缺血预处理大鼠自体肝移植后剩余肝细胞的凋亡和增殖

Effects of ischemic preconditioning on liver cell apoptosis and proliferation following autologous liver transplantation in rats

背景:肝脏缺血再灌注损伤后肝细胞受到增殖和凋亡的双重调控。肝大部切除后,余肝经历缺血再灌注损伤,其肝再生受到明显抑制,缺血预处理可减轻此损伤,促进肝再生,其机制是否也与这有关,目前未见相关报道。目的:探讨缺血预处理对大鼠自体肝移植后余肝肝细胞凋亡和增殖的影响。设计、时间及地点:随机对照动物实验,于2006-09/2007-07在中南大学湘雅医学院实验动物中心完成。材料:雄性SD大鼠144只,随机分为3组:单纯肝叶切除组、自体肝移植组、缺血预处理组,48只/组。方法:单纯肝叶切除组大鼠只行肝左、中叶切除,不阻断肝右、尾叶血流。自体肝移植组大鼠结扎切断肝后与食管腹段之间的静脉交通支,游离liberate尾状叶,游离第一肝门、肝上及肝下下腔静脉后,阻断并经门静脉持续低温灌注保存液,同时进行无血肝切除(切除肝左、中叶),肝脏在体持续低温灌洗15min。解除肝门阻断,完全恢复肝脏血流。快速复温肝脏表面,并冲洗腹腔,缝合关腹。缺血预处理组大鼠在门静脉灌注前先阻断肝右、尾叶血流10min,然后开放血流10min,余步骤同自体肝移植组。各组大鼠分别于肝叶切除后0,1,3,6,12,24,48,72h时取材。主要观察指标:生化分析仪测定血清丙氨酸氨基转移酶及天冬氨酸氨基转移酶的水平,流式细胞仪检测肝细胞凋亡指数,通过Ki-67抗原的表达检测肝细胞增殖情况。结果:与自体肝移植组比较,除肝叶切除后0h外,其余各时间点单纯肝叶切除组、缺血预处理组血清丙氨酸氨基转移酶和天冬氨酸氨基转移酶水平均明显降低(P<0.05)。各组在肝叶切除后0h(即正常肝组织)基本不存在凋亡细胞;单纯肝叶切除组肝大部切除后余肝肝细胞凋亡指数稍有升高;自体肝移植组复灌注后肝细胞凋亡指数急剧升高,约在12h达到高峰,而后逐渐下降;与自体肝移植组比较,缺血预处理组肝细胞凋亡指数明显降低(P<0.05)。各组Ki-67表达率在肝叶切除后均明显升高,约在24h达高峰,而后逐渐下降。与单纯肝叶切除组比较,自体肝移植组Ki-67表达率明显降低(P<0.05);与自体肝移植组比较,缺血预处理组Ki-67表达率明显增高(P<0.05)。结论:缺血预处理可减轻自体肝移植后肝缺血再灌注损伤所致肝细胞凋亡,促进肝细胞增殖,这可能是其促进肝再生的机制之一。

BACKGROUND：Liver cells are regulated by proliferation and apoptosis following ischemia/reperfusion injury,and the liver regeneration is obvious inhabited after ischemia/reperfusion injury,which can be relieved by ischemic preconditioning.However,the mechanism is still poorly understood.OBJECTIVE：To explore the effects of ischemic preconditioning on remained liver cell apoptosis and proliferation following autologous liver transplantation.DESIGN,TIME AND SETTING：The randomized controlled animal experiment was performed at the experimental animal center of Xiangya Medical College of Central South University from September 2006 to July 2007.MATERIALS：Totally 144 male Sprague Daweley rats were randomly divided into hepatic resection,auto-transplantation,ischemic preconditioning groups,with 48 animals in each group.METHODS：Rats in the hepatic resection group were underwent left hepatic lobe and median lobe resection without blocking blood current at the right hepatic lobe and caudal lobe.In the auto-transplantation group Venous traffic branches of rats were broke,with liberating caudate lobe,first porta hepatis,and inferior vena cava,followed by blocking and continuous hypothermical perfusion preservative fluid via porta hepatic,simultaneously,anemia hepatectomy was performed （left hepatic lobe and median lobe was resected）.The liver was washed and preserved in cold preservation solution for 15 minutes.At the end,portal triad clamping was removed and underwent abdominal closure.The procedure of rats in the ischemic preconditioning group was identical to auto-transplantation group except 10 minutes blocking and 10 minutes recover the blood flow at right hepatic lobe and caudal lobe prior to portal vein perfusion.Liver tissues were harvested at hours 0,1,3,6,12,24,48 and 72 after hepatic resection in 3 groups.MAIN OUTCOME MEASURES：Contents of aspartate aminotransferase and alanine aminotransferase were calculated by biochemical analyzer.The index of cell apoptosis was detected by flow cytometry.In addition,proliferation of liver cells was measured by Ki-67 expression.RESULTS：Compared to the auto-transplantation group,the levels of alanine aminotransferase and aspartate aminotransferase were dramatically decreased in hepatic resection and ischemic preconditioning groups at each time point exception with 0 hour after operation （P〈0.05）.Few apoptosis cells existed in each group at 0 hour after operation.The index of cell apoptosis increased slightly after resection in the hepatic resection group,which was sharp increased in the auto-transplantation group after reperfusion,reached a peak at 12 hour,and then gradually decreased.Compared auto-transplantation group,the index of cell apoptosis in ischemic preconditioning group was significantly decreased （P〈0.05）.The expression of Ki-67 in 3 groups increased after hepatic resection,peaked at 24 hour after hepatic resection,then decreased lower and lower.Compared to the hepatic resection group,the expression of Ki-67 in auto-transplantation group was significantly lower after hepatic resection （P〈0.05）.Compared to the auto-transplantation group,the expression of Ki-67 in ischemic preconditioning group was significantly increased after hepatic resection （P〈0.05）.CONCLUSION：Ischemic preconditioning can decrease cell apoptosis and promote cell proliferation after rat＇s liver auto-transplantation,which may be one mechanisms of ischemic preconditioning in promoting liver regeneration.