诱生型一氧化氮合酶在阻塞性黄疸肝损害中的调控作用

目的：通过体内、外实验，探讨诱生型一氧化氮合酶（iNOS）在阻塞性黄疸肝损害中的调控作用。方法：（1）　体外实验：采用胶原酶原位肝灌注法分离大鼠肝细胞，行原代培养后，用iNOS抑制剂SMT作用于肝细胞，50μmol/L 甘氨鹅脱氧胆酸钠(GCDC) 作用后用流式细胞术（FCM）及原位末端标记法（TUNEL）检测肝细胞凋亡情况。（2）体内实验：结扎大鼠胆总管, 结扎后3，7，14，21d, 分别用TUNEL法及免疫组化SABC法检测大鼠肝组织细胞凋亡状态及iNOS蛋白的表达。结果：（1） 随SMT浓度的增加，肝细胞的凋亡明显减少。（2）大鼠胆总管结扎后随结扎时间的延长细胞凋亡指数(AI)升高，结扎14d后AI达高峰。iNOS蛋白表达越强, 则AI越高。结论：iNOS参与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。

The regulating mechanism of inducible nitric oxide synthase in the hepatic injury of obstructive jaundice

Abstract:Objective： To explore the regulating mechanism of inducible nitric oxide synthase(iNOS) in hepatic injury of obstructive jaundice (OJ) in vivo and in vitro experiments. Methods：(1) Rat hepatocytes were isolated by in situ collagenase perfusion and primary culture. Hepatocytes were pretreated with various concentrations of iNOS inhibitor SMT for 20 min. After pretreatment, 50μM GCDC was added for an additional 24hr. Cells were next detected by FCM and TUNEL.(2) Experimental obstructive jaundice (BDL) was induced by double ligation of the bile duct in rats. After BDL for 3d、7d、14d、and 21d, the apoptotic status in liver of all rats were determined with TUNEL, and iNOS protein in liver of OJ was ditermined with immunohistochemistry method. Results：(1) SMT decreased GCDC-induced apoptosis in a concentration - dependent manner. (2) The apoptotic rate of liver was related to length of time of OJ. Apoptosis index (AI) was highest from rats with 14d bile duct ligation. The stronger the iNOS expression, the higher was the number of apoptotic cells that was found in OJ. Conclusions：iNOS is involved in the regulation and the occurrence and progression of hepatic injury of obstructive jaundice.