Inhibitory Effects Of Recombinant Of Phosphatase And Tensin Homolog Tumor Suppressor Gene Transfered On Vascular Restenosis

1 Objective:Nearly, percutaneous transluminal coronary intervention (PCI) has been developed in many hospitals in China with rapid development and wide apply of intervention cardiopathy. But coronary artery restenosis, that is major lasting combination, happens frequently. It is major obstacle in intervention treatment of coronary heart disease and affects lasting curative effect badly.In the form mechanism of restenosis after PCI, the theory of vascular smooth muscle cells(VSMC) proliferating and moving is very important. Drug therapy for RS is not satisfactory. Toxicity of medicine restricts its clinical application. Therefore it is very imperative to find new ways to control RS.Since 1990s a new technique has been born.that is gene therapy, opening up a new years of modern therapy.PTEN gene is a new gene gained by Li etal in 1997, and its 175 amino acid sequence is in common with cell franework albumen(tensin).The gene is named by cell tensility albumen, phosphorate enzyme gene deleted on chromosome 10. The gene could induce cell cycle block by regulating PI3K/Akt signal transfer approach, and regulating cell cycle evolvment and cell survival. It could induce cell death and restrain tumor metastasis by affecting FAK and MAPK bypass. It could restrain vascular formation. The level of mRNA of vascular endothelium growth factor(VEGF) was reduced by activated LY294002(the inhibitor of PI3K), what hints that PI3K is very important on vascular formation and regulation of VEGF expression. It is reported that PTEN could regulate a number of signals in correlative with proliferation and restrain cell to span G-S point.Over express of PTEN restrained the reaction of VSMC that appeared key funtion on internal proferation and restenosis. It has been valid method of preventing and curing many vascular diseases including restenosis.In the same time, PTEN appeared negative control on cell spliting and moving. Koide reported that these effctions might due to restraining inflammation reaction, including restraining the incursion of macrophage cell and the express of inflammation cell factor.Currently, the research is limited on the role of PTEN in RS. Scholars only observed the role of PTEN in RS morphologic cases. The role of PTEN in correlation index and process mechanism of RS has not been reported. Accordingly, firstly PTEN eukaryotic expression vector is constructed in our experiment. In vitro studies, we observed the proliferation induced by PDGF conditioned medium is inhibited on PTEN gene-modified VSMC cells. In vivo studies, We also observed PTEN transfered by artery cavity injection restrains RS in model rabbits.2 contentFirst PTEN gene were cloned from pGEM-T-PTEN plasmid into the eukaryotic expression vector pcDNA4/myc-His, recombinant plasmid pcDNA4/myc-His-PTEN was constructed. The truth of gene was tested by application digestion and DNA sequencing. VSMC cells were transfered with pcDNA4/myc-His-PTEN eukaryotic expression vector by Liposome. After G418 selection, MTT assay observed transfected VSMC proliferation induced in human PDGF conditioned medium. Then the gene expression of RS model rabbit was detected in the arteries by injection into the artery cavity, and the the restraining role to RS was studied.The results show that we constructed the eukaryotic expression vector pcDNA4/myc-His-PTEN successfully. By identifing and sequencing the plasmid we found PTEN was same as the presence of the gene sequence in Genebank. PTEN was transfered into VSMC cells by liposome and expressed stable protein, which could be suggested through RT-PCR and Western blot techniques. The proliferation of VSMC cells induced by PDGF conditioned medium could be inhibited after transfection through MTT assay.On the other hand,in vivo Liposome-induced pcDNA4/myc-His-PTEN could be expressed stably by injecting in rabbits' artery cavity, and inhibited new intermal form significantly also.3 Major achievements(1)Internal and external we observed the proliferation of human VSMC cells in PDGF conditioned medium after PTEN gene was transferred into cells by liposome first experim-entally,and we observed the changes of its signal transfection bypass. In conclusion PTEN could inhibit VSMC cell proliferation in this condition.(2)Internal and external we observed the eukaryotic expression vector pcDNA4/myc-His-PTEN was injected into rabbits'artery cavity by liposome and expressed stable protein in the arteries, at the same time the compound could inhibit RS significantly first experim-entally4 Significance and value of the research achievements:Since 1997 PTEN gene was found, a great deal of research has been done by scholars. As a endogenous tumor inhibition factor PTEN has good histocompatibility and could resist angiogenesis significantly. Internal and external it was reported that PTEN gene can be transferred to the target cells by virus vector, or the compound AV-PTEN could be injected into artery cavity in vivo,and the results suggested that PTEN protein content increased in arteries obviously, and in vivo could survival more time to devote treatment effect, had broad prospects for the future.Although detecting morphological changes in RS cases found PTEN could inhibit new internal form, it was not explicit about the mechanism of PTEN in RS and it was not reported about appropriate research study. We tested PTEN could inhibit VSMC cell proliferation induced with PDGF conditioned media first experimentally, that was due to the gene translation and express of Akt/NF-κB signal convey bypass partly. And PTEN gene was transferred in arteries could also inhibit RS significantly. So this reseach laid a foundation for further study about PTEN roles in RS and its application in the RS prevention.