【作者】 陈菡子；

【导师】 钱菊英；

【作者基本信息】 复旦大学 ， 内科学， 2011， 硕士

【摘要】 研究背景置入部位支架贴壁不良(stent malapposition, SM)是指在支架置入术后,至少有1处或以上的支架梁与动脉管壁内膜未能完全贴合(非覆盖于边支的支架),而且在支架梁后存在血流。晚期获得性支架贴壁不良(late-acquired stent malapposition, LASM)是指在支架置入术后即刻贴壁良好,随访发现贴壁不良。药物洗脱支架(drug-eluting stent, DES)术后LASM发生率明显高于裸金属支架(bare-metal stents, BMS),为0%-25%(平均约10%～12%)比0～6%(平均约4%)。研究显示无论在DES还是BMS的LASM的发生中,局部血管正性重构,即血管外弹力膜面积(external elastic membrane area, EEMA)的增加起了主要作用。但是目前关于支架术后血管正性重构的机制仍不明确。研究目的1.通过建立兔髂动脉粥样硬化模型后行支架置入术,研究BMS和DES不同支架组术后血管正性重构发生率及程度的差别；2.研究支架术后血管组织的炎症反应、血管平滑肌细胞(vascular smooth muscle cell, VSMC)增殖与凋亡、基质金属蛋白酶(matrix metalloproteinase, MMPs)与基质金属蛋白酶组织抑制剂(tissue inhibitor of metalloproteinase, TIMPs)变化与血管正性重构的关联,探讨支架置入术后血管正性重构的相关因素,以期揭示LASM的可能发生机制。材料和方法雄性新西兰大白兔60只,高脂饮食1周,施行双侧髂动脉球囊导管损伤术,术后高脂饮食饲养8周后,行髂动脉造影,并予以支架置入术,及给予术前、术后即刻血管内超声(intravascular ultrasound, IVUS)检查,在动脉粥样硬化病变部位分别置入BMS(商品名Mustang,微创公司),涂层不可降解雷帕霉素洗脱支架(permanent polymer sirolimus-eluting stent, PPSES,商品名Firebird,微创公司),涂层可降解雷帕霉素洗脱支架(biodegradable polymer sirolimus-elutingstent,BPSES,商品名Excel,吉威公司)、无涂层雷帕霉素洗脱支架(polymer-free sirolimus-eluting stent, PFSES,吉威公司)。术后给予普通饮食,并喂食阿司匹林25mg QD+氯吡格雷12.5mg QD联合抗血小板至8周。至终点存活动物53只,其中BMS组12只,PPSES组14只,BPSES组13只、PFSES组14只。支架植入术后8周复查髂动脉造影及血管内超声检查,比较随访与术后即刻EEMA变化,定义随访／术后即刻EEMA为重构比值,以重构比值>1.05为血管正性重构组(vascular positive remodeling, VPR),重构比值<0.95为负性重构,重构比值≥0.95且≤1.05为无重构,负性重构及无重构均归入非正性重构组(vascular non-positive remodeling, NPR)。并取髂动脉血管标本行组织形态学测量、Verhoeff-Van Geison染色、病理积分、炎症反应程度及免疫组织化学方法检测增殖细胞核抗原(proliferating cell nuclear antigen, PCNA)、基质金属蛋白酶-2(matrix metalloproteinase-2, MMP-2)、基质金属蛋白酶组织抑制剂-2 (tissue inhibitor of metalloproteinase-2, TIMP-2)表达、脱氧核糖核苷酸末端转移酶介导的原位末端标记法(terminal deoxynucleotidyl transferase-mediate dUTP-biotin nick end labeling assay, TUNEL)方法检测细胞凋亡情况,分析上述指标与血管正性重构关系。研究结果(一)各支架组出现血管正性重构发生率分别为：BMS组25.00%(3/12), PPSES组42.86%(6/14), BPSES组30.77%(4/13), PFSES组28.57%(4/14),各组发生率差异无统计学意义。随访-术后即刻EEMA变化(△EEMA)分别为-0.51±0.54mm2、0.86±0.38mm2、0.67±0.41mm2、0.05±0.27 mm2, PPSES组、BPSES组相比BMS组血管正性重构程度增加,PFSES组与BMS组、PPSES组比BPSES组相比之间差异均无统计学意义。VPR组中,BMS组、PPSES组、BPSES组、PFSES组△EEMA分别为：0.96±0.45mm2、2.47±0.35mm2、2.59±0.57mm2、1.23±0.24mm2, PPSES组、BPSES组比BMS组血管正性重构程度增加,PFSES组与BMS组、PPSES组比BPSES组相比之间差异均无统计学意义。共有2个支架处出现LASM,其中1个支架为PPSES,其EEMA、△EEMA、LSM面积分别为13.03mm2、2.92mm2、4.30mm2;另1个支架为BPSES,其EEMA、△EEMA、LSM面积分别为13.86mm2、3.32 mm2、2.20mm2。(二)各支架组弹力-胶原纤维、炎症反应、细胞增殖／凋亡、MMPs/TIMPs指标比较1.各支架组弹力、胶原纤维均未见明显断裂、降解表现。2. BMS、PPSES、BPSES、PFSES组炎症面积分别为：0.03±0.03 mm2、0.14±0.11 mm2、0.05±0.04 mm2、0.00±0.00 mm2;炎症细胞密度分别为：2.87±1.22×103个/mm2、7.49±5.85×103个/mm2、4.38±4.05×103个/mm2、0.00±0.00×103个/mm2; PPSES组比其他三组炎症面积及炎症细胞密度均增加。3. BMS、PPSES、BPSES、PFSES组细胞增殖指数(proliferation index, PI)分别为：61.35±8.98%、29.50±5.47%、28.59±7.05%、16.85±5.30%; BMS组比PPSES组、BPSES组、PFSES组均高；DES组中,PPSES组、BPSES组比PFSES组PI高。4. BMS、PPSES、BPSES、PFSES组凋亡细胞指数(apoptosis index, AI)分别为：14.16±3.48%、24.50±3.17%、24.69±2.58%、60.20±2.40%,PPSES组、BPSES组、PFSES组分别较BMS组AI增高；DES组中, PFSES组与PPSES组、BPSES组比较AI也较高。5. BMS、PPSES、BPSES、PFSES组MMP-2平均光密度值分别为：0.16±0.07、0.15±0.21、0.13±0.01、0.07±0.03、PFSES组平均光密度值均较其他三组明显减少,其余三组两两比较无明显差异。6. BMS、PPSES、BPSES、PFSES组TIMP-2平均光密度值各组两两比较差异无统计学意义。(三)各支架组内VPR组与NPR组炎症反应、细胞增殖／凋亡、MMPs/TIMPs指标比较1. PPSES组、BPSES组的VPR组比其对应的NPR组炎症面积及炎症细胞密度均增加；余两组VPR组比其对应的NPR组无明显差异。2.PPSES组的VPR组比NPR组PI明显降低,BPSES组、PFSES组的VPR组比其对应NPR组PI有所升高,BMS组VPR组比NPR组PI无明显差异。3. PPSES组、BPSES组、PFSES组的VPR组比NPR组AI明显升高,且以PPSES组差异最为明显。BMS组VPR组比NPR组AI差异无统计学意义。4. PPSES组、BPSES组的VPR组比NPR组MMP-2表达均明显升高；BMS组、PFSES组的VPR组与NPR组两者比较均无明显差异。5. PPSES组的VPR组比NPR组TIMP-2表达增加,BMS组、BPSES组、PFSES组的VPR组与NPR组比较无明显差异。(四)各支架组AEEMA与细胞凋亡、MMPs指标相关分析1.细胞凋亡指数(AI)与△EEMA具有正相关关系,相关系数r=0.275,P<0.05；2.MMP-2平均光密度与△EEMA具有正相关关系,相关系数r=0.550,P<0.05。研究结论1.支架置入术后,有涂层(包括涂层可降解及涂层不可降解)雷帕霉素洗脱支架血管正性重构程度比无涂层雷帕霉素洗脱支架和裸金属支架显著。2.支架置入术后血管正性重构过程与血管平滑肌细胞凋亡和MMP-2表达上调机制相关,血管平滑肌细胞凋亡／增殖失衡与雷帕霉素促凋亡作用和支架聚合物涂层局部炎症反应二者有关,MMPs/TIMPs失衡与支架聚合物涂层局部炎症反应有关。更多还原

【Abstract】 BackgroundThe late-acquired stent malapposition (LASM) is an finding which has separation of at least 1 stent strut from the arterial wall intima that does not overlap a side-branch, with evidence of blood flow (speckling) behind the strut, where the immediate postimplantation intravascular ultrasound (IVUS) revealed complete apposition of the stent to the vessel wall. LASM may occur both after drug-eluting stent (DES) and bare-metal stent (BMS) implantation. The incidence of LASM is significantly higher in DES compared with BMS (0%～25% vs.0～6%, respectively). It has proved that the main cause of LASM is regional positive remodeling, which means a regional increase in external elastic membrane area (EEMA). However, the mechanism of regional positive remodeling after stent implantation still remains unknown.Objective1. To study the incidence and degree of vascular positive remodeling after BMS or DES implantation via performing iliac arterial stent implantation in atherosclerotic rabbit models.2. To observe the inflammatory reaction, vascular smooth muscle cell (VSMC) apoptosis and proliferation, matrix metalloproteinase (MMPs) and tissue inhibitor of metalloproteinase (TIMPs) expression of vascular tissue after BMS and DES implantation, investigate those indicators in their relation to vascular positive remodeling, in hope to reveal the mechanism of vascular positive remodeling and LASM.Methods60 male New Zealand rabbits fed on high fat diet for 1 week and then had bilateral iliac artery balloon catheter injury. Immediately following the operation, all subjects were maintained on high fat diet for 8 weeks and then had iliac arterial stent implantation. IVUS was applied pre-operatively and post-operatively. One types of 4 stents, including BMS, permanent polymer sirolimus-eluting stent (PPSES), biodegradable polymer sirolimus-eluting stent (BPSES) and polymer-free sirolimus-eluting stent (PFSES), was implanted at the detected atherosclerotic stenotic lesions in an animal subject. Post-operatively, all subjects received both ordinary diet and a dual antiplatelet therapy of aspirin and clopidogrel for 8 weeks. At the end of the experiment, the total of 53 survived animals is composed of 12 in BMS,14 in PPSES,13 in BPSES and 14 in PFSES. Iliac arterial angiography and IVUS as follow ups were conducted on all survived animal subjects. The ratio of follow-up EEMA to post-stent implantation was defined as remodeling ratio, which with a value beyond 1.05 is categorized into the vascular positive remodeling (VPR) group. Any remodeling ratio value lower than 0.95 was catagorized into the vascular negative remodeling group. Any remodeling ratio value in between 0.95 and 1.05 was considered no remodeling. The negative remodeling group and the group considered as no modeling were combined into the vascular non-positive group (NPR).Meanwhile, histomorphometry measurement, Verhoeff-Van Geison staining, pathological scores, and inflammatory reaction on a specimen of iliac artery, and immunohistochemistry to detect proliferating cell nuclear antigen (PCNA), matrix metalloproteinase-2 (MMP-2), and tissue inhibitor of metalloproteinase-2 (TIMP-2) expression, and terminal deoxynucleotidyl transferase-mediate dUTP-biotin nick end labeling assay (TUNEL) to detect cell apoptosis were all analyzed in relation to vascular positive remodeling.ResultsThe incidence of vascular positive remodeling was 25.00%(3/12) in BMS group, 42.86%(6/14) in PPSES group,30.77%(4/13) in BPSES group, and 28.57%(4/14) in PFSES group. There was no statistical significance among the 4 groups. The follow-up EEMA minus post-stent implantation (△EEMA) was-0.51±0.54mm2, 0.86±0.38mm2,0.67±0.41mm2,0.05±0.27 mm2, respectively. As compared with BMS group, PPSES group and BPSES group demonstrated significantly larger△EEMA, while PFSES group had no significant difference. The difference between PPSES group and BPSES group was not significant differently. For the VPR groups,△EEMA was 0.96±0.45mm2、2.47±0.35mm2.2.59±0.57mm2、1.23±0.24 mm2 in BMS group, PPSES group, BPSES group, PFSES group, respectively. As compared with BMS group, PPSES group and BPSES group presented significantly larger△EEMA, while PFSES group had no significant difference. The difference between PPSES group and BPSES group was not significant differently. LASM occurred in 2 stents, one was PPSES; the other was BPSES. The EEMA, AEEMA, LSM cross section area of PPSES was 13.03mm2,2.92mm2,4.30mm2, respectively; while the EEMA,△EEMA, LSM cross section area of BPSES was 13.86mm2,3.32mm2,2.20mm2, respectively.The rupture and degradation of elastic and collagen fibers was not observed in each group. The inflammatory area was 0.03±0.03 mm2,0.14±0.11 mm2,0.05±0.04 mm2,0.00±0.00 mm2 in BMS group, PPSES group, BPSES group, PFSES group, respectively; The number of inflammatory cells per inflammatory area was 2.87±1.22×103/mm2,7.49±5.85×103/mm2,4.38±4.05×103/mm2,0.00q0.00×103/mm2, respectively. As compared with BMS, BPSES and PFSES group, PPSES presented larger inflammatory area and more inflammatory cells. The proliferation index (PI) was 61.35±8.98%,29.50±5.47%,28.59±7.05%,16.85±5.30% in BMS group, PPSES group, BPSES group and PFSES group, respectively. PPSES, BPSES and PFSES group demonstrated lower PI value as compared with BMS group; PPSES and BPSES group demonstrated higher PI as compared with PFSES group. The apoptosis index (AI) was 14.16±3.48%,24.50±3.17%,24.69±2.58%,60.20±2.40% in BMS group, PPSES group, BPSES group and PFSES group, respectively. PPSES, BPSES and PFSES group presented higher AI value as compared with BMS group; PPSES and BPSES group presented lower PI as compared with PFSES group. The average optical density (AOD) of MMP-2 was 0.16±0.07,0.15±0.21,0.13±0.01,0.07±0.03 in BMS group, PPSES group, BPSES group and PFSES group, respectively. PFSES group demonstrated lower AOD value of MMP-2 as compared with the other 3 groups. There was no significant difference in AOD of TIMP-2 among all the groups.The VPR group in both PPSES and BPSES presented larger inflammatory area and more number of inflammatory cells per inflammatory area than their NPR rival groups. The VPR group in PPSES demonstrated lower PI value than its NPR rival group, while the VPR group in both BPSES and PFSES group demonstrated higher PI than their NPR rival groups. The VPR group in PPSES, BPSES and PFSES group demonstrated higher AI than their NPR rival groups, especially in the PPSES group. The VPR group in PPSES and BPSES group presented lower MMP-2 expression than their NPR rival groups. The VPR group in PPSES group presented increased TIMP-2 expression than its NPR rival group.There was statistically positive correlation between AI and△EEMA(r=0.275, P＜0.05), so was between AOD of MMP-2 and△EEMA (r=0.550, P＜0.05)Conclusions 1. The degree of vascular positive remodeling was more prominent in SES with polymer coating than SES without polymer coating after stent implantation.2. The course of vascular positive remodeling was associated with VSMC apoptosis and increased MMP-2 expression after stent implantation. VSMC apoptosis/ proliferation imbalance was related to the effect of rapamycin and local inflammatory reaction induced by polymer coating, MMPs/TIMPs imbalance was related to the local inflammatory reaction induced by polymer coating.更多还原