72,96 and 168 h postoperation. Six animals were used per time-point. The liver function was evaluated by serum ALT,AST and HA levels. Morphology was observed under light microscopy and TEM. Additionally,the incidence of apoptosis (AI) in hepatocytes and sinusoidal endothelial cells (SECs) were measured separately by TUNEL method.Results The survival rate at 168 h in UW 12-h group was 50 %,significantly lower than that in UW 1-h group (F= 6.39 ,P< 0.05 ). The levels of serum ALT,AST and HA were significantly higher in UW 12-h group than those in UW 10 h group (P< 0.05 ). In UW 12-h group,massive heaptocyte necrosis was evident at 24-h time-point,but there were no significant histological alterations in UW 1-h group at any time. The AI of SECs was significantly higher in UW12h group than that in UW1h group (F= 63.58 ,P< 0.01 ) and peaked at 6 h time-point postoperation in both groups. In addition,the AI of SECs was significantly higher than that of hepatocytes.Conclusions Injury to hepatocytes and SECs resulting from cold preservation/reperfusion was associated with the length of preservation time. In comparing with hepatocytes,SECs was more sensitive to this kind of injury via an apoptosis mechanism.