Connective Tissue Growth Factor In The Mechanism Research Of Retinal Pigment Epithelial Cells

PurposeTo investigate the role of connective tissue growth factor(CTGF) in the retinal pigment epithelial cell (RPE) of proliferative vitreoretinopathy (PVR).Methods1. Culture ARPE-19cells then detect the influence CTGF to the extracellular matrix in Western Blot.2. Construct the p3xFLAG-CMV14/CTGF eukaryotic expression vector. Design and synthesize three pairs of targeted CTGF siRNA. Screen effective target points in Western Blot. Synthesize effective target of Oligo DNA, then anneal and synthetic double-stranded DNA, connect with lined pGC-LV-GFP vector, screen positive cloning in PCR and sequence; The recombinant plasmid was transfected into293T cell. Then transfer ARPE-19cell in packaging lentivirus, then investigate CTGF expression.3. Investigate the influence of TGF-β2producing CTGF, FN and α-SMA in the ARPE-19cell and the production of targeted interferenced by siRNA targeted CTGF.4. Investigate the influence of CTGF to ARPE-19cells migration and the influence of targeted siRNA CTGF.Results1. CTGF can stimulate the multiplication of ARPE-19cells obviously.2. Through the double enzyme cut and gene sequencing, we can confirm that the CTGF shRNA lentiviral vector was built successfully. CTGF protein decreased in ARPE-19cells infected by lentivirus.3. TGF-β2can stimulate the expression of CTGF, FN and α-SMA in ARPE-19cell, while the effect can be inhibited by targeted siRNA CTGF.4. In wounding test, the migration of siCTGF decreased obviously.ConclusionThe results indicate that CTGF significantly stimulate the proliferation, migration and epithelial-mesenchymal transformation (EMT) of ARPE-19cells while CTGF siRNA exhibits inhibitory effects.