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Downregulation of lentivirus-mediated ILK RNAi on tractional force generation in human retinal Müller cells.

Zheng YP, Liu H, Zeng H, Xiong L, Feng ZH, Sun NX - Acta Pharmacol. Sin. (2009)

Bottom Line: Significant decreases in ILK mRNA and protein expression were detected in Müller cells carrying lentiviral ILK-shRNA vector.Lentivirus-mediated ILK RNAi decreased cell migration and contractile force generation by inhibiting alpha-SMA stress fiber formation in human retinal Müller cells.This tool might be useful to treat ocular fibroproliferative diseases associated with transdifferentiated Müller cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, 2nd Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an, China.

ABSTRACT

Aim: To investigate the effect of lentivirus-mediated integrin-linked kinase (ILK) RNA interference (RNAi) on human retinal Müller cells transdifferentiation into contractile myofibroblasts.

Methods: A lentiviral vector expressing ILK-specific shRNA was constructed and introduced into cultured retinal Müller cells. Silencing of the ILK gene was identified by real time RT-PCR and Western blot. The Müller 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 Müller 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 Müller cells. This tool might be useful to treat ocular fibroproliferative diseases associated with transdifferentiated Müller cells.

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Related in: MedlinePlus

Identification of cultured hRMCs. The primary cultured retinal Müller cells (RMCs) were identified morphologically by phase-contrast images (A) and positive immunofluorescence staining for glutamine synthetase (GS, B). (Scale bar=100 μm).
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fig2: Identification of cultured hRMCs. The primary cultured retinal Müller cells (RMCs) were identified morphologically by phase-contrast images (A) and positive immunofluorescence staining for glutamine synthetase (GS, B). (Scale bar=100 μm).

Mentions: Phase-contrast images of primary cultured retinal Müller cells on the 3rd day after dissociation are shown in Figure 2A. Cells proliferated from isolated single cells or cell clusters and displayed two types of morphology; most of them were polygonal and flat, while a few were elongated. The cells were identified by staining for glutamine synthetase (GS), a specific marker of Müller cells. Müller cells are the only cells in the retina that express the enzyme glutamine synthetase (GS)28, 29, but expression of GS in cultured Müller cells is transient. Expression appears two days after dissociation and decreases when cells achieve confluence23. Figure 2B shows GS expression in cultured Müller Cells on the fourth day after dissociation. Positive GS staining was observed in the nuclei of small cells that appeared to be proliferating, while expression appeared to decrease in cells that were large and confluent.


Downregulation of lentivirus-mediated ILK RNAi on tractional force generation in human retinal Müller cells.

Zheng YP, Liu H, Zeng H, Xiong L, Feng ZH, Sun NX - Acta Pharmacol. Sin. (2009)

Identification of cultured hRMCs. The primary cultured retinal Müller cells (RMCs) were identified morphologically by phase-contrast images (A) and positive immunofluorescence staining for glutamine synthetase (GS, B). (Scale bar=100 μm).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4007506&req=5

fig2: Identification of cultured hRMCs. The primary cultured retinal Müller cells (RMCs) were identified morphologically by phase-contrast images (A) and positive immunofluorescence staining for glutamine synthetase (GS, B). (Scale bar=100 μm).
Mentions: Phase-contrast images of primary cultured retinal Müller cells on the 3rd day after dissociation are shown in Figure 2A. Cells proliferated from isolated single cells or cell clusters and displayed two types of morphology; most of them were polygonal and flat, while a few were elongated. The cells were identified by staining for glutamine synthetase (GS), a specific marker of Müller cells. Müller cells are the only cells in the retina that express the enzyme glutamine synthetase (GS)28, 29, but expression of GS in cultured Müller cells is transient. Expression appears two days after dissociation and decreases when cells achieve confluence23. Figure 2B shows GS expression in cultured Müller Cells on the fourth day after dissociation. Positive GS staining was observed in the nuclei of small cells that appeared to be proliferating, while expression appeared to decrease in cells that were large and confluent.

Bottom Line: Significant decreases in ILK mRNA and protein expression were detected in Müller cells carrying lentiviral ILK-shRNA vector.Lentivirus-mediated ILK RNAi decreased cell migration and contractile force generation by inhibiting alpha-SMA stress fiber formation in human retinal Müller cells.This tool might be useful to treat ocular fibroproliferative diseases associated with transdifferentiated Müller cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, 2nd Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an, China.

ABSTRACT

Aim: To investigate the effect of lentivirus-mediated integrin-linked kinase (ILK) RNA interference (RNAi) on human retinal Müller cells transdifferentiation into contractile myofibroblasts.

Methods: A lentiviral vector expressing ILK-specific shRNA was constructed and introduced into cultured retinal Müller cells. Silencing of the ILK gene was identified by real time RT-PCR and Western blot. The Müller 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 Müller 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 Müller cells. This tool might be useful to treat ocular fibroproliferative diseases associated with transdifferentiated Müller cells.

Show MeSH
Related in: MedlinePlus