Construction Of The Recombinant Lentiviral Vectors Encoding Human TRADD Gene And The Research About TRADD Lentiviruses On Inhibiting Proliferation Selectively Of Hypertrophic Scar Fibroblast

Background and Objectives:Restenosis of esophagus after stent implantation with a high ratio of incidence is one ofthe most common diseases, which affects the clinical application of stent implantationgreatly[1]. Restenosis mainly expressed as fibroblast activation and over proliferation, thengranulation and fibrosis[2]. The key factor for restenosis is abnormality of cellular proliferationand apoptosis in fibroblast. TRADD may play an important role in the TNF-α-mediatedapoptosis which could be one of the main apoptotic signaling pathways. Biological activity ofTNF-α is realized mainly by TNFR1-mediated pathways. The combination of TRADD andTNFR1is an important step in TNF/TNFR1mediated apoptotic signaling pathways. TRADDconnected to TNFR1by joining their DD domain together, transmits the signal ofprogrammed cell death through activation of downstream molecules. Sayah[3]discovered thatthere were8down-expressed gene including the TRADD in64apoptosis associated genes inkeloid as compared with normal scar tissue. Over proliferation of hyperplastic scar fibroblast(HSFb) is induced by down-expressed of TRADD via inhibition of TNF-α mediatedapoptosis.Lentiviruses which use HIV-1as a resource have superiority over the other transgenictechnology in infection efficiency, stable expression, weak antigenicity, high degree ofmaneuverability, capability of containing a large quantity outside source purpose genefragment、infection cells either in dividing phase or not[4,5]. In recent years the study of genetherapy using Lentiviruses as the gene vector has become the hot research areas[6,7].The aim of this study was to construct a recombinant lentiviral vector carrying human TRADD gene, and to observe the expression of fusion protein after hypertrophic scarfibroblasts that were used as research object, normal fetal fibroblasts (NFFb) and Hacat cellsthat were used as the control, were infected by TRADD Lentiviruses. MTT was used toexplore the role of TRADD Lentiviruses with or without10ng/ml TNF-α on inhibitingproliferation selectively of hypertrophic scar fibroblast, which could provide scientific basisfor experimental studying of TRADD as a potential therapeutic target preventing restenosisafter stent implantation.Methods:1. To construct the recombinant lentiviral vector carrying human TRADD gene: TheTRADD specific fragment was amplified by PCR technique and cloned into the EcoRⅠsiteof the lentiviral vector pLVX-EGFP-3FLAG-Puro. Recombinant Lentiviruses were producedafter the293FT packing cells were contransfected with pLVX-TRADD-EGFP-3FLAG-Puroand lentiviral packaging plasmids, while titer of the virus was detected by Real-time PCR.2. The expression of the TRADD in HSFb and NFFb was analyzed by the immunofluo-rescence method and Western blot.3. Chose the best MOI of HSFb, NFFb and Hacat, then transfect HSFb, NFF band Hacatwith TRADD Lentiviruses useing the best MOI as optimum condition. Western-blot was usedto analyze the expression level of TRADD-GFP-FLAG fusion protein.4. Effect of TRADD Lentiviruses with or without10ng/ml TNF-α on proliferation andapoptosis of HSFb, NFFb and Hacat were analyzed by MTT method. The HSFb, NFFb andHacat were divided into three groups independently: transfection group (experiment group),transfection+TNF-α group (experiment group+TNF-α); empty vector transfection group(control group), empty vector transfection+TNF-α group(control group+TNF-α); non-transfec-tion group (blank group) and on-transfection+TNF-α group (blank group+TNF-α).Results:1. The recombinanted lentiviral vector pLVX-TRADD-EGFP-3FLAG is constructedsuccessfully with the titers of3.22×10~8IU/ml.2. The expressions of TRADD that decreased markedly in HSF compared with those inNFF verified by the immunofluorescence method and Western blot.3. MOI100of lentiviruses was found to infect more than80%of HSFb, NFFb and Hacat.Western blot demonstrated that fusion protein was effectively expressed in HSFb, NFFb and Hacat cells.4.(1) NFFb cells were treated with empty vector transfection displayed a faster growthrate in contrast to the other groups, while there is no difference in growth rate between blankgroup and experiment group24h later;10ng/ml TNF-α showed no significant effects ongrowth activity of NFFb.(2) The fastest growth of HSFb was control group and it was followed by blank groupand experiment group;10ng/ml TNF-α showed no significant effects on growth activity ofHSFb.(3) Growth rate of Hacat cells in blank group was much faster than that in control groupand experiment group, while there is no difference in growth rate between control group andexperiment group48h later;10ng/ml TNF-α showed significant promotive effect on growthactivity of Hacat within72-96hours.Conclusions:1. The recombinanted lentiviral vector pLVX-TRADD-EGFP-3FLAG is constructedsuccessfully and the TRADD-GFP-FLAG fusion protein holding biological activity in HSFb,NFFb and Hacat is demonstrated by Western-blot.2. The expressions of TRADD decreased markedly in HSFb compared with those inNFFb, suggesting that TRADD might be involved in the formation of hyperplastic scar.3.10ng/ml TNF-α showed no significant effects on growth of fibroblast but promotiveeffect on growth of Hacat. These results suggest that TNF-α had distinct effects on differentspecies cells.4. Proliferation of fibroblast, but not Hacat, was inhibited by the overexpression ofTRADD-GFP-FLAG fusion protein, indicating that TRADD plays a very important role inapoptotic pathway mediated by TNF-α in fibroblast, but not in Hacat.5. The expression of TRADD protein were markedly upregulated after HSFb, NFFb wereinfected by TRADD Lentiviruses, but there was only inhibitory effects on growth of HSFb,which suggest that there is a certain balancing mechanism between cell apoptosis andactivation of NF-κB mediated by TNF-α in fibroblast. The balances of the two signalingpathways guarantee the normal state of cell growth activity.