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Xiaoshuang Jiang, Yingqin Ni, Gezhi Xu; Overexpression of CCR2 Gene Promotes Efficient Recruitment of Retinal Microglia. Invest. Ophthalmol. Vis. Sci. 2012;53(14):784.
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Retinal microglia, playing protective roles in early retinal degeneration, can be activiated and recruited by chemokines. CCL2 (MCP-1) and it’s receptor, CCR2, have been implicated as key mediators for microglial cells traffic and accumulation in the lesion tissue. The purpose of our study was to explore whether overexpression of CCR2 gene in retinal microglia cells can increase the ability and efficiency of their recruitment towards CCL2.
Primary microglial cells were isolated from retina of newborn SD rats and were transfected with CCR2-GFP recombinant lentiviral vectors. CCR2 antibody was used to identify whether microglia transfected can express CCR2 by immunofluorescence. Overexpression of CCR2 was assessed by western blot analysis and fluorescence-activated cell sorting (FACS). The chemotaxis of microglia toward CCL2 was investigated using BD chemotaxis chamber assay: Microglia transfected with CCR2-GFP lentiviral vectors were compared with cells undergoing transfection with GFP-lentiviral vectors and cells without any intervention by the rate of migration from upper to lower chamber adding CCL2(20ng/ml).
Three days after transfection, Microglia transfected with CCR2-GFP and GFP both showed green under fluorescence microscopy. FACS suggested 97.6% cells were transfected with CCR2 gene. And the cells overexpressing CCR2 still sustain microglial morphology and showed a significant overexpression of CCR2. Chemotaxis assay revealed the number of microglia cells overexpressed CCR2 was statistically significant (P<0.05) increased than other two groups after 24 hours coculture.
Microglia can be transfected with CCR2-GFP recombinant lentiviral vectors and the rate of transfection is high. Constitutive overexpression of CCR2 in retinal microglia can be recruited to chemokines faster and more and these microglia present targets for cell-based therapeutic manipulation in retinal disease.
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