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The Recombinant Psilencer1.0-U6-siRNA-STAT3 Inhibits the Neointima Hyperplasia of Arterialized Venous of Rat in Vitro and in Vivo

Author: SunJiangBin
Tutor: LiuKeXiang
School: Jilin University
Course: Surgery
Keywords: The recombinant Psilencer1.0-U6-siRNA-STAT-3 plasmid VSMCs STAT3 proliferation of cells
CLC: R543
Type: PhD thesis
Year: 2010
Downloads: 62
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Cardiovascular diseases due to atherosclerosis are the main reasons for deaths in the western world. For many patients with severe coronary disease,bypass-surgery is required. Veins are frequently used and have far better results compared with synthetic conduits. But Veins are not ideal bypass conduits which are reflected by the incidence of vein graft failure. Recent studies demonstrated that the primary patency of vein grafts was not better than 60-80 % within the first year after surgery treatment,and by the time when in the tenth year,the failure rate is 50%. A large amount of clinical and experimental studies have demonstrated that intimal thickening may lead to the restenosis of the vein graft. Proliferation and migration of vescular smooth muscle cells(VSMCs) may be a key contributor to neointimal formation. It was demonstrated in a canine vein graft model that the SMCs were transformed from contrictile to a secretory phenotype in response to vessel wall injury. By the switch of phenotypes the SMCs enter a state enabling proliferation,migration,and gain synthetic capacity,which is a pre-requisite for neointimal formation. It has been a well- supported opinion,that the source for the intimal cell population is SMCs from the media. Accumulating evidence sμggests that neointima formation is mediated by a complex interaction of a variety of growth regulatory molecules, such as growth factors,vasoactive peptides,inflammatory cytokines and chemokines.However,it remains largely unknown which signaling pathways are responsible for neointima formation. STAT3,as one of the candidate signaling pathways,plays an important role in the SMCs proliferation and migration. STAT3 contains a conserved amino-terminal,aDNA-binding domain that binds specific interferon-activated DNA sequences,a SH-2 domain for receptor recruitment and STAT dimerization,and a transactivation domain. STAT3 protein is activated by a variety of stimuli. It is also involved in contradictory responses such as proliferation and apoptosis. Following activation,STAT3 protein changes conformation,forms homo- or hetero-dimers,and translocated from the cytoplasm to the nucleus. In the nucleus,STAT3 binds cis elements,which induce growth responsive and inflammatory genes; including junB,IRF-1,CyclinD1 and anti-apoptotic factors Bcl-xL and Bcl-2. In cultured rat SMCs , the STAT3 activation is substantially involved in the angiotensin II(AII)-or platelet-derived growth factor (PDGF)–induced proliferation.To prevent restenosis of vein graft,extensive research has been conducted including standard technique of vein harvest and drug therapy et al,but these methods were not an ideal therapy. Two decades ago,gene therapy emerged since the first description of the site-directed transfer of exogenous genetic material into the vascular system .In initial experiments,vascular gene transfer were used to evaluate hypothesis by targeted overex- pression of selected proteins in the vasculature.Therefore,gene therapy as a potential strategy for the treatment of restenosis after angioplasty and vascular bypass graft has been extensively studied. Although effective delivery tools are remain to be solved,these reports sparked the anticipation that such approaches would be used for the therapeutic modulation of vascular responses,including stenosis after injury and restenosis.In this study,the potential anti-proliferation effects of the recombinant plasmid- Psilencer1.0-U6-siRNA-STAT-3 on VSMCs in vitro and in vivo were investigated to find potential novel strategies for the treatment of restenosis after vein grafting. This plasmid is constructed by RNAi technique to aim at inhibiting mRNA expression and phosphorylation of STAT3. Following this process,Psilencer1.0-U6-siRNA-STAT-3 block the tranicription and translation of the downstream target genes-CyclnD1 and Bcl-2. By this mechanism,proliferation of VSMCs and the neointima formation are inhibited,and restenosis of transpl- anted vein graft is lessen or even blocked. Our study afford a theory base for anti-restenosis after coronary artery bypass graft on clinic.Psilencer1.0-U6-siRNA-STAT-3 inhibited the proliferation of rat vascular smooth muscle cells in vitroMethods:RSMCs (2×105 cells per well) seeded in 6-well culture plates were divided into 3 groups: the control group that no transfection were done; the scramble plasmid group in which the scramble plasmid transfected RSMCs and recombinant plasmid group in which RSMCs were transfected with Psilencer1.0-U6-siRNA-STAT-3. The lipofectamine 2000,as the vector,was mixed with plasmid (recombinant plasmid group or scramble)or itself at a proportionality of 0.5μl:0.2μg and added to the RSMCs .After coincubation for 72h,we use contrast phase microscope to observe the shape change of RSMCs; we added MTT solution (5 mg/mL in phosphate-buffered saline (PBS) to observe the proliferation capability of RSMCs; RT-PCR were examined to observe the mRNA expression of STAT3 and its downstream target gene CyclnD1 and Bcl-2; Western-blot were performed to observe expression of their fuctional products-protein.Results:Observed with contrast phase microscope,Most RSMCs of the control group and scramble plasmid group are clostridial、anomalous triangle or fan shape with clear and intermediate nucleis and proliferate vigorous;however,RSMCs of Psilencer1.0-U6-siRNA- STAT3 group lost their intrinsical form and proliferated slowly with a pycnosis nucleis and some of them even floated in the culture bottle. By MTT assays,the proliferation of RSMCs in Psilencer1.0-U6-siRNA-STAT-3 group was significantly inhibited compared with the control group. RT-PCR detection show significantly decreased mRNA expression of STAT3、CyclnD1 and Bcl-2 in the Psilencer1.0-U6-siRNA-STAT-3 group,whereas the mRNA exepression of them were indistinguishable between the control and scramble plasmid group. Western-blot detection show that STAT3、Bcl-2 and CyclinD1 phosphorylation in the Psilencer1.0-U6-siRNA-STAT-3 group significantly declined compared with those of the control group.Psilencer1.0-U6-siRNA-STAT-3 inhibited the proliferation of vascular smooth muscle cells in vivoMethods:we constructed vein graft model and the Psilencer1.0-U6- siRNA- STAT-3/ lipofectamine2000 mixture were mixed with bioprotain gel (volume 80 ul) and sprayed to adventitia of vein graft. 168 free male wistar rats (250-350g) were divided into two groups: the control group (n=84) and recombinant plasmid(experiment)group (n = 84). On day 3、7、14 and 28 after operative procedure,the rats were killed. 5 of these interposed vein graft containing an adjacent section of the left carotid artery were harvested for morphological examination(HE) and immuno- histochemistry(PCNA); 8 of these interposed vein graft were harvested for RT-PCR detection and 8 for Western-blot assay.Results:The intimal thickening on 3d post operative procedure had not much difference in the two groups,and it peaked on day 7 in the scramble plasmid group,while that in the recombinant plasmid group on day 7 had only scabrosity,on day 14 and 28,it had even no thickening in the experiment group. The thickness of intima had significant difference on day 7(12.3±0.21 and 6.7±0.25 respectively) and 14(7.2±0.31 and 4.6±0.14 respectively); PCNA index in the control group on day 7 and 14 were 31.3±4.7 and 27.2±5.7 respectively and that of experiment group were 23.3±2.8 and 15.6±0.4 respectively; there are significant deviation between the control and experiment group on day 7 and 14 of PCNA positive cells. RT-PCR detection shows that from day 3 after operative procedure,mRNA exepression of STAT3 and its downstream target gene CyclnD1 and Bcl-2 were showed in both the control and exeperiment group,and all peaked at 14d and declined to insignificant level at 28d. Compared with those of the control group,the mRNA expression of the experiment group on 7d decreased significantly. Immunoblotting studies showed that a transient STAT3 induction was accompanied with constitutive expression in the control group,The constitutive STAT3 phosphorylation was detected in the arterialized vein graft after day 3,peaked at day 7,and declined to insignificant levels at day 14. while in the experiment group,although STAT3 phosphorylation detected on day 3 was nearly the same as that in the control group,it was far lower than that of the control group on the 7d; and also that on day 7,CyclnD1 and Bcl-2 proteins level in the experiment group declined significantly compared with that of the control group.

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CLC: > Medicine, health > Internal Medicine > Heart, blood vessels ( circulatory ) disease > Vascular disease
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