拟炎症高NO大鼠原代培养肝细胞的信号转导途径研究  

Signal transduction pathways induced by nitric oxide in rat hepatocytes

作　　者：陈奕[1] 王红莉[1] 楼宜嘉[1] 

机构地区：[1]浙江大学药学院,浙江杭州310031

出　　处：《浙江大学学报（医学版）》2002年第6期424-428,共5页Journal of Zhejiang University(Medical Sciences)

基　　金：国家自然科学基金资助项目 (30 0 70 90 4 )

摘　　要：目的 :通过对信号分子 NO在原代培养大鼠肝细胞所经由的信号转导途径研究 ,探索炎症急性期肝细胞高 NO的生物学意义 ,及药物可能作用的新靶点。方法 :采用 NO供体硝普钠 (SNP)模拟肝脏急性炎症期高 NO环境 ,Griess法测定 NO的特异氧化终产物 NO2 - / NO3- 。测定肝细胞中 c GMP的浓度和肝细胞培养液中 GSNO的浓度 ,由此分别了解在原代培养大鼠肝细胞模型 ,NO经由 c GMP通路信号转导 ,还是经由非 c GMP通路信号转导的途径。结果 :1.5 4 3mmol/ L SNP释放 NO达峰值分别为 ,以 2 5 mmol/ L GSH为 NO释放通路 ,30 min时为 (0 .6 3± 0 .0 6 ) mmol/ L ;以 2 5 mmol/ L L - Cys为活性巯基通路 ,2 5 min时为 (0 .98± 0 .11) mmol/ L。 SNP与原代培养大鼠肝细胞共孵育过程中 ,随着孵育时间的延长及 SNP浓度的增高 ,细胞内 c GMP含量与对照组相比有显著性差异 (P<0 .0 5 ) ;培养液中 GSNO浓度也有增高。与对照组比均有显著差异 (P<0 .0 5 )。结论 :NO供体 SNP在含有巯基(GSH和 Cys)的肝细胞培养液中可持续释放 NO,通过 c GMP依赖性通路和 c GMP非依赖性通路—— GSNO通路进行信号转导 。Objective: To study signal transduction pathways in cultured rat hepatocytes in the high nitric oxide (NO) environment of hepatitis. Methods: NO levels were assessed by measurement of its stable oxidative pro ducts nitrite (NO 2 -) and nitrate (NO 3 -) using the Griess method with or without thiols (GSH or L Cys). Rat hepatocytes were incubated with Sodium Nitroprusside (SNP) to produce a high NO environment and the intracellular cGMP and s nitrosoglutathione (GSNO) in the culture media were measured using radioimmunoassay or with the MTT assay absorbed at 334nm respectively. Results: After incubation of 1.543 mmol/L SNP for 30 minutes 0.63±0.06 mmol/L and at 25 minutes 0.98±0.11 mmol/L of NO was released in containing 25 mmol/L GSH and L Cys condition. The levels of both cGMP and GSNO were significantly increased ( vs control P < 0.05 ) in a dose related manner. Conclusion: Signal transduction of cultured rat hepatocytes in a high NO environment could be a cGMP dependent as well as a non cGMP dependent pathway.

关 键 词：一氧化氮 肝炎 原代培养肝细胞 信号转导 环磷酸鸟苷 亚硝基硫醇 

分 类 号：R363[医药卫生—病理学]