【摘要】 目的:探讨阻抑核心岩藻糖基化修饰对肾小管上皮细胞间充质转化(EMT)过程的影响。方法:利用转化生长因子β1(TGF-β1)建立肾小管上皮HK-2细胞EMT的模型,应用RNAi技术沉默HK-2细胞的α-1,6-岩藻糖基转移酶(FUT8)基因表达,光镜下观察FUT8基因沉默后细胞形态变化,免疫印迹及免疫细胞化学方法测定细胞表型标记物蛋白E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、α-平滑肌肌动蛋白(α-SMA)和成纤维细胞特异性蛋白-1(FSP-1)的表达变化,流式细胞仪测定细胞凋亡。结果:TGF-β1孵育48 h后,HK-2细胞失去原有的上皮细胞形态,呈现纤维细胞形态,纤维细胞表型标记蛋白α-SMA、FSP-1及N-cad-herin表达明显升高,而上皮细胞表型标记蛋白E-cadherin表达明显下降,同时伴有FUT8基因表达上调,细胞凋亡增加,而提前转染FUT8 siRNA能明显减弱上述这些反应。结论:FUT8催化的核心岩藻糖基化修饰参与HK-2细胞的EMT过程;阻断核心岩藻糖基化修饰,能有效阻断肾小管上皮细胞的EMT过程。更多还原

【Abstract】 AIM: To investigate the role of inhibiting core fucosylation in the process of epithelial mesenchymal transition(EMT) in HK-2 cells.METHODS: An EMT cell model with transforming growth factor-β1(TGF-β1) was established and RNAi technique was used to silence the expression of α-1,6-fucosyltransferase(FUT8) gene which is responsible to catalysation of core fucose.The morphological changes of HK-2 cells were observed under light microscope.The epithelial cell marker E-cadherin and fibrotic cell markers N-cadherin,fibroblast-specific protein-1(FSP-1) and α-smooth muscle actin(α-SMA) were detected by Western blotting and immunocytochemistry.The apoptosis induced by TGF-β1 was determined by flow cytometry.RESULTS: After incubated with TGF-β1 at the concentration of 5 μg/L for 48 h,HK-2 cells lost epithelial morphology and showed fibrotic morphology.The expression of α-SMA,FSP-1 and N-cadherin was markedly increased,while E-cadherin was decreased.Meanwhile,the expression of FUT8 was up-regulated,and the apoptosis of the cells increased.However,pre-incubation of the cells with FUT8 siRNA inhibited these changes above.CONCLUSION: The core fucosylation involves in the process of EMT in HK-2 cells.Blockage of core fucosylation results in the inhibition of EMT in HK-2 cells.更多还原