| Part I Construction of rat SOCS3recombinant adenovirus vector and its expression in rat primmary vascular smooth muscle cellsObjective With the aim of establishing the foundation for experimental research for gene therapy of vein graft diseases, it is performed that the recombinant adenovirus vector which can carry rat SOCS3gene is constructed and used to assess its expression efficiency in vascular smooth muscle cells.Methods The rat SOCS3plasmid(pUC57-Simple-rSOCS3) and advenovirus shuttle plasmid(pYr-adshuttle-4) which contain green fluorescent protein(GFP) reporter gene were cleaved by restriction endonuclease BamHI and EcoRI. The target gene fragments were connected together to generate a recombinant plasmid pYr-ads-4-rSOCS3and then transfected into E.coli DH5a. The plasmid is confirmed to be constructed as expectation by enzyme digestion and sequence reaction. The plasmid pYr-ads-4-rSOCS3and5Type adenovirus backbone plasmid pAd/PL-DEST were reconstructed by homologous recombination processes to obtain rat SOCS3recombinant adenovirus vector which named pYrAd-rSOCS3.The plasmid pYrAd-rSOCS3was linearized by Pac Ⅰ and subsequently transfected into HEK293cells for packaging and amplification. After purifying, virus titer was determined by tissue culture infectious dose50(TCIDso). Polymerase Chain Reaction(PCR) was used to confirm the existence of recombinant rat SOCS3gene. The primary rat vascular smooth muscle cells were infected by pYrAd-rSOCS3. After24hours, the expression of GFP was observed by fluorescent microscopy. SOCS3mRNA and protein expression were determined by real time-PCR and Western blot.Results Restriction endonuclease and PCR analysis demonstrated that the recombinant adenovirus vector was constructed correctly. hEF1a and CMV promoter existed in the expression vector. The virus titer reached2x1010pfu/ml. Infection efficiency of recombinant adenovirus in vascular smooth muscle cell was more than80%.The results of real time-PCR and Western blot showed that SOCS3mRNA and protein expression were up-regulated significantly in the infected vascular smooth muscle cells.Conclusion In this study, we successfully constructed a recombinant adenovirus vector that carries rat SOCS3gene and can be helpful for further research of the effects and mechanisms of the SOCS3gene on the vein graft diseases. Part IIRegulation of SOCS3on neointimal hyperplasia of vein graft in rat modelObjective To explore the regulation and mechanism of SOCS3on neointimal hyperplasia in vein graft disease.Methods The external jugular vein to carotid artery grafts model in rat was established. Ninety rats were randomly divided into three groups: pYrAd-rSOCS3transfected group (n=30), pYrAd-GFP transfected group (n=30), and control group (n=30). The rats were sacrificed on day1, day3, week1, week2, or week4after the operation (6rats per time point). The normal veins were detected as blank controls.The real time-PCR and western blotting were used to detected the IL-1β, IL-6, MCP-1, ICAM-1, TNF-a, STAT3(only protein), P-STAT3(only protein), and SOCS3mRNA and protein expression levels in samples harvested on day1, day3, and week1. Samples collected at weeks2and4were used for histological and immunohistochemical analyses. The measurement of intimal thickness was performed in hematoxylin-eosin or Verhoeff-Van Gieson staining sections.The CD68and PCNA-positive cells were counted in CD68and PCNA immunohistochemical sections.Results (1)Compared with normal veins, IL-1β, IL-6, MCP-1, ICAM-1, TNF-a, STAT3, P-STAT3, and SOCS3mRNA and protein expression levels were significantly higher in the graft samples within1week after surgery.(2) Compared to the pYrAd-GFP transfection group and control group,in pYrAd-rSOCS3transfected group,the SOCS3mRNA and protein expression of grafted veins were further up-regulated and peaked on third day,however, IL-1β, IL-6, MCP-1, ICAM-1, TNF-a, STAT3, P-STAT3mRNA and protein level were markedly down-regulated and decreased to the baseline level.(3)Compared to the pYrAd-GFP transfection group and control group,the neointimal thickness of vein grafting two weeks and four weeks after surgery in pYrAd-rSOCS3transfected group were decreased.(4)Two and four weeks after operation, a weaker PCNA staining signal was observed in the intima and media of the vein grafts in the pYrAd-rSOCS3treated group compared with the pYrAd-GFP treated group and control vessels.(5)Mac-2(CD68) staining showed that, compared with pYrAd-GFP transfection group and control group,the number of macrophages which is the primary inflammatory cells were significantly decreased in pYrAd-rSOCS3treated veins.Conclusions These results imply that an acute inflammatory response emerged in the graft vein that was grafted into artery. Similarly, JAK2/STAT3signaling may be activated and SOCS3may play a role in pro-inflammatory signaling because the expression of STAT3, P-STAT3, and SOCS3was up-regulated in the graft vessels. Furthermore, local over-expression of SOCS3in the graft vein can inhibit neointimal hyperplasia and VSMCs proliferation by down-regulating pro-inflammatory cytokines expression and alleviating inflammatory response in the grafted veins, suggesting that SOCS3may have a negative regulatory role in graft vein stenosis through negatively regulating the JAK2/STAT3pathway. Part ⅢRegulation of SOCS3in inflammation,migration and proliferation of vascular smooth muscle cell in vitroObjiective To study the effection of SOCS3on the inflammation, migration and proliferation of VSMCs in vitro.Methods The small interference RNA plasmid targeting rat SOCS3(SiRNA-rSOCS3) and the empty plamid as control(SiRNA-control) were constructed.The rat VSMCs were cultured.There were two large groups of A(SOCS3up-regulated):control, PDGF-BB group,PDGF-BB+pYrAd-rSOCS3group, PDGF-BB+pYrAd-GFP group and B(SOCS3down-regulated):control,PDGF-BB+SiRNA-rSOCS3group and PDGF-BB+SiRNA-control group. The pYrAd-rSOCS3and SiRNA-rSOCS3were transfected into VSMCs induced by PDGF-BB. After24hour, real time reverse transcription polymerber chain reaction(RT-PCR) and western blotting were used to analyse the mRNA and protein expression of SOCS3、STAT3(only by western blotting). P-STAT3(only by western blotting)、IL-1β、IL-6、TNF-α、MCP-1and ICAM-1. The MTT/BrdU, Transwell and flow cytometry assay were used to detect VSMCs proliferation, migration and cell cycle progression, respectively.Results Compared with control group, the mRNA and protein expression of SOCS3%STAT3、P-STAT3. IL-1β、IL-6. TNF-α、 MCP-land ICAM-1were significantly up-regulated in VSMCs stimulated by PDGF-BB. But VSMCs were transfected with pYrAd-rSOCS3before treated with PDGF-BB, the expression of mRNA and protein SOCS3were further up-regulated, STAT3,P-STAT3,IL-1β,IL-6,TNF-a,MCP-1and ICAM-1were significantly down-regulated as compared with PDGF-BB stimulation alone and pYrAd-GFP transfection. However, the expression of those related-cytokines in SiRNA-rSOCS3group was markedly increased as compared with PDGF-BB group and PDGF-BB+SiRNA-control group. The OD values, BrdU-positive cells, cells migrated to lower chamber, percentage of cells in the G2/M+S phase were increased in VSMCs with PDGF-BB stimulation. Before PDGF-BB stimulation,with pYrAd-rSOCS3and SiRNA-rSOCS3incubation for30minutes, the OD values, BrdU-positive cells, cells migrated to lower chamber, the percentage of cells in the G2/M+S phase were significantly decreased and increased, respectively.Conclusions These results imply that PDGF-BB, a strong stimulator, can promote transformation of VSMCs phenotype form a quiescent contractile state to a synthetic state by activating JAK2/STAT3pathway. Over-expesssed SOCS3might inhibit pro-inflammatory effect, migration and growth of VSMCs by blocking STAT3activation and phosphorylation. These data in vitro confirm that SOCS3may play a negatively regulatory role in development and pregression of vein graft failure.These conclusions can provide a novel strategy for clinical treatment with vein graft diseases and a new theoretic clue to for related drug development. |
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