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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。
Objective: To investigate the effect of inhibition of core fucosylation on renal tubular epithelial cell mesenchymal transition (EMT). Methods: The model of renal tubular epithelial HK-2 cells was established by transforming growth factor-β1 (TGF-β1). RNAi technique was used to silence the expression of α-1,6-fucosyltransferase (FUT8) The morphological changes of FUT8 gene were observed under light microscope. The expression of E-cadherin, N-cadherin, α The expressions of α-SMA and FSP-1 were detected by flow cytometry. Apoptosis was measured by flow cytometry. RESULTS: After 48 hours of TGF-β1 incubation, HK-2 cells lost their epithelial morphology and showed fibroblast morphology. The expression of fibronectin, FSP-1 and N-cad-herin were significantly increased , While the expression of E-cadherin, a marker of epithelial cell phenotype, was significantly decreased. At the same time, FUT8 gene expression was up-regulated and apoptosis was increased. However, the early transfection of FUT8 siRNA significantly attenuated these responses. CONCLUSION: FUT8-catalyzed core fucosylation is involved in the EMT process of HK-2 cells. Blocking the core fucosylation can effectively block the EMT of renal tubular epithelial cells.