转化生长因子β1对肝星状细胞活化及跨膜信号转导影响与舒肝颗粒的干预  

Effects of Shugan granule on hepatic stellate cells activation and its trans-membrane signal transduction stimulated by transforming growth factor beta 1

作　　者：吕沐瀚[1] 李晓云[1] 李昌平[1] 

机构地区：[1]泸州医学院附属医院消化内科,四川省泸州市646000

出　　处：《中国组织工程研究与临床康复》2009年第50期9898-9902,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家人事部留学回国人员基金(030079);四川省卫生厅资助项目(川卫125号)~~

摘　　要：背景:肝纤维化是一种可逆性的病变,及时地干预肝纤维化的病程能够减少肝硬化及其致命并发症的发生。肝星状细胞在肝纤维化发病机制中起主要作用。目的:采用舒肝颗粒作用于体外培养的大鼠肝星状细胞,观察其是否对转化生长因子β1促进肝星状细胞活化及跨膜信号转导有影响,以探讨其抗纤维化的作用机制。设计、时间及地点:对照观察细胞学实验,于2008-06/2009-02在泸州医学院分子中心实验室完成。材料:肝星状细胞株HSC-T6购自上海中医药大学肝病研究所,其表型为活化的肝星状细胞。舒肝颗粒由泸州医学院附属医院药物研究所提供,批号:20071120。方法:四甲基偶氮唑盐法测不同质量浓度(0.01,0.02,0.04,0.08g/L)舒肝颗粒对HSC-T6细胞增殖情况的影响,选择对细胞增殖无影响的剂量(0.01g/L)。再将HSC-T6细胞种板培养后,分为4组,空白对照组培养液中不加其他处理因素。转化生长因子β1组培养液中加入5μg/L转化生长因子β1液。舒肝颗粒组培养液中加入前面选择的0.01g/L舒肝颗粒药液;联合用药组培养液中同时加入舒肝颗粒药液与转化生长因子β1液,剂量同上。主要观察指标:①肝星状细胞的形态。②免疫组织化学观察肝星状细胞表达Smad3和Smad7。③反转录-聚合酶链反应观察肝星状细胞活化及跨膜信号转导结果。结果:①转化生长因子β1组细胞形态类似于对照组,且伸展更明显,联合用药组,细胞形态更接近于转化生长因子β1组,各组均未见核固缩或凋亡现象。②免疫组织化学法测Smad3和Smad7在对照组分别表达较高;转化生长因子β1能轻度上调Smad3和Smad7的表达(P<0.05);而舒肝颗粒组和联合用药组与对照组相比,Smad7表达上调更为显著,为对照组的1.99倍(P<0.01)。③反转录-聚合酶链反应结果显示与对照组相比,舒肝颗粒组上调Smad7mRNA表达(P<0.05),而Smad3mRNA的表达无明显变化。给予BACKGROUND： Hepatic fibrosis is a reversible disease, interfering in course of disease promptly can decrease hepatic cirrhosis and fatal complication. Hepatic stellate cells （HSCs） is a key factor in pathogenesy of hepatic fibrosis. OBJECTIVE： To investigate the effects of Shugan granule on HSCs activation and trans-membrane signal transduction stimulated by transforming growth factor beta 1（TGF-β1） in rats, and to explore the anti-fibrosis mechanism. DESIGN, TIME AND SETTING： Controlled observational trials based on cytology were performed in the Central Laboratory of Molecules, Luzhou Medical College between June 2008 and February 2009. MATERIALS： HSC-T6 cell line was purchased from Institute of Liver Diseases, Shanghai University of Traditional Chinese Medicine, its phenotype was the activated hepatic stellate cells. Shugan granule was offered by Drug Institute, Affiliated Hospital of Luzhou Medical College at a Batch No. 20071120. METHODS： The influence of different concentrations （0.01, 0.02, 0.04, 0.08 g/L） of Shugan granule on HSCs proliferation was determined by MTT. 0.01 g/L was defined as the dose of Shugan granule contributing no influence on ceil proliferation. HSCs were cultured in a culture ptate and then divided into 4 groups： control group without management, TGF-β1, group with 5μg/L TGF-β1 solution in culture medium, Shugan granule group with 0.01g/L Shugan granule in a culture medium, and TGF-β1 ＋ Shugan granule group with 5μg/L TGF-β1 solution and 0.01g/L Shugan granule in culture medium. MAIN OUTCOME MEASURES： Morphological features of HSCs were detected by microscopic. Immunohistochemical method was used to detect the expression of Smad3 and Smad7 in HSCs. RT-PCR was applied to observe the HSCs activation and trans-membrane signal transduction. RESULTS：①The cell morphology of TGF-β1 group was similar with that in the control group, and the extension was more obvious In the TGF-β1 ＋ Shugan granule group, the cell morphology was close to that in TGF-β

关 键 词：舒肝颗粒 肝星状细胞 细胞因子 肝纤维化 TGF-β1 SMAD3 SMAD7 

分 类 号：R392.114[医药卫生—免疫学]