【作者】 徐尚华；

【导师】 李庚山；

【作者基本信息】 武汉大学 ， 内科学， 2004， 博士

【摘要】 血管平滑肌细胞（VSMC）迁移、增生和细胞外基质（ECM）在内膜过度沉积形成新生内膜是支架内再狭窄的主要原因。与PTCA相比，冠脉内支架的应用再狭率窄已明显降低，但支架内再狭窄（in-stent restenosis，ISR）仍然是一个严重的问题。目前全身用药都未能减少ISR发生率。近距离放射治疗是预防ISR的有效方法，但最大缺点是内皮化延迟和晚期血栓形成。药物包被支架（drug-coated stent）因具有靶血管局部治疗的优点而备受关注。 大蒜作为一种中草药已经应用了数千年，三硫二丙烯（diallyl trisulfide，DT）是大蒜的有效成分之一，具有很强的抗炎、抗血小板聚集、抑制血栓形成、预防动脉粥样硬化等多方面药理作用。体外研究结果显示DT能明显抑制兔主动脉VSMC的增殖；我们实验室已先后成功进行了放射性核素³²P胶体和DT包被支架的动物实验，证明血浆蛋白涂膜可良好携带³²P和DT，抗血流冲刷能力强，能有效预防支架术后内膜增生。 本实验应用血浆蛋白作为载体，制作DT、³²P包被支架以及二者联合包被支架，分别植入犬冠状动脉，进一步研究DT预防支架术后再狭窄的剂量效应关系；研究DT与³²P联合支架预防再狭窄的疗效；探讨DT抑制再狭窄的可能作用机制。 实验内容包括三部分。 第一部分，观察DT包被支架对犬冠状动脉损伤后内膜增生的量效关系。制作蛋白包被的DT包被支架，剂量分别为0μg（对照组）、60μg（小剂量组）、130μg（中剂量组）和210μg（大剂量组）。在犬左冠状动脉分别置入过大的单纯蛋白包被支架和DT包被支架，于术后28天和6个月冠状动脉造影后处死动物，取出带支架血管组织分为三份，一份固定在2.5％戊二醛中，用于制作扫描电镜标本，观察内皮覆盖情况；一份塑料包埋，碳化钨钢刀切片，分别行elastic-van Gieson和Masson染色，形态学观察，另一份用于第三部分分子生物学指标检测。结果发现，术后28天，对照组血管内膜表面已完全内皮化，210μg DT包被支架组＞90％内皮化；光镜结果显示，各组血管损伤程度无显著性差异（P＞0.05）。内弹力板层包绕面积在各组间无差别。支架置入后28天，对照组内膜明显增厚，ECM面积占内膜总面积的40％，6个月后增加到91％（P＜0.001）。DT呈剂量依赖性减少内膜ECM比例。血管参数分析显示，各指标在小剂量组与对照组差异均无显著性（P＞0.05）；术后28天，大、中剂量组的平均新生内膜厚度、新生内膜面积无差别（P>0.05），但均大于小剂量组和对照组（P<0.OOI）;DT呈浓度依赖性增加管腔面积（P<0.OI）和抑制面积狭窄率（P<0.001）。术后6个月，所有指标大、中剂量组与小剂量组、对照组之间均有统计学差别;DT呈浓度依赖性抑制内膜增生（P<0.01）;管腔面积、新生内膜面积及面积狭窄率在大、中剂量组间无差别（P>0.05）。上述结果表明，60林gDT对预防再狭窄无效，在支架置入术后6个月内，中、高剂量DT（130陀，210林g）包被支架呈剂量依赖性地抑制支架术后内膜增生，其机制与DT降低内膜SMC聚集和减少ECM沉积有关。 第二部分，设计DT与32P联合包被支架，置入犬无病变的冠状动脉，期望经两个不同途径，更彻底地抑制内膜增生。分别制作蛋白涂层的DT包被支架（210林留支架），’ZP包被支架（20林ei/支架）和DT与犯P联合包被支架（210林g+20林ei/支架）置入犬冠状动脉，于术后28天和6个月行冠状动脉造影后处死动物，取材，分别制作扫描电镜标本和组织切片，行elastic一van Gieson和Masson染色，形态学观察，进行参数测量、计算。结果发现，术后6个月，DT包被支架组血管内膜表面完全内皮化，联合包被支架组内皮化程度与32P支架组相似（80%）。Masson染色显示，各治疗组抑制内膜增生的同时伴有ECM含量的降低，联合组最低。血管参数分析发现，术后28天和6个月，各指标在治疗组与对照组差异均有显著性意义（尸<0.05）;联合包被组的平均新生内膜厚度、新生内膜面积和面积狭窄率均小于32P和DT组（尸<0.05），但管腔面积大于32P和nT组（尸<0.05）;各指标在32P和DT两组间无差别（P>.05）。术后28天，联合包被组的平均新生内膜厚度虽小于32P组（尸<0.05），但与DT组无差别（P>.05）;6个月后不仅联合包被组对内膜增生的抑制程度大于32P和DT组（尸<0.05），而且DT组对内膜增生的抑制程度大于32P组（尸<0.05）。6个月后冠状动脉造影结果显示，邻近参考血管直径在各组间差异无显著性。其余指标在治疗组与对照组差异均有显著性意义（P<0.05）。支架段血管直径狭窄程度，联合包被组小于犯P组和DT组（尸<0.05）。支架边缘段血管直径狭窄程度，DT组和对照组无差别，但32P组和联合组直径狭窄程度均大于对照组（p至0.016）。冠造结果未发现动脉瘤及局部血栓形成。上述结果表明，DT和犯P联合包被支架抑制内膜增生程度大于单纯犯P和DT包被支架，但内皮化程度没有进一步减少，提示联合包被支架预防ISR疗效更显著，但不增加副作用。联合包被支架的应用为开发即能有效预防内膜增生，又能快速内皮化的药物提供了新的思路。 第三部分，观察DT包被支架对基质金属蛋白酶一9，一2（MMP一9，一2）和金属蛋白酶组织抑制剂一1，一2（TIMP一1，一2）基因表达的影响，并探讨血管?更多还原

【Abstract】 Intimal hyperplasia due to migration and proliferation of smooth muscle cells (SMCs) from the media to the intima and extracellular matrix (ECM) deposition is a major component of restenosis after stent implantation. Although intracoronary stent placement reduces restenosis compared with conventional coronary angioplasty alone, in-stent restenosis (ISR) still remains a serious problem in interventional cardiology. No pharmacologic agents were effective in reducing the incidence of ISR. At present, vascular brachytherapy has been shown to be an effective treatment option. Nevertheless, this therapy is not 100% effective and recurrent ISR after brachytherapy occurs. Antirestenosis therapy offered by drug-coated stents has obvious merits in the ability to target therapy locally.Garlic has been used in herbal medicine for thousands of years. Some reports have shown that diallyl trisulfide (DT), a component of garlic, has protective effects against atherosclerosis as well as inhibits platelet function and thrombus formation. Studies in vitro revealed that DT exhibited a profound antiproliferative effect in SMCs. Our previous studies had shown that plasma-coated stent with DT or 32P significantly inhibited intimal hyperplasia in the canine coronary restenosis model.The purpose of this study was thus to test whether DT-coated stent prevented neointimal proliferation in a concentration-dependent manner, and to evaluate the effect of stents coated with DT and 32P concomitantly on neointimal proliferation in canine coronary arteries injury model. In addition, we established a cascade-based study model to explore the mechanism of the effect of DT-coated stents on neointima formation. This experiment included three parts.The first part was designed to investigate the efficacy of DT-coated coronary stents in preventing neointimal proliferation in canine coronary artery injury model. DT-coated stents (0, 60, 130, or 210 ug/stent) were deployed with mild oversizing in both left circumflex coronary arteriy (LCX) and left anterior descending coronary artery (LAD) of 20 dogs. Arteries were harvested 28 days (n=18) and 6 months (n=22) after stenting, One third of the stented region was cut off each one, and fixed in 2.5% Glutaral for scanning electron microscopic (SEM) analysis. One third of stented region was embedded in acrylic plastic, sectioned with a tungsten carbide knife and either stained with elastic-van Gieson or Masson for light microscopic examination. The remaining third of stented region was stored at liguid nitrogon. At 28 days after stenting, SEMshowed >90% stent surface endothelialization in DT (210ug) coated stents group, whereas complete endothelization was exhibited in control group. Masson staining showed that intimal extracellular matrix (ECM) content increased with time after stenting. ECM consisted of 40% of total intimal volume at 28 days and increased to 91% at 6 months (P<0.001), which markedly reduced by DT in a concentration-dependent manner. There was no significant difference in injury score among all groups (P>0.05). Histomorphometric analysis at day 28 indicated that the mean neointimal hyperplasia and neointimal area were similar in high-dose and immediate-dose group (P>0.05), but were less than those in low-dose or control group (.P<0.001). The percent area stenosis deceased with increasing dose (P<0.001), whereas the luminal area increased with escalating dose (P<0.01) in three DT-coated stents groups. After 6 months, the mean neointimal hyperplasia markedly reduced in high-dose group compared with immediate-dose group (P=0.008), low-dose group as well as control group (P<0.001). The neointimal area, the percent area stenosis and the luminal area were no significant difference between in high-dose group and immediate-dose group (P>0.05), but those indices in either group were significantly different from either low-dose or control group (P<0.001). There were no cases of aneurysm or thrombosis. In summary, in a canine model of restenosis, the low-dose DT (60ug) failed to reduce restenosis, w更多还原