小鼠肝纤维化肝脏细胞上皮-间质转换的研究

Epithelial mesenchymal transition in experimental hepatic fibrosis of mice

目的探讨小鼠肝纤维化(hepatic fibrosis,HF)时,肝细胞发生的上皮-间质转换(epithelialmesenchymal transitions,EM T)的变化。方法建立小鼠CCl4诱导HF模型,随机分为模型组与正常对照组,每周2次,分别于第2、4、6、8、10、12周分批处死大鼠。用密度梯度离心和贴壁培养相结合的方法分离培养小鼠肝细胞,免疫荧光方法对细胞表型进行鉴定,采用H-E及Massion染色等观察E-钙粘附素(E-cadherin,E-cad)、成纤维细胞特异性蛋白-1(fibroblast specific protein-1,FSP-1)、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SM A)的表达。用免疫荧光显微镜及激光共聚焦定位分析观察EM T蛋白[FSP-1、白蛋白(albumin)、α-SMA、波形蛋白(vimentin,VM)、胶原Ⅰ(collagen Ⅰ)、纤维连接蛋白(fibronectin,FN)、N-钙粘附素(N-cadherin,N-cad)、E-cad]表达情况。结果小鼠HF时,肝细胞存在EMT现象。H-E、Massion染色及免疫荧光均显示,随着HF的程度加重,小鼠肝上皮细胞标志(E-cad、Albumin)逐渐减低,肝间质细胞标志(N-cad、FSP-1、VM、Collagen Ⅰ、FN及α-SMA)逐渐升高(P<0.05)。结论实验性小鼠HF的进程中,肝细胞发生EMT现象。

Objective To observe the epithelial mesenchymal transition （EMT） in experimental hepatic fibrosis （HF） of mice. Methods Eighty mice were randomly divided into fibrosis group and control group. Liver fibrosis was induced by intraperitoneal injection of carbontetrachloride, mice were sacrificed in batches after 2, 4, 6, 8, 10, 12 weeks. Liver cells were isolated and cultured, the cell morphology was observed by phase contrast microscope; the cell phenotype was identified with fluorescence immunoassay method. Cell differentiation was induced in the cultured hepatic cells, hematoxylin eosin （H-E） and Massion staining methods were used to analyze the E-cadherin （E-cad）, fibroblast specific protein-1 （FSP-1） and a-smooth muscle actin （α-SMA）. The expression of EMTproteins FSP-I, albumin, α-SMA, vimentin （VM）, collagen I , fibronectin （FN）, N-cadherin （N- cad） and E-cad were examined by immunofluorescence microscopy and laser confocal microscopy. Results After HF induced by CC14, the epithelial markers E-cad and albumin decreased gradually, while liver mesenchymal cell markers N-cad, FSP-1, VM, collagen I, FN and α-SMA increased gradually （P 〈 0.05 ）. Conclusion Liver cells present epithelial mesenchymal transition with the process of experimental hepatic fibrosis in mice.