摘要
目的 探讨阻塞性黄疸肝损害的调控机制。方法 采用胶原酶原位肝灌注法获取大鼠肝细胞 ,行原代培养 ,用蛋白激酶 (PK)C激动剂帕斯酶埃 (PMA )、拮抗剂切勒斯埃作用于肝细胞 ,再用 5 0 μmol/L甘氨鹅脱氧胆酸钠 (GCDC)作用后行流式细胞术 (FCM )及用末端脱氧核苷酸转移酶 (TdT)介导的脱氧核苷酸 (dUTP)缺口末端标记技术 (TUNEL)检测肝细胞凋亡情况。结扎大鼠胆总管后 3、7、14、2 1d处死大鼠 ,用TUNEL技术及免疫组织化学方法检测阻塞性黄疸大鼠肝脏组织细胞凋亡状态及PKC蛋白的表达。结果 随PMA浓度的增加 ,肝细胞的凋亡明显增加 ,随Chelerythrine的增加 ,肝细胞的凋亡明显减少。大鼠胆总管结扎后随结扎时间的延长细胞凋亡指数 (AI)增加 ,结扎 14d后AI达高峰。PKC表达越强 ,AI就越高。结论 PKC信号通道参与了阻塞性黄疸肝细胞凋亡的调节 ,并在阻塞性黄疸肝损害的发生和发展中起重要作用。
Objective To explore the modulatory mechanism of hepatic injury in obstructive jaundice.Methods Rat hepatocytes were isolated by in situ collagenase perfusion and subjected to primary culture.After heptocytes were pretreated with various concentrations of PKC agonist PMA and inhibitor Chelerythrine for 20 min, 50 μmol/L GCDC was added for an additional 24 h.The hepatic apoptosis was detected by FCM and TUNEL.Experimental obstructive jaundice (BDL) was induced by double ligation of the bile duct,and the rats were killed on the day 3,7,14,21.The apoptotic status and the expression of PKC proteins in liver of all rats with obstructive jaundice were detected by using TUNEL and immunohistochemistry respectively.Results (1) PMA increased GCDC induced apoptosis and chelerythrine decreased GCDC induced apoptosis in a concentration dependent manner.(2) The apoptotic rate of liver was related to times of obstructive jaundice.AI reached the peak at 14th day after bile duct ligation.The stronger the PKC expression,the higher the AI in obstructive jaundice.Conclusion PKC might take part in the regulation of hepatic apoptosis and play an important role in pathogenesis of hepatic injury in obstructive jaundice.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2004年第12期1459-1460,i021,共3页
Chinese Journal of Experimental Surgery
基金
湖北省科技厅攻关计划项目 (2 0 0 2AA30 1C30 )
国家"863计划"重大项目基金 (2 0 0 2AA2 1 4 0 61 )
