四氯化碳肝纤维化大鼠肝组织差异蛋白质组的动态变化

Dynamic proteomic analysis of liver tissue in the carbon tetrachloride-induced liver fibrosis rat model

目的探索肝纤维化形成过程中肝组织蛋白质组的变化，部分解析肝纤维化发生、发展的病理生理机制。方法采用四氯化碳（CCl4）诱导大鼠肝纤维化模型，应用双向凝胶电泳技术分离大鼠肝组织总蛋白质，经考马斯亮蓝染色、图像分析、识别差异表达的蛋白质点，应用基质辅助激光解吸电离飞行时间串联质谱及数据库查询鉴定差异蛋白质点。采用Westemblot、免疫组织化学方法对细胞角蛋白（CK）8、CK18进行蛋白质表达水平的验证。计量资料采用单因素方差分析中的LSD法或非参数检验中的H检验进行统计学分析。结果大鼠造模后，随时间的推移，肝纤维化评分逐渐增加（3周组〈6周组〈9周组），建立了正常大鼠和大鼠CCl4肝纤维化形成过程3、6、9周肝组织双向凝胶电泳图谱，质谱鉴定了44种差异蛋白质；采用Westemblot、免疫组织化学方法对CK8、CK18的验证结果与双向凝胶电泳结果基本一致。正常组及3、6、9周模型组CK8／CKl8蛋白相对表达量分别为：0．113士0．005／0．170±0．030、0．473±0．046／0．530±0．070、0．682±0．087／0．780±0．080、0．837±0．096／1．390±0．130，各组间差异均有统计学意义旷值分别为196．085／74．088、13．870／16．115、75．800／75．900，（P值均〈0．01）。结论CCl4大鼠肝纤维化形成与发展过程中差异表达蛋白质的功能主要涉及细胞生长、发育与分化，细胞增殖与凋亡，血管新生或重构，氧化应激，物质代谢及转运，信号转导等。

Objective To elucidate the dynamic physiopathologic mechanisms of liver fibrosis by investigating the differential proteome of liver tissue during progression of liver fibrosis in a chemically induced rat model. Methods Following treatment with carbon tetrachloride （CCl4）, livers were harvested from rats at various time points. The respective total protein extracts were resolved by two-dimensional gel electrophoresis （2-DE） and compared to identify differentially expressed protein spots, which were then analyzed by matrix-assisted laser desorption/ionization two-stage time-of-flight mass spectrometry （MALDI-TOF/TOF- MS） and identified by database querying. The differential expression of selected proteins was validated by Western blotting and immunohistochemical methods. Statistical analyses were carried out by the least significant difference method of one-way ANOVA for parametric data or by the H test for non-parametric data. Results The severity scores of liver fibrosis increased in a time-dependent manner following CC14 exposure （post- induction weeks： 3 〈 6 〈 9）. Forty-four protein spots were different on the 2-DE maps for the different time points, among which the CK8 and CK18 proteins were identified and verified as significantly differentially expressed as liver fibrosis progressed. Protein expressions of CK8/CK18 were enhanced upon CC14 exposure and increased over time （untreated controls： 0.113 ± 0.005/0.170 ± 0.030; CC14-induced rats at week 3：0.473 ± 0.046/0.530 ± 0.070, at week 6：0.682 ± 0.087/0.780 ± 0.080, and at week 9：0.837 ± 0.096/1.390 ± 0.130）. Moreover, the rate of ＂a＂ determinant mutations for CK8/CK18 was also significantly differently between weeks 3, 6, and 9 （F= 196.085/74.088, 13.870/16.115, and 75.800/75.900, P 〈 0.01）. Conclusion Dynamic proteomic analysis of liver tissue can indicate physiopathologic changes in protein expressions that are related to liver fibrosis induced by CCl4. Proteins with differential expression in CCl4-darnaged fibrotic liver are associated with cell growth, development and differentiation, cell proliferation and apoptosis, angioneogenesis or reconstitution, oxidative stress, substance metabolism and transport, and signal transduction.