酒精性脂肪肝中库普弗细胞表型变化的探讨

发布时间：2019-02-13 14:43

【摘要】：目的通过建立小鼠酒精性脂肪肝(AFL)的模型,并采用肝脏原位灌流,分离出形态良好、具有生物学活性的库普弗细胞(KCs),研究其在AFL中的作用。方法采用Lieber-DeC arli液体饮食加一次急性酒精灌胃,建立小鼠AFL模型。造模周期为16 d,于第16天灌胃9 h后处死小鼠,取小鼠肝脏、血清及KCs。检测各组血清谷丙转氨酶/谷草转氨酶(ALT/AST)、肝匀浆、血清中总胆固醇/三酰甘油(TG/TC)水平变化和肝脏病理切片HE、油红染色。选取原位灌流的方法分离KCs,采用流式细胞术分析KCs表型及组成的改变。荧光实时定量PCR(qRT-PCR)检测组织及提取细胞中各细胞因子水平。结果 ALT/AST、TG/TC等反应肝损伤的指标模型组显著高于对照组,HE及油红染色结果与之一致,表明小鼠AFL模型建立成功。肝脏原位灌流每只小鼠细胞得率约1.5×106~2.0×106个。以小鼠巨噬细胞表面标记分子F4/80及白细胞共同抗原CD45双标设门,流式细胞术分析F4/80和CD45双阳性细胞,在模型中肝脏固有CD68+细胞显著降低,并出现大量的浸润单核细胞。qRTPCR结果显示,在肝组织及原代KCs中细胞因子,肿瘤坏死因子(TNF-α)、白介素-6(IL-6)、单核细胞趋化蛋白(MCP-1)水平显著升高。结论小鼠AFL模型建立成功,肝脏原位灌流法细胞得率较高,AFL的发病可能与KCs构成、表型改变,与细胞因子升高介导外周单核细胞浸润有关。
[Abstract]:Objective to establish a mouse model of alcoholic fatty liver (AFL) and to isolate the well-formed and biologically active Kupffer cells (KCs),) in AFL by in situ perfusion of the liver. Methods Lieber-DeC arli liquid diet and acute alcohol were used to establish AFL model in mice. The model period was 16 days. The mice were killed 9 hours after gavage on the 16th day. The liver, serum and KCs. were taken from the mice. Serum alanine aminotransferase / alanine aminotransferase (ALT/AST), liver homogenate, serum total cholesterol / triglyceride (TG/TC) levels and HE, oil red staining in pathological sections of liver were detected. KCs, was isolated by in situ perfusion. The phenotype and composition of KCs were analyzed by flow cytometry. Fluorescence real-time quantitative PCR (qRT-PCR) was used to detect the levels of cytokines in tissues and extracted cells. Results the results of HE and oil red staining showed that the model group with ALT/AST,TG/TC and other indexes of liver injury was significantly higher than that of the control group, which indicated that the AFL model of mice was established successfully. In situ perfusion of liver, the cell yield of each mouse was about 1.5 脳 10 ~ (6) ~ (2) 脳 10 ~ (6) ~ (2) 脳 10 ~ (6). Mouse macrophage surface marker F4 / 80 and leukocyte common antigen (CD45) were double labeled. F480 and CD45 double positive cells were detected by flow cytometry. In the model, the inherent CD68 cells in liver decreased significantly. The results of qRTPCR showed that the levels of cytokines, tumor necrosis factor (TNF- 伪), interleukin-6 (IL-6) and monocyte chemotactic protein (MCP-1) in liver tissue and primary KCs were significantly increased. Conclusion the AFL model of mice was established successfully, and the rate of cells obtained by in situ perfusion of liver was higher. The pathogenesis of AFL may be related to the composition and phenotype of KCs and the increase of cytokines to mediate the infiltration of peripheral monocytes.
【作者单位】：安徽医科大学药学院;安徽医科大学肝病研究所;安徽省创新药物产业共性研究院;
【基金】：国家自然科学基金(编号:81273526) 安徽省教育厅基金项目(编号:KJ2012A156) 安徽省自然科学基金(编号:1308085MH145) 安徽医科大学博士科研启动基金(编号:XJ201118)
【分类号】：R575.5

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