摘要
目的探寻肝移植大鼠肝窦内皮细胞（SEC）损伤的详细过程、方式及机制，为冷保存再灌注损伤的保护研究开辟新的途径。方法雄性SD大鼠随机分为假手术组（n=6）、uw1h肝移植组（n=48）、UW12h肝移植组（n=48）。大鼠原位肝移植采用双袖套法，分别于术后不同时相点采取血液及组织标本，检测血清丙氨酸氨基转移酶（ALT）及透明质酸（HA）水平；HE染色观察肝脏病理学变化；TUNEL法检测凋亡，免疫组化法检测Bcl-2及Cleaved Caspase-3的表达状况。结果uw1h、UW12h组肝移植后血清ALT、HA均较假手术组明显升高（P〈0．05），UW12h组又明显高于UW1h组（P〈0．05）。UW12h组ALT水平于术后6h达高峰，而HA水平却在术后1h、24h呈双峰表现。UW12h组首先出现SEC的凋亡继而出现肝细胞的坏死，且UW12h组细胞凋亡指数（apoptosls index，AI）明显高于uw1h组（P〈0．01）。两组大鼠SEC的AI均于术后6h达高峰，与血中ALT的高峰时相点一致。肝移植术后Bcl-2表达明显减弱（P〈．01）而Cleaved Caspase-3表达上调。UW12h组SEC的AI与Bcl-2表达呈显著负相关（r=-0．982，P=0．018），与Cleaved spase-3表达呈显著正相关（r=0.954，p=0．046）。结论SEC凋亡是冷保存再灌注损伤的首要靶点及关键环节，其机制可能与Bcl-2表达的下调及Cleaved Caspase-3表达的上调有关。针对Bcl-2及Cleaved Caspase-3的抗凋亡措施可能是临床上提高肝移植疗效的有效途径。
Objective To investigate the detailed process and mechanisms of injury to sinusoidal endo thelial cells （SECs） after cold-preserved transplantation of rat liver. Methods Male rats were randomized into three groups： sham-operated group, UW 1 h group and UW 12 h group. Orthotopic liver transplantation （OLT） was performed using the two-cuff technique devised by Kamada with some modifications. Liver tissue specimens and blood samples were collected at different time points after operation. The liver function was evaluated by serum ALT and HA levels. Morphology was observed with light microscopy. The incidence of apoptosis was determined by TUNEL method. Additionally, the expression of Bcl-2 and Cleaved Caspase-3 was examined by immunohistochemistry. Results The levels of serum ALT and HA were significantly high er in UW 12 h group than those in UW 1 h group （P〈0. 05）. The serum ALT level reached to its peak at 6 h time-point postoperation in both groups. However, in UW 12 h group, the HA level had two peaks at 1 h and 24 h after transplantation separately. In UW 12 h group, the apoptosis of SEC was induced in the early stage of reperfuslon followed by the necrosis of hepatocytes in the later period. The apoptosis index （AI） in UW 12 h group was significantly higher than that in UW 1 h group （P〈0. 01）, and the AI of SEC in both groups also reached to its peak at 6 h time-point postoperation, which was in coincided with the peak of ALT level. Meanwhile, the expression of Bcl-2 declined significantly but Cleaved Caspase-3 increased after transplantation. In UW 12 h group, the AI of SEC was negatively correlated with the expression of Bcl-2 （r=-0. 982, P=0. 018） and positively correlated with the expression of Cleaved Caspase-3 （r= 0. 954, P= 0. 046） significantly. Conclusion SEC is more sensitive to cold preservation/reperfusion injury （CP/RI） in comparison to hepatocyte via an apoptotic way. The apoptosis of SEC was induced by both down-regulatlon of Bcl-2 and up-regulation of Cleaved Caspase-3. Anti-apoptosls measures aiming at regulating the expression of Bcl-2 and Cleaved Caspase-3 may be effective to improve the outcome of clinical liver transplantation.
出处
《中华肝胆外科杂志》
CAS
CSCD
2008年第6期400-403,共4页
Chinese Journal of Hepatobiliary Surgery
基金
国家自然科学基金资助项目（30571834）
全军医药卫生科研基金资助项目（01Z077）
关键词
肝移植
肝窦内皮细胞
凋亡
Liver transplantatlon, Sinusoldal endothelial cell
Apoptosis
