Inhibition Of HGF Expression Reduces RPE Cell Proliferation In Proliferative Vitreoretinopathy Via RNA Interference

Proliferative vitreoretinopathy (PVR), as the complication of hegmatogenous detachment of retia, is the common failure cause of the operation on retinal detachment.Most scholars think that the beginning of PVR due to the migration and proliferation of cells,including retinal pigment epithelium and neuroglia cells,and it is controlled by a confused network that has not been clear to us. Because the molecular mecharism of PVR is very comlex,the optimal therapy for patients with these disease has yet to be defined. Some researchers applied medicine in treating PVR,however these medicine may damage the normal tissues when suppressing the proliferation of pathology,and it's popularization was restricted.Hepatocyte growth factor(HGF) is a kind of polypeptide that secrete from many types of cells and can stimulate the proliferation of glial cells. The physiological activity is very extensive,and it'abundance expression in the central neural system act on neuron and glial cells's growth and development. Nowadays, more and more research have shown that the HGF play a importment role in inducing the RPE migration and leading to PVR in the last, We focused on HGF to design and synthesis siRNA and its'eukaryotic expression vector inhibiting the HGF's expression in vitro and in vivo, providing experimental basis for the eventual treatment of PVR.objective:To determine the role of HGF in PVR, we successfully down-regulated HGF's expression by RNA interference (RNAi) technology, in HRPE D407 cell line.methods:1.The expression of HGF in HRPE D407 cell line;2.Detecting the interference effect of 3 pairs chemical synthesis dsRNA; Selection and synthesis of RNAi targets;3.Construction of shRNA-Expression Vector;4.Detecting the interference effect of 3 pairs shRNA-Expression Vector; Verification of RNAi targets;5. Detecting the inhibitive effect of PHGF2 in RPE proliferation;6. Detecting the inhibitive effect of PHGF2 in PVR Rat. Results:1. The expression of HGF in induced RPE is positive;2. The interference effect of 3 targets of chemical synthesis dsRNA were different, the target1 was most effective;3.Construction of shRNA-Expression Vector;4.Cell transfection and identification:After transfection of RNAi eukaryotic expression vectors into RPE D407 cell line using LipofectAmineTM 2000 reagent, the interference effect was in correspondence with the second part.5.The PHGF1 can downregulate the proliferation and migration of RPE.6.The PHGF1 can inhibite PVR's progression in animal model.Conclusions:1.The shRNA-expression vector of HGF was constructed successfully.2.RT-PCR and Western Blot results showed that HGF expression was significantly inhibited by siRNA transfectants in HRPE cells at mRNA and protein levels.3.The results of functional experiments showed that downregulation of HGF expression via RNA interference could influence the biological behaviour of HRPE cells.It could be presented in the following aspects:a. The expressions of HGF in the transfected cells were inhibited significantly compared with those in control by RT-PCR and western-blot assay. It mainly focused on the decrease of HGF in quantity.b. The suppression of HGF expression in human RPE cells could reduce the potential proliferation and migration of cells in vitro.Taken together, downregulation of HGF expression via RNA interference could inhibit significantly the secretions of HGF in the transfected cells. It could decrease the proliferative capability of HRPE. It was demonstrated that targeting HGF could potentially be a new experimental approach for PVR gene therapy. Alteration the biological behavior of RPE cells by RNAi technology is a trend for the treatment of PVR.