【作者】 杨长春；

【导师】 温进坤； 韩梅；

【作者基本信息】 河北医科大学 ， 中西医结合基础， 2002， 博士

【摘要】 经皮腔内冠状动脉成形术（percutaneous transluminal coronaryangioplasty,PTCA）作为治疗缺血性心脏病的一个重要方法已在世界范围内得到了广泛的应用，但限制其远期疗效的重要原因是其术后6个月内30％-60％的再狭窄发生率。目前认为血管再狭窄的发生与血管平滑肌细胞（vascular smooth muscle cell，VSMC）增殖和迁移以及细胞外基质（extracellular matrix，ECM）大量合成和重构有关。粘着斑激酶（focal adhesion kinase，FAK）是一种非受体型酪氨酸蛋白激酶，起介导细胞外信号由整合素向细胞内转导的作用，FAK的磷酸化激活以及由此所产生的下游一系列蛋白质的磷酸化，是ECM与细胞相互作用并产生一系列生物学效应的关键环节，参与细胞增殖、迁移与凋亡的调节过程。然而作为重要的信号转导分子，FAK是否参与血管再狭窄发生的病理过程，目前尚不清楚。 由于血管再狭窄的发生机理尚不十分清楚，迄今为止，尚未找到理想的防治药物和方法。中药具有整体调节及多层次、多位点发挥作用的独特优势，有望使该病的治疗取得进展。中医理论认为，血管再狭窄属于“血瘀症”的范畴，其病因病机为血管内皮损伤导致的瘀血阻滞，血脉不通，心脉痹阻。中医药防治PTCA术后再狭窄宜采用益气活血化瘀为治则，以求得治病求本，标本兼治。本室以往对益气活血化瘀方药（黄芪、丹参、当归、生蒲黄、徐长卿）及单味药黄芪、当归防治大鼠血管再狭窄的分子机制进行了一系列研究，证明该类方药可以通过调整TXA₂／PGI₂和tPA／PAI的平衡减轻血瘀的病理表现；通过调节ECM代谢及诱导VSMC凋亡而化解VSMC过度增殖和ECM异常积聚之“瘀”。本实验在上述研究的基础上，选用黄芪及当归作为益气活血化瘀中药的代表，在揭示血管再狭窄发生与FAK表达和活化的关系的基础上，从整体、细胞和分子水平上探讨黄芪、当归防治再狭窄的作用机制，以寻找中药抗血管再狭窄的新靶点，为中药防治血 中文摘要管再狭窄提供实验依据。1．FAI表达和活化与血管再狭窄的关系 采用球囊导管建立大鼠主动脉内皮剥脱动物模型，从整体水平动态观察再狭窄发生过程中血管壁病理形态学、血流动力学及血浆一氧化氮＊itric oxide，NO)含量的变化，探讨 FAK表达和活化与内膜增生之间的关系。实验结果如下：1．1 正常对照组大鼠主动脉壁各层结构完整，内膜光滑，中膜VSMC排列整齐。内皮剥脱后3d，内膜变粗糙，局部出现细胞增生；剥脱后7d，中膜VSMC核排列紊乱，新生内膜中可见局灶性VSMC增殖；术后14士 内膜呈进行性弥漫性增厚，管腔变小，增厚的内膜中以VSMC为主。 对组织切片进行图像分析的结果表明，主动脉内皮剥脱后7d，血管内径减小，内膜／中膜口／M)厚度和内膜／中膜（I／M）面积比值增大（P＜ O．05）；7d至14d期间，血管内径进一步缩小，I／M厚度和U：MU：M面积比值进一步增大；至引d时，I／M厚度和FM面积比值分别达1．97和 1．13。提示内皮剥脱成功地引发了血管再狭窄的形成。l二超声影像学分析结果显示，最大血流速度及最小血流速度从内皮剥脱后第 3d开始降低，ZId达最低值。搏动指数0I)和阻力指数（RI）从内皮剥脱后第 3 d开始升高，分别于 ZId和 14d达峰值。1．3在正常大鼠血管中FAK基因有一定程度的表达，FAK主要以非磷酸化的形式为主；内皮剥脱后 3d，FAK InRNA、蛋白含量及磷酸化水平均明显升高，分别比对照组增加了 36．4％，35％和 60％；内皮剥脱后7d，FA K InRNInRNA和蛋白含量达到峰值；第14d，磷酸化型FA K达到峰值；之后，该基因表达活性及表达产物的活化程度均逐渐降低，但至Zld，仍高于对照组水平。 免疫组化结果显示，在正常血管中，FAK在内膜、中膜和外膜均有表达，在新生内膜中FAK阳性染色颗粒显著增多。l．4正常大鼠血浆中 NO含量为 44．sll．15 u，mol几，随着内皮剥脱时间的延长，NO含量持续下降，第 3d，7d，14d和 Zld的NO含量分别为引二7士1二7umol／L，28．33士1．42umol／L，23．90土1．77umol／L，16．86土 1．47 u mol／L，各时间点均比对照组大鼠显著性降低(P＜0刀5人 2 中文摘要 上述结果表明，在血管内皮损伤所致的内膜增生过程中，伴有FAK 表达活性和 NO含量的变化，提示 FAK和 NO参与血管再狭窄的发生 与发展过程。 2．黄蔑、当归防治血管内皮剥脱后再狭窄的实验研究 中医理论认为，内皮剥脱后血管再狭窄属于“血瘀症”范畴，瘀 血阻滞，血脉不通，心脉痹阻是其主要的病理表现。根据再狭窄的病 因病机，其治疗应以益气活血化瘀为原则。本部分实验从整体水平观 察黄蔑、当归单独及联合应用对血管 VSMC FAK表达与活化的影响及 对大鼠血管内皮剥脱后再狭窄的防治作用，以探讨中药抗血管再狭窄 的作用机理。结果如下?更多还原

【Abstract】 Percutaneous transluminal coronary angioplasty (PTCA), an important method for treatment ischemic heart diseases, has been used extensively. But the high incidence of restenosis after PTCA is the vital limitation for its long term effect. Although the exact mechanism in restenosis is still not very clear, it is believed to be related to many events such as vascular smooth muscle cell (VSMC) proliferation and migration, excessive synthesis of extracellular matrix (ECM) as well as ECM remodeling and so on. Increasing studies have demonstrated that focal adhesion kinase (FAK), a non-receptor tyrosine kinase, involves in the intracellular signal transduction mediated by integrin. Phosphorylation of FAK itself and following proteins is the key link in the interactions between ECM and cells which in rum show a series of biological effects, including cell proliferation, migration and apoptosis. But up to date whether or not FAK as an important signal molecule plays a role in pathogenesis of restenosis after endothelium injury still remain uncertain.There are no satisfactory methods and medicines to treat restenosis so far because of the complicated mechanisms in the development of restenosis. Many studies showed that the functional features of Chinese Traditional Medicine (TCM) reflect its superiority in preventing and treating vascular restenosis. According to the theories of TCM, restenosis is a kind of "blood stasis", and supplementing Qi, activating blood circulation to eliminate stasis should be used as therapeutic principles. Our previous experiments have showed that the recipe of supplementing Qi, activating blood circulation to eliminate stasis, including Astragalus Membranaceus(Ast) and Angelica Sinensis (Ang), could effectively prevent intimal hyperplasia by adjusting the balances of tPA/PAI and TXA2/PGI2, inhibiting proliferation of VSMC and excessive formation of ECM. On the basis of our previous studies, we used Ast and Ang, as two therapeutic medicines, and investigated their effects on FAK expression and activation. So, we could further explore the mechanisms in intimal hyperplasia caused by de-endothelialization and provide theoretical basis for finding new therapeutic targets in arresting restenosis by TCM.1. FAK expression and activation in the development of restenosis after vascular de-endothelializationThe restenosis model was established by denuding the rat aorta endothelium with balloon catheter. The pathological features of arterial wall, changes of hemodynamics, NO content in plasma as well as activities of FAK expression and phosphorylation were observed. The results are as follows:1.1 Compared with smooth intima and orderly arranged medial VSMC of aorta in the normal rats, rough intima was first seen on day 3 after de-endothelialization, and on day 7, focal VSMC proliferation in neointima and disordered medial VSMC nuclei were found in injured area. On day 14 to 21, progressive and widespread intimal thickening and lumen stenosis were observed.Image analysis showed that both thickness ratio and area ratio of intima/media (I/M) increased, and the diameter of lumen decreased on day 7 after de-endothelialization. On day 21, the two ratios reached 1.97 and 1.13, respectively. It implied that restenosis was successfully elicited by endothelial denudation.1.2 The maximal and minimal blood velocity began to decrease on day 3 after endothelial denudation and got to the lowest point on day 21. In contrast, pulsatile index (PI) and resistance index (RI) increased from day 3 after de-endothelialization, and the highest values were obtained on day 21 and 14, respectively.1.3 Compared with low level expression and low-phosphorylation of FAK in aorta of control rat, amount of FAK mRNA and protein as well as degree of FAK phosphorylation increased by 36.4%, 35% and 60%, respectively, on day 3 after de-endothelialization. On day 7 the amount of FAK mRNA and protein reached the peak, and began to decrease on day 14. On day 21, the activity of FAK gene expression and level of p更多还原