【摘要】 目的　研究刀豆蛋白A诱导下T细胞介导的小鼠免疫性肝纤维化模型形成过程中细胞因子mRNA表达水平的变化。方法　Balb/c小鼠随机分为正常对照组、模型组。模型组小鼠静脉注射刀豆蛋白A 15mg/kg ,每周 1次 ,共 2 0周 ,正常对照组给予等量生理盐水。于第 1、5、12和 2 0次注射后 2 4h分别处死 8只小鼠 ,取肝脏组织低温保存 ,常规苏木精 伊红染色、果莫里法 (Gomori)纤维染色及冰冻切片CD4+ 、CD8+ T细胞染色。抽提小鼠肝脏组织总RNA ,RT PCR扩增肝组织IL 2、IL 4、IL 10、转化生长因子 (TGF) β1和 β 肌动蛋白 (β actin)的mRNA ,凝胶图像分析系统扫描扩增产物的灰度值 ,以 β actin作为内参照 ,计算细胞因子mRNA的相对表达量。 结果　模型组小鼠苏木精 伊红染色和Gomori染色提示实验中晚期发生肝纤维化 ,冰冻切片免疫组织化学染色提示肝组织内淋巴细胞浸润以CD4+ 细胞为主。IL 2mRNA水平仅在第 1次注射后上升 ,随后迅速降低 ;IL 4、IL 10和TGF β1mRNA表达水平则在模型形成过程中保持较高的表达水平。结论　反复刀豆蛋白A刺激可以诱导T淋巴细胞介导的小鼠免疫性肝纤维化模型的发生 ,IL 4、IL 10和TGF β1等细胞因子mRNA在模型形成过程中持续高水平表达 ,在模型的发病过程中起着重要作用。更多还原

【Abstract】 Objective To explore the fluctuation of cytokine mRNA expression level in a novel T-cell-mediated immune hepatic fibrosis model induced by repeatedly injections of Concanavalin A in BALB/c mice. Methods BALB/c mice were divided into different groups. Model group mice were injected weekly up to 20 weeks with Concanavalin A (15mg/kg), via retro-orbital venous plexus under ether anesthesia. Normal control group mice were treated in the same manner weekly with normal saline. Twenty-four hours after Concanavalin A challenge at 1, 5, 12 and 20 week, 8 mice from each time were killed by cervical dislocation, repectively. The livers of different group were excised and fixed in 10% formalin for HE staining and Gomori Ag staining or frozen in optimal cutting temperature (O.C.T.) media in liquid nitrogen for immunohistochemical staining for CD4 +T or CD8 +T cell. After extracting total RNA from liver tissues, IL-2, IL-4, IL-10 and transforming factor β1 messenger RNA were amplified by reverse transcription polymerase chain reaction. PCR products were electrophoresed on agrose containing ethidium bromide and visualized under ultraviolet light. Densitometric RT-PCR data were standardized with β-actin signals. Results The histological change of HE staining and Gomori Ag staining indicated the fibrogenesis in model group mice. Immunohistochemical staining for CD4 + or CD8 + T cell indicated that the infiltrating lymphocytes in liver parenchyma were mainly CD4 +T lymphocytes. IL-2 mRNA expression level only increased after the first injection of Concanavalin A. The expression levels of IL-4, IL-10 and transforming growth factor β1 mRNA significantly increased over the whole experiment period as compared with control group. Conclusions Repeated administration of Concanavalin A can induce T-cell-mediated immune hepatic fibrosis model in BALB/c mice. The expression levels of IL-4, 10 and TGF-β1 increase over the whole experiment period and may play an important role in creating mouse fibrotic model.更多还原