| Objective: To study the effects and mechanism of Osteopontin (OPN) on humanproximal tubular epithelial cells (HK-2) to the transition ofmesenchymal cells (EMT).Methods: The HK-2cells were divided into six groups depending on differentconcentrations of added recombinant human osteopontin (rhOPN).The concentrations of rhOPN were:0,0.04,0.08,0.12,0.16,0.20μg/ml. After treated with rhOPN for48hours, the cells werecollected and observed the morphological changes using Confocalmicroscope.The protein and mRNA expressions of E-cadherin,α-SMA, Fibronectin were measured by Real-time PCR and Westernblot assay. The apoptosis of different groups cells were detected byFlow Cytometry.Results: OPN could change the shape of HK-2cells to the phenotype ofmesenchymal cells. Real-time PCR and Western blot analysis showedthat epithelial cell surface marker E-cadherin was reduced andmesenchymal cell markers α-SMA, Fibronectin were graduallyincreased as the concentration of rhOPN increased.The relative abundance of E-cadherin mRNA by Real-time PCR were:1947±301,1114±187.4,668.7±180.4,436.7±121.8,242.7±105.2,223.5±73.40;α-SMA mRNA were:0.01465±0.003384,0.03428±0.01200,0.06034±0.005653,0.09205±0.01068,0.1136±0.01892,0.1863±0.02175; Fibronectin mRNA were:0.0008176±0.0001026,0.001341±0.0002026,0.001708±0.0001278,0.002170±0.0001611,0.003369±0.0003143,0.004961±0.0003636. The relative abundanceof E-cadherin protein by Western blot analysis were:1.719±0.159,1.370±0.0159,1.238±0.031,0.916±0.056,0.520±0.065,0.456±0.073;α-SMA protein were:0.623±0.127,0.640±0.031,0.865±0.0453,1.015±0.107,1.290±0.103,1.381±0.040; Fibronectinprotein were:0.475±0.044,0.499±0.062,0.904±0.066,0.981±0.052,1.272±0.093,1.614±0.056(all p <0.05). The capacity of resistance tocell apoptosis was gradually increased by rhOPN in a dose-dependentmanner.Conclusion: Osteopontin could not only induce HK-2cells to mesenchymal celltransition。，but also inhence antiapoptotic action of HK-2cells in adose-dependent manner. |
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