光学相干断层成像结合病理评价药物洗脱支架术后延迟的内膜修复愈合情况以及阿托伐他汀改善新生内膜愈合情况的研究

【作者】 王天杰；

【导师】 杨跃进；

【作者基本信息】 北京协和医学院 ， 内科学， 2012， 博士

【摘要】 目的：药物洗脱支架(drug-eluting stent, DES)术后发生的晚期(late stent thrombosis,LST)和晚晚期支架内血栓(very late stent thrombosis, VLST)具有很高的死亡率和急性心肌梗死率,危害极大。和金属裸支架(bare metal stent, BMS)相比,DES术后延迟的内膜修复愈合以及不完全的再内皮化被认为和LST和VLST发生密切相关。有相关研究证实阿托伐他汀可以通过激活PI3K/Akt通路增加内皮型一氧化氮合酶(endothelial nitric oxide synthase, eNOS)和一氧化氮(nitric oxide, NO)表达增强内皮细胞功能,最近研究发现阿托伐他汀还可以促进骨髓中的内皮祖细胞(endothelia progenitor cell, EPC)的动员,而EPC是内皮细胞的前体细胞,损伤发生时,EPC可以从骨髓中动员出来并归巢到血管内膜损伤处,通过增殖分化为成熟内皮细胞修复损伤。因此本课题利用中华小型猪BMS和DES支架植入模型,通过活体光学相干断层成像技术(optical coherence tomography, OCT)检查评价支架处内膜覆盖情况,对比病理中的内膜修复和再内皮化情况,评价DES术后内膜延迟愈合的情况,并给予阿托伐他汀进行干预治疗,观察阿托伐他汀对DES术后内膜和内皮修复的影响,并探讨其机制。方法：健康中华小型猪随机分为BMS组(n=15),DES组(n=15),DES+阿托伐他汀组(n=15)。每组又分为7天组,14天组和28天组三个亚组(n=5/亚组)。DES+阿托伐他汀组术前给予50mg阿托伐他汀,术后改为20mg/天。通过OCT评价支架植入即刻、7天、14天、28天的支架覆盖率、新生内膜厚度、新生内膜面积等指标,并和病理结果进行比较。术后7天、14天、28天通过免疫组化和扫描电镜评价支架处再内皮化情况、血小板粘附聚集和eNOS表达情况。通过流式细胞仪检测支架植入术前和支架术后7天、14天、28天外周血单个核细胞中的EPC比例。结果：OCT发现DES组内膜修复愈合较BMS组明显减慢,7天(56.8±5.7%vs44.4±1.0%,P<0.05)和14天(45.3±2.8%vs20.3±3.3%,P<0.05)时支架未覆盖率明显增高,DES+阿托伐他汀组术后7天(42.7±1.3%)和14天(24.8±4.3%)时支架未覆盖率明显降低,但28天时三组支架内膜均基本完全覆盖,覆盖率没有差异。OCT和病理检查测得的内膜覆盖率和扫描电镜检测的内皮覆盖率存在明显差异(P<0.05),再内皮化情况要明显延迟于内膜覆盖情况,尤其是在DES组差异更加明显。而DES+阿托伐他汀组再内皮化情况明显改善,内皮覆盖率较DES组明显增加,即使是在28天各组内膜化基本完全时,三组内皮覆盖率仍有明显差异(BMS组97.0±4.0%,SES组54.4±13.0%,SES+阿托伐他汀组77.3±10.3%)。流式细胞仪检测发现SES+阿托伐他汀组外周血EPC增加(0.21±0.02%vs0.11+0.03%,P=0.022),免疫组化染色发现支架上覆盖内皮细胞表达eNOS增加,扫描电镜结果提示单位面积内皮细胞上粘附的血小板数目也减少。结论：DES术后内膜修复和再内皮化并不同步,再内皮化更加延迟,阿托伐他汀可以促进DES术后内膜修复和内皮覆盖,并改善支架处新生内皮功能,其机制可能和促进骨髓EPC动员以及激活内皮细胞eNOS有关,这些可能有助于降低LST发生风险。目的：药物洗脱支架(drug-eluting stent, DES)术后发生的晚期支架内血栓(late stent thrombosis, LST)具有很高的死亡率和心梗率,危害极大,其机制主要认为和DES术后内膜修复愈合延迟,支架长期裸露有关,而过早的停用阿司匹林和氯吡格雷的双联抗血小板治疗可以明显增加LST的发生风险。但临床实践中阿司匹林可以导致胃溃疡和出血,有时植入DES后的患者不能耐受,需停用阿司匹林改为单纯服用氯吡格雷进行抗血小板治疗,这一经验性抗血小板治疗的效果尚无研究考证。在前期动物实验中,我们已经通过光学相干断层成像(optical coherence tomography, OCT)和病理检查证实阿托伐他汀可以改善DES术后延迟的内膜修复和促进再内皮化,囚此,本研究试图通过小型猪动物模型评价单纯服用氯吡格雷在内膜尚未完全修复的药物洗脱支架中的安全性,以及观察阿托伐他汀改善的内膜和内皮修复是否能够减低支架内血栓的发生。方法：18头中华小型猪(25+5kg)随机分为3组：BMS组(n=6),SES组(n=6),SES+阿托伐他汀组(n=6)。各组均在术前一晚给予300mg阿司匹林和300mg氯吡格雷,然后进行支架植入,BMS组植入无药物涂层的Firebird-2金属裸支架,SES组和SES+阿托伐他汀组植入携带西罗莫司的Firebird-2支架,术后改为100mg阿司匹林和75mg氯吡格雷治疗,持续一周,然后停用阿司匹林,仅给予氯毗格雷治疗。支架术后和14天、21天、28天进行冠脉造影和OCT检查,评价内膜修复情况和支架内血栓发生情况,同时抽取外周血进行光学比浊法检查血小板聚集率。对发生血栓的动物处死行病理检查,并通过扫描电镜评价血小板粘附情况。结果：术后28天各组累计支架内血栓发生率分别为BMS组0%(0/6),SES组16.7%(1/6),SES+阿托伐他汀组33.3%(2/6),但SES组和SES+阿托伐他汀组血栓发生率并无差异(P=1.0)。14天时支架新生内膜覆盖率BMS组(77.3±8.0%)和SES+阿托伐他汀组(75.2±9.5%)要优于SES组(54.7±6.2%,P<0.001),但21天和28天时,三组间支架覆盖率并无差异。病理检查发现支架内血栓基本均发生在支架裸露部位,扫描电镜检测各组血小板计数分别为BMS组9.7±1.8,SES组21.9±3.9,SES+阿托伐他汀组18.0±3.5,SES组和SES+阿托伐他汀组单位面积内皮细胞粘附血小板数目要高于BMS组(SES vs BMS, P=0.014;SES+阿托伐他汀vs BMS, P=0.052)。结论：在小型猪支架模型中,DES术后支架内膜尚未完全修复愈合时,单独氯吡格雷抗血小板治疗不能完全阻止小型猪支架模型中的血栓形成,其支架内血栓的发生机制可能与裸露的支架丝以及损伤的内皮功能有关。而阿托伐他汀虽然可以改善DES术后早期内膜修复,但并未减少支架内血栓的发生。目的：药物洗脱支架(drug-eluting stent, DES)术后延迟的内膜修复愈合情况被认为和晚期支架内血栓(late stent thrombosis, LST)的发生密切相关。光学相干断层成像技术(optical coherence tomography, OCT)是近年来新兴的冠脉内成像技术,具有10um的超高分辨率,可以准确评价DES术后的新生内膜覆盖情况。既往研究主要关注DES植入后6月以后以及更长期的内膜覆盖情况,DES术后极早期的内膜修复愈合情况尚未见报道。近年来研究显示新一代药物洗脱支架依维莫司洗脱支架(everolimus-eluting stent, EES)同原来的西罗莫司洗脱支架(sirolimus-eluting stent,SES)相比能够进一步减少MACE事件和LST的发生,但尚无对比EES和SES对极早期内膜修复愈合影响的研究。因此,本研究通过OCT评价DES术后7天以及3个月左右的新生内膜修复情况,了解DES术后内膜修复愈合规律,并探讨可能影响早期内膜修复愈合的因素。方法：回顾性分析了2009年9月至2011年9月间,37名存在冠心病多支病变的患者,这些患者均接受了分次DES植入,并在支架植入即刻和随访中接受OCT检查。根据第二次OCT检查时间分为7天组(n=18例,36DES)和3月组(n=19例,33DES)。利用OCT专用分析软件(QIVUS2.1Bate Version with OCT Module)检测术后即刻和随访中的支架覆盖率、贴壁不良率、管腔面积、支架面积、附壁血栓发生率、新生内膜厚度等指标。并根据患者临床表现和植入支架类型不同进行多元线性回归分析,观察各因素对支架新生内膜覆盖率和支架贴壁不良率的影响。结果：7天时18.7±16.6%的支架丝已被覆盖,3月时77.3±16.0%的支架丝已被覆盖。多元线性回归分析发现SES(p系数-14.9%,95%可信区间：-23.2%～-6.6%,P=0.001)和高血压(p系数-9.3%,95%可信区间：-17.4%～-1.3%,P=0.025)两个因素和支架内膜覆盖率负相关,3月时植入EES患者覆盖率85.6±5.9%,而植入SES组仅有69.8±3.7%(P=0.003)；高血压患者组平均覆盖率64.5±4.2%,非高血压组80.9±4.2%(P=0.02)。在支架贴壁不良方面,回归分析发现术后即刻贴壁不良(p系数0.3%,95%可信区间：0.1%～0.5%,P=0.002)和慢性完全闭塞病变(chronic total occlusion, CTO)(β系数1.3%,95%可信区间：0.2%～2.4%,P=0.018)能够影响支架植入术后3月的贴壁不良率。结论：DES植入术后一周即检测到支架处新生内膜覆盖,但覆盖率较低,大部分支架丝裸露。在术后极早期的内膜修复方面,新一代的EES和SES相比支架新生内膜覆盖率更高,高血压患者和非高血压患者相比术后3月内的支架新生内膜覆盖率下降。而术后即刻支架贴壁不良和CTO病变可能会增加术后早期的随访支架贴壁不良。更多还原

【Abstract】 Background:Delayed neointimal coverage and re-endothelialization after drug-eluting stent implantation are thought to be the underlying mechanisms of late stent thrombosis (LST).Methods and Results:Forty-five Chinese minipigs were divided into three groups (n=15each):bare metal stent (BMS), SES, and SES and atorvastatin treatment (SES+ator). Neointimal coverage and endothelium coverage were evaluated separately by optical coherence tomography (OCT), pathology, and scanning electron microscopy (SEM) at days7,14and28. OCT showed that SES significantly delayed neointimal coverage compared with BMS and the percentage of uncovered struts in the SES+ator group was significantly decreased on days7(42.7±1.3%vs56.8±5.7%, P<0.01) and14(24.8±4.3%vs45.3±2.8%, P<0.01) compared with the SES group. However, re-endothelialization was even more seriously delayed than the neointima formation after SES deployed (P<0.05). Pathology and SEM revealed improved re-endothelialization on the neointima by atorvastatin therapy in terms of more struts covered by endothelium, less platelet adhesion, and higher eNOS expression of the endothelial cells in the SES+ator group. Flow cytometry illustrated that the SES+ator group had more mobilized EPCs compared with the SES group at day7(0.21+0.02%vs0.11±0.03%, P=0.022).Conclusions:Atorvastatin pretreatment can accelerate both neointimal coverage and re-endothelialization after SES implantation which may be mediated by the mobilization of EPC and enhancement of the endothelial function of the neointima. Objective:To investigate whether monotherapy by clopidogrel without aspirin after drug-eluting stent implantation (DES) was effective to inhibite platelets activation and prevent stent thrombosis formation.Methods:Eighteen Chinese mini-porcine were randomized into3groups of bare metal stent (BMS)(n=6), sirolimus-eluting stent (SES)(control; n=6), and SES plus Ator group(atorvastatin; n=6). Each animal accepted300mg aspirine and300mg clopidogrel before stent implantation and100mg aspirin and75mg clopidogrel per day for7days after stent implantation. Then aspirine therapy was stopped and only75mg clopidogrel was given. Inhabitation of platelet was evaluated by platelet affregation test before PCI and at7days,14days,21days, and28days after PCI. The rate of stent thrombosis was observed by coronary angiography and optical coherence tomography (OCT). Stent thrombosis was then confirmed by pathology and the adhension of platelets on stents was evaluated by scanning electron microscopy (SEM).Results:The percentage of stent thrombosis was0%in the BMS group (0/6),16.7%in the SES group (1/6),33.3%in the SES+ator group (2/6), respectively. Although the BMS group and the SES+ator group showed better neointima coverage compared with the SES group at14days after PCI, there was no signigicant difference about the rate of stent thrombosis between the SES group and the SES+ator group (P=1.0). the percentage of stent coverage was77.3±8.0%,54.7±6.2%, and75.2±9.5%in the BMS, SES, and SES+ator group respectively. The result of pathology showed that the stent thrombosis was mainly formed on the stent struts without neointimal coverage. SEM revealed that the number of platelet adhension was9.7±9.7,21.9±17.9and18.0±20.9in the three groups respectively. The SES group and the SES+ator group had more platelets adhension than the BMS group at28days(P=0.014and P=0.052). there was no significant difference between the SES and the SES+ator group (P=0.41).Conclusion:mono antiplatelet therapy by clopigrel could not completely prevent stent thrombosis formation after DES implantation. although acceralted neointimal coverage was observed by atorvastatin therapy, no decrease of stent thrombosis was shown. Objective:To evaluate the neointimal coverage at the very early phase after sirolimus-(SES) and everolimus-eluting stent (EES) implantation using optical coherence tomography (OCT).Background:Previous studies usually focus on the delayed neointimal coverage at long term follow-up after drug-eluting stent (DES) implantation. The very early neointimal hyperplasia is seldom studied.Methods:Thirty-seven multi-vessel coronary artery disease patients who accepted OCT examination both at immediately post-intervention and follow-up were classified into either the7-day group (n=18patients,36stents) or the3-month group (n=19patients,33stents). The stent coverage and malapposition was analyzed using OCT.Results:18.7±16.6%and77.3±16.0%of the struts were covered at7days and3months, respectively. SES and hypertension were associated with a significantly lower percentage of stent coverage at3months (69.8±3.7%in SES vs85.6±5.9%in EES, P=0.003;64.5±4.2%vs80.9±4.2%, P=0.02, respectively). The stepwise linear regression analyses showed that the percentage of immediately malapposed struts (beta coefficient:0.3%,95%confidence interval:0.1%to0.5%, P=0.002) and chronic total occlusion (CTO)(beta coefficient:1.3%,95%confidence interval:0.2%to2.4%, P=0.018) could increase the percentage of struts malapposition in the3months follow-up.Conclusion:Neointimal coverage could be detected at one week after DES implantation by OCT. However, EES showed better neointimal coverage compared with SES. Hypertension could impact the neointimal coverage in the first3months. The rate of stent malapposition immediately after intervention and CTO may increase struts malapposition in the very early phase after DES implantation.更多还原