酒精性脂肪肝中库普弗细胞表型变化的探讨

The study of kupffer cells phenotypic changes in alcoholic fatty liver

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-DeCarli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。 qRT-PCR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。

Objective Establish the model of alcoholic fatty liver in mice and isolate the biological activity kupffer cells （KCs）, in order to study its role in the alcoholic fatty liver disease. Methods C57BL/6 mice were fed with the Lieber-DeCarli diet for 16 days plus one time of acute alcohol lavage,to establish the model of alcoholic fatty liver. Tissue specimens were collected on the sixteenth day after gastric lavage 9 h later. To verify whether the model was established successfully by detecting the level of serum alanine aminotransferase/glutamic oxalacetic transaminase（ ALT/AST） and total cholesterol/triglycerides（ TG/TC） , the level of TG/TC of liver tissue homoge-nate and liver pathological section HE and oil red staining. KCs were isolated by in situ perfusion, and the cell yield and cell changes were detected by flow cytometry. Cytokine levels of organization and the primary cell were detected by qRT-PCR. Results The model response to liver injury index of ALT/AST was higher than that of con-trol group,meanwhile HE and oil red staining results were consistent with that. So the alcoholic fatty liver model was successfully established in mice. Cell yield of each mouse was about 1. 5 × 106 ~2. 0 × 106 , KCs were double-labeled by murine macrophage cell surface marker F4/80 molecule and white blood cells surface antigen molecules CD45, F4/80 and CD45 double positive cells were selected to flow analysis. Data showed that natural CD68 ＋ was significantly lower in the model, a large number of the infiltration of mononuclear cells were elevated. Florescent real-time quantitative RT-PCR（qRT-PCR） results showed that cytokine,tumor necrosis factor （TNF-α）,interleu-kin-6 （ IL-6 ） , monocyte chemoattractant protein （ MCP-1 ） level increased significantly in liver tissue and primary cell. Conclusion The model of alcoholic fatty liver is successfully established and there is a high yield of cells in situ perfusion. The incidence of alcoholic fatty liver may be relative to hepatic macrophages constitute and pheno-typic changes, associated with elevated cytokine mediated infiltration of peripheral mononuclear cells.