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Zheng YP, Liu H, Zeng H, Xiong L, Feng ZH, Sun NX: Downregulation of lentivirus-mediated ILK RNAi on tractional force generation in human retinal Muller cells. Acta Pharmacol Sin. 2009 Dec;30(12):1625-33. Epub 2009 Nov 16.
AIM: To investigate the effect of lentivirus-mediated integrin-linked kinase (ILK) RNA interference (RNAi) on human retinal Muller cells transdifferentiation into contractile myofibroblasts. METHODS: A lentiviral vector expressing ILK-specific shRNA was constructed and introduced into cultured retinal Muller cells. Silencing of the ILK gene was identified by real time RT-PCR and Western blot. The Muller cell phenotype change was confirmed by immunodetection of alpha-smooth muscle actin (alpha-SMA) stress fiber formation. The generation of tractional force was assessed using a tissue culture assay with cells incubated in three-dimensional collagen gels; cell migration was determined by the Boyden chamber method, using 10% FBS as a chemotactic factor. RESULTS: Significant decreases in ILK mRNA and protein expression were detected in Muller cells carrying lentiviral ILK-shRNA vector. Cells treated with anti-ILK siRNA showed less alpha-SMA stress fiber formation under hypoxic conditions or cell subcultivation. Lentiviral ILK-shRNA vector transfection also significantly reduced cell migration and cell-mediated gel contraction. CONCLUSION: Lentivirus-mediated ILK RNAi decreased cell migration and contractile force generation by inhibiting alpha-SMA stress fiber formation in human retinal Muller cells. This tool might be useful to treat ocular fibroproliferative diseases associated with transdifferentiated Muller cells. |
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