【作者】 王军；

【导师】 景华；

【作者基本信息】 南京大学 ， 外科学， 2011， 博士

【摘要】 在冠脉搭桥手术(CABG)中,大隐静脉(great saphenous vein)血管桥被广泛使用,文献报道约80%的CABG使用了大隐静脉桥作为血管桥。较之于乳内动脉(internal thoracic artery)等动脉血管桥,大隐静脉血管桥的远期通畅率较低,术后1年通畅率85%,而术后10年通畅率只有50-60%。临床上许多病人因为大隐静脉血管桥远期阻塞需要接受二次搭桥手术。提高大隐静脉血管桥的远期通畅率能减少患者需要接受二次手术的几率以及接受二次手术所带来的风险和花费。大隐静脉移植桥的闭塞可以分为早期、中期和晚期三个阶段。早期闭塞主要的原因是缝合技术不规范、患者血液高凝状态等,中期闭塞的主要原因是移植静脉内膜的增生,晚期闭塞主要是由于移植血管粥样硬化。目前对于移植静脉中期的内膜增生再狭窄尚没有有效的治疗方法。移植静脉内膜的增生来源于血管平滑肌细胞的增生,研究表明造成内膜增生的血管平滑肌细胞是来源于局部的吻合血管的。暴露于动脉血流动力学环境之下的移植静脉中膜血管平滑肌细胞在动脉血流的剪切力作用下发生表型转变(phenotypic switching)，从收缩型平滑肌细胞(contractile phenotype)转变为合成型的平滑肌细胞(synthetic phenotype)。合成型血管平滑肌细胞向内膜区迁移、增生,并分泌大量细胞外基质。这一过程造成了移植静脉血管内膜的增生,而增生的内膜组织较之正常内膜更易发生粥样硬化的改变。移植静脉内膜的增生是造成静脉移植物晚期狭窄的一个重要原因,目前对于移植静脉内膜的增生尚没有有效的治疗办法。以往的研究表明移植静脉内膜增生的过程涉及许多基因的异常高表达。C-myc基因在移植静脉中的表达情况与移植静脉内膜增生的发生发展情况是一致的,被认为启动并维持了移植静脉内膜的增生。既往使用AS-ODN抑制c-myc基因表达的研究证实了抑制c-myc基因的表达可以抑制移植血管中膜平滑肌细胞的增生,缓解移植静脉的狭窄。RNAi技术提供了简单高效的基因沉默的方法。本研究使用体外培养的血管平滑肌细胞筛选合适的干扰序列,并建立大鼠自体动静脉移植模型,首次研究c-myc siRNA在体内抑制移植静脉内膜增生的情况。第一部分：C-myc siRNA抑制体外培养血管平滑肌细胞增生的研究目的：人工合成c-myc siRNA序列,通过转染体外培养的血管平滑肌细胞检测其体外抑制血管平滑肌细胞增生的能力。方法：使用基因软件筛选出合适干扰序列,采用化学合成法人工合成3对c-myc siRNA序列和一对SCR序列。取SD大鼠胸主动脉,按照组织块培养法获取体外培养的血管平滑肌原代细胞并传代培养。使用阳离子脂质体为转染试剂进行体外转染实验。使用荧光标记的SCR序列筛选最佳转染条件,使用MTT法、WB、q-rt-PCR法检测各干扰序列的抑制效率,筛选出具有最强干扰效率的c-myc siRNA序列。结果：最佳干扰序列为：ACAUCAUCAUCCAGGACUGdTdT。转染48h后,筛选出的干扰序列对体外培养的血管平滑肌细胞细胞增殖的抑制为40%,抑制VSMCs c-myc mRNA的表达约为75%,抑制c-myc蛋白表达大约55%。结论：使用组织块培养法可以简便有效的获取体外培养的血管平滑肌细胞。序列ACAUCAUCAUCCAGGACUGdTdT能明显抑制体外培养的血管平滑肌细胞的增生。其对血管平滑肌细胞的增生是来源于对c-myc mRNA和c-myc蛋白质表达的抑制。第二部分：RNAi抑制大鼠自体移植静脉内膜增生的研究目的：体外实验部分筛选出了合适的干扰序列,本部分研究的目的是验证这一干扰序列在体内情况下是否能有效抑制移植静脉内膜的增生。方法：建立大鼠自体颈外静脉-颈总动脉移植模型。32只SD大鼠随机分成四组：空白对照组(No treatment),阴性干扰序列组(SCR),转染试剂组(Gel), c-myc siRNA组。术后3周处死大鼠并切取移植静脉。分别检测移植静脉内膜增生、c-myc基因表达以及血管平滑肌细胞PCNA表达的情况。结果：相对于其它对照组,c-myc siRNA组的内膜增生被明显抑制,增生内膜厚度减少约75%。Q-rt-pcr检测显示c-myc siRNA抑制c-myc基因的表达约50%,免疫组化染色显示c-myc蛋白表达在c-myc siRNA组也明显减少,同时细胞增殖能力的指标PCNA表达也明显下调。结论：本实验使用的c-myc siRNA能有效缓解移植静脉内膜的增生。C-myc siRNA能抑制移植静脉中膜血管平滑肌细胞的增值能力。C-myc siRNA对移植静脉内膜增生的抑制作用来源于其对c-myc基因表达的抑制。更多还原

【Abstract】 Autologous saphenous vein is one of the most commonly used vascular conduits for coronary heart disease. It was reported that the great saphenous vein are used in almost about80%of all the CABG However, the long-term treatment outcome is far from satisfactory due to the gradually diminished patency rate of the vein grafts over time. One year, and10years following CABG surgery, the patency rate was about85%and50-60%, respectively. It is benefit to improve the long-term patency rate of vein grafts.Three distinct phases of vein grafts failure have been recognized till now. Early graft occlusion relates to technical complications and thrombosis. Mid-term graft occlusion is due to the development of fibrotic intimal hyperplasia. And atherosclerotic degeneration contributes to the late time restenosis.Vascular intimal hyperplasia was found to be one of the major causes for occlusion of the vein grafts. It was reported that the smooth muscle cells play a central role in the vein grafts’ intimal hyperplasia. Evidences suggested that most of the smooth muscle cells come from the local vessel wall in the progress of vein graft’s intimal hyperplasia. After reperfusion, VSMCs of the vein grafts would gradually change from contractile to synthetic phenotype. The phenotypic switching of the VSMCs will make the cells immigrate to the tunica intima of the vein grafts, enter the cell cycle and produce extracellular matrix. These contribute to the vein grafts stenosis. While traditional pharmacotherapy has no benefits to intimal hyperplasia, gene therapy provides a possible treatment for inhibiting intimal hyperplasia.Many genes are involved in the progress of intimal hyperplasis. The progress of the intimal hyperplasia of the vein grafts was consistent with the expression of the c-myc gene. It is believed that the continuous high expression of the c-myc gene is partly responsible for the development of intimal hyperplasia.The use of synthetic siRNA has been established as a technique for gene silencing. It was reported that antisense oligonucleotides to c-myc gene could reduce c-myc gene expression and inhibit VSMCs growth. Here, we employed siRNA as a medium to silence c-myc gene expression in order to inhibit VSMCs proliferation and protect vein grafts from late restenosis in a rat autologous vein graft model.Part Ⅰ:Small interfering RNA to c-myc inhibits VSMCs proliferation in vitroObjective:Three c-myc siRNAs and one SCR were synthesized. The effects of siRNAs on VSMCs proliferation in vitro were examined both in cell proliferation and c-myc geng expression.Methods:VSMCs from Sprague-Dawley rat were isolated and harvested. Cells were identified by morphology and immunohistochemistry for α-actin, and were used for experiments at passage5. VSMCs were transfected with the different siRNAs by using Lipofectamine2000reagent (Invitrogen Life, Biotechnologies, Carlsbad, CA). MTT assay, WB and q-rt-PCR were used to detecte the inhibition efficiency in cell proliferation.Result:The sequence ACAUCAUCAUCCAGGACUGdTdT was selected as the most effective siRNA in our study. In vitro, at48hours after transfection, this c-myc siRNA inhibited cell proliferation by40%, reduced c-myc mRNA level by70%and c-myc protein level by50%as compared with the controls.Conclusions:The VSMCs proliferation in vitro could be reduced effectively by downregulation c-myc gene expression utilizing c-myc siRNA. Part Ⅱ:Small interfering RNA to c-myc inhibits vein graft restenosis in a rat vein graft modelObjective:In vitro study, the c-myc siRNA was selected. To investigate the inhibition efficiency of this c-myc siRNA on grafted veins’ intimal hyperplasis, a rat vein graft model was used in this study.Methods:Autologous jugular vein to carotid artery reverse interposition grafts were performed in male Sprague-Dawley rats.32rats were divided into four groups as follows:nontreated group, receiving no treatment (n=8); only gel group,200ul of25%pluronic F-127gel (sigma) in5%glucose solution was applied to the surface of the vein grafts immediately after arteriovenous anastomosis (n=8); scrambled siRNA treated group, treated with200ul of above gel containing50ug scrambled siRNA (n=8); c-myc siRNA treated group,200ul gel containing50ug c-myc siRNA was applied to the surface of the grafts(n=8). Rats were sacrificed3weeks after surgery. The intimal hyperplasis and the c-myc gene expression were detected. Stains for PCNA were also preformed.Result:In vivo, the c-myc siRNA treatment reduced intimal hyperplasia by75%. Compared to the controls, the c-myc gene expression was also reduced in c-myc siRNA treatment group as well as the PCNA expression.Conclusions:Small interfering RNA to c-myc can inhibit vein graft restenosis in a rat vein graft model.更多还原