【作者】 李辉；

【导师】 刘闺男；

【作者基本信息】 中国医科大学 ， 循环内科， 2010， 硕士

【摘要】 前言经皮冠状动脉腔内介入(percutaneous coronary intervention, PCI)术是目前治疗冠心病最有效的手段之一,但PCI术后3到6个月内血管重建部位再狭窄(restenosis,RS)的发生在一定程度上限制了其应用。大量研究表明,RS的发生是一个多因子参与的复杂病理过程,其中包括血管弹性回缩、血小板黏附聚集及血栓形成、新生内膜形成等多个环节。其中主要机制是血管损伤局部中膜平滑肌细胞(smooth muscle cell, SMC)过度增殖,向内膜迁移及细胞外基质形成引起的内膜增生。因此寻找有效的手段抑制SMC增殖和迁移对临床预防RS具有重要的指导意义。骨桥蛋白(osteopontin, OPN)是细胞外基质中一种重要的功能性蛋白,差异显示技术证明OPN是血管平滑肌细胞(vascular smooth muscle cell, VSMC)表型转化的标志基因,其表达决定了VSMC的表型。多项研究表明,OPN在血管损伤后新生内膜中的表达明显上调,而且它能促进VSMC和外膜细胞的增殖、迁移,被认为是血管损伤修复过程中重要的始动因素。尽管OPN在促进VSMC增殖的过程中起着重要的作用,但其具体机制研究尚少。本课题拟通过初步探讨OPN反义核苷酸对大鼠血管SMC增殖的影响及其机制,进而为OPN作为防治RS的新靶点提供理论基础和实验依据。材料和方法一、大鼠A10主动脉VSMC的培养用含10%胎牛血清的DMEM培养基,在37℃、5%CO2、饱和湿度的培养箱中培养大鼠A10主动脉平滑肌细胞。用0.25%胰酶消化、传代。传代以后使用含10% FBS、100U／ml青霉素、100μg／ml链霉素,pH7.4的DMEM维持细胞生长,2～3天换液一次,选取3～8代细胞进行实验。二、OPN反义核苷酸的转染委托Takara公司合成OPN反义核苷酸,并应用转染试剂FuGENE6对VSMC进行瞬时转染。操作步骤严格按转染试剂FuGENE6使用说明书进行。三、实验方法MTT比色法测定OPN反义核苷酸转染对VSMC增殖的影响；FCM分析OPN反义核苷酸转染对VSMC细胞周期分布的影响；RT-PCR检测OPNmRNA、PCNAmRNA;四、统计学分析采用SPSS11.0统计软件进行数据分析。One-way ANOVA分析不同组间参数比较,两两比较采用LSD方法。p<0.05为统计学上有显著性差异。实验结果1、MTT结果显示：OPN反义核苷酸转染24、48、72h后,细胞增殖抑制率分别为24%、19.6%、17%,与对照组相比,有明显差异(p<0.05),说明OPN反义核苷酸对VSMC的增殖有明显的抑制作用,并且随时间延长对细胞增殖抑制率下降。2、FCM分析显示：OPN反义核苷酸主要作用于G1／S限制点,能阻止VSMC由G0／G1期向S期推进,从而将VSMC阻滞于G0／G1期,抑制VSMC的增殖。3、RT-PCR结果显示：VSMC转染OPN反义核苷酸后,PCNAmRNA的表达水平较对照组降低(p<0.05)。结论大鼠VSMC中,转染OPN反义核苷酸可以抑制VSMC的增殖,其机制可能为转染OPN反义核苷酸能够阻止VSMC由G0／G1期向S期推进,从而将VSMC阻滞于G0／G1期,抑制VSMC的增殖。更多还原

【Abstract】 ObjectivePercutaneous coronary intervention (PCI) is now one of widely used methods to treat coronary artery disease, but restenosis following PCI during 6 months limits its long-term benefits.The biological mechanisms are complex, involving elastic recoil in the first instance, followed by extracellular maxtrix production, smooth muscle cell proliferation and migration, and remodeling of the vessel. Therefore the hunt for efficient inhibitors of restenosis remains an ongoing challenge.Therefore, search available road to inhibit SMC proliferation and migration have important significance for preventing RS. OPN is an important functionality protein in extracellular matrix. Differential display technique demonstrate, OPN is a marker gene on VSMC phenotype transformation, it is expression determine VSMC phenotype. Many research show that OPN obviously up-regulation in vascular injury neointimal, and it could facilitate SMC and adventitial cell proliferation and migration.So it is known as an important initiating agent in vascular injury neointimal.In spite of OPN has an important role on facilitate SMC proliferation process,but specific mechanism research has not known. The aim of this study was to approach OPN antisense nucleotide on proliferation of rat vascular smooth muscle cells(VSMCs)and study its mechanism of inhibiting proliferation, and then provide rationale and experiment evidence for OPN preventing and curing RS.Methods1. Rat A10 aortic VSMCs were culturedRat A10 aortic VSMCs were purchased from ATCC. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) at 37℃in a humid atmosphere of 5% CO2. 2. OPN antisense nucleotide transfectionTo entrust Takara company synthesis OPN antisense nucleotide, and to utilize oligofectamine reagent FuGENE6 transient transfection on VSMC.3. Experiment methodsThe effect of OPN antisense nucleotide on proliferation of VSMC was observed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)metabolism measuring. FCM was performed to track cell cycle progression. RT-PCR was done to detect the mRNA level of OPN antisense nucleotide, PCNA.4.Statistical analysesAll values were expressed as mean±SD. SPSS11.0 software was used for all statistical analysis. Data were analyzed using one-way analysis of variance followed by a least significant difference test (LSD) for multiple comparisons. Differences were considered significant if p<0.05.Results1. The optical density (OD) of MTT decreased significantly in VSMCs transfected with OPN antisense nucleotide compared with that in VSMCs non-transfected.The inhibition ratio of VSMC proliferation was 24%,19.6%,17% respectively after transfecting of OPN antisense nucleotide on the 24th,48th and 72th hour. OPN antisense nucleotide can significantly inhibit 10% FBS induced VSMC proliferation.2. FCM analysis indicated that the G0/G1 phase fraction ratio of the OPN antisense nucleotide group was higher than the control group, while its S-phase fraction ratio was lower than the control group, the proliferation index of the OPN antisense nucleotide group decreased significantly. The result suggested that OPN antisense nucleotide blocked VSMC cycle in G0/G1 phase, inhibited the proliferation of VSMC.3. The results of RT-PCR showed that the PCNA was decreased obviously in the OPN antisense nucleotide group compared with the control group.ConclusionIn the rat vascular smooth muscle cell, OPN antisense nucleotide inhibited the proliferation of VSMC, its mechanism maybe is OPN antisense nucleotide blocked VSMC cycle in G0/G1 phase.更多还原