Inhibition Of PDGF-A 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.Now operation is the most main treatment for patients with PVR.Despite the medical treatment level in invasive techniques including vitrectomy,posterior retinotomy,use of intraocular silicone or gas,and scleralbuckling,has been progressed rapidly in this era,and the apparatus used in operation have been more and more sophisticated.But the visual and anatomical outcomes are disappointing with these more complex and invasive techniques and the risk of recurrence for these patients is remain very high.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. platelet-derived gowth factor(PDGF)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 PDGF play a importment role in inducing the RPE migration and leading to PVR in the last.With the development of molecular biology,appling the gene technology in preventing and treating PVR has been the hot spot of many ophthalmologic researchers.RNA interference(RNAi)is a phenomenon displayed by most eukaryotic cells to get rid of foreign double-stranded RNA molecules from themselves.RNAi technology has become one of methods to gene silence. RNAi has now been demonstrated that it can close specific gene's expression in mRNA level,post transcription through double-stranded RNA(dsRNA). the target gene'mRNA can be degradated efficiently and specifically when the double-stranded RNA(dsRNA)which has homologous sequences with the target gene's mRNA is transfected into cells,and the gene's expression is inhibited.We focused on PDGF-A isoforms of PDGF,to design and synthesis siRNA and its' eukaryotic expression vector inhibiting the PDGF-A's expression in vitro and in vivo,providing experimental basis for the eventual treatment of PVR.[objective]To determine the role of PDGF-A in PVR,we successfully downregulated PDGF-A's expression by RNA interference(RNAi)technology,in HRPE D407 cell line. [methods]1.The expression of PDGF-A 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 PIP1 in RPE proliferation;6.Detecting the inhibitive effect of PIP1 in PVR Rat.[Results]1.The expression of PDGF-A in induced RPE is positive;2.The interference effect of 3 targets of chemical synthesis dsRNA were different,the targetl was most effective;3.Construction of shRNA-Expression Vector;Three pairs of oligonucleotides were designed and synthesized which encoding short hairpin transcripts directed against three different parts on PDGF-A mRNA.These three pairs of oligonucleotides were then ligated into the Linearized plasmid pGPU6/GFP/Neo.The constructed vectors were confirmed by enzyme cutting:restriction endonuclease BamHâ… and Pstâ… were employed to cutting the recombinant plasmid,then the cutting product were electrophoresis in 1%agarose gel,and identified by DNA sequencing at last: the plasmid were sequenced by shanghai yingjun biotechnology limited company.The recombination plasmid was construct succeed.4.Cell transfection and identification:After Transfection of RNAi eukaryotic expression vectors into RPE D407 cell line using LipofectAmine^TM2000 reagent,,the interference effect was in correspondence with the second part.5.the PIP1 can downregulate the proliferation and migration of RPE. 6.the PIP1 can inhibite PVR's progression in animal model.[Conclusions]:1.The shRNA-expression vector of PDGF-A was constructed successfully.2.RT-PCR and Western Blot results showed that PDGF-A 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 PDGF-A expression via RNA interference could influence the biological behaviour of HRPE cells.It could be presented in the following aspects:a.The expressions of PDGF-A in the transfected cells were inhibited signifi-cantly compared with those in control by rt-pcr and western-blot assay. It mainly focused on the decrease of PDGF-A in quantity.b.The suppression of PDGF-A expression in human RPE cells could reduce the potential proliferation and migration of cells in vitro.Taken together,downregulation of PDGF-A expression via RNA interference could inhibit significantly the secretions of PDGF-A in the transfected cells.It could decrease the proliferative capability of HRPE.It was demonstrated that targeting PDGF-A 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.