【作者】 肖丽；

【导师】 王贵学；

【作者基本信息】 重庆大学 ， 生物医学工程， 2007， 博士

【摘要】 经皮腔内冠脉介入(percutaneous coronary inteventional,PCI)是冠心病治疗的重要手段,但术后引起的损伤反应—再狭窄是困扰介入治疗的难题。冠脉内支架植入术的广泛应用使PCI术后再狭窄率明显降低,但支架植入又会引起支架内再狭窄(in-stent restenosis;ISR),其发生率有(13-20)%。目前,防治ISR的研究主要集中在药物涂层支架和血管内放射治疗,它们都能抑制或破坏DNA合成,从而显著抑制新生内膜增生,但同时也会影响内皮细胞再生,有迟发血栓形成和使支架裸露的危险。因此,两者都不是最佳的防治ISR的治疗方法。人们现在普遍认为对支架表面进行内皮化修饰可能会促使内皮功能及完整性的恢复,似乎成为解决ISR的良好途径。近年来的研究发现,内皮祖细胞(endothelial progenitor cells,EPCs)具有发育为内皮样细胞的能力,其分化的内皮细胞比成熟内皮细胞具有更高的增殖潜能;更有利于细胞的自我保护,这为实现血管内支架内皮化提供一种新的途径。目前研究支架内皮化的研究主要集中于种子细胞来源、支架内皮化时间以及抗再狭窄的能力等方面。本文通过筛选能特异性粘附EPCs的基质,将其涂层于支架表面,利用抗原抗体特异性结合特性捕获循环血液中的EPCs,体内分化为成熟内皮细胞参与损伤内皮的修复。模拟生理条件,在流场条件下检测特异性粘附基质对EPCs的数量,及增殖、粘附和分泌NO能力的影响。同时,抗体支架植入早期,植入部位有炎症及急性血栓形成,如果在抗体涂层支架表面包被抗炎、抗血栓形成的药物,可能会更好地发挥上述支架捕获EPCs和防治ISR作用。因此,本文还对抗再狭窄的药物进行筛选,并且研究该种药物对EPCs增殖、粘附和NO分泌能力的影响。本文中采用的主要研究方法和技术如下:①采用Percoll梯度离心法和贴壁法分离大鼠骨髓中EPCs,通过免疫荧光、免疫细胞化学和流式细胞仪鉴定分离的EPCs。②应用单微管吸吮技术,根据EPCs与不同基质(明胶、VEGFR2抗体、CD34抗体和CD133抗体)裱衬表面的粘附力比较,筛选得到能特异性结合EPCs的抗体基质。③利用平行平板流动腔技术,体外模拟定常流条件,研究剪应力对EPCs结合基质能力的影响,运用噻唑蓝(MTT)法、细胞计数、NO试剂盒等实验手段测定研究EPCs生物学性能的影响。④采用球囊拉伸损伤大鼠颈总动脉,施加药物治疗,筛选抗再狭窄药物;然后提取筛选药物苦参的有效部位作用于EPCs,采用MTT法、NO表达量测定、流式细胞仪等实验手段研究了苦参总碱对EPCs数量和功能的影响⑤体外模拟流动场,形成循环EPCs液,将支架置入细胞悬液中,采用光镜观测和细胞计数实验手段,检测4h后支架表面特异性基质涂层表面EPCs捕获情况。通过上述研究方法和手段,得到下面的实验结果:①获得了纯度较高的EPCs,流式细胞仪检测纯度在70%以上,其鉴定结果为分离的EPCs是VEGFR2+CD34+CD133+细胞。②单微管吸吮实验发现:明胶、抗体VEGFR2、CD34、CD133四种基质中,EPCs均呈浓度依赖性,但是对明胶浓度依赖性差异不显著;(3)EPCs对抗体CD133的粘附力最强,相比较内皮细胞,EPCs对抗体CD133的特异性粘附显著,在EC表面没有CD133抗原,CD133抗体对EPCs的更具有特异性;EPCs对不同基质的粘附力,以及对基质浓度的依赖性。③EPCs在用CD133裱衬的Chamber上受到剪应力作用,其保留率、增殖率都显著高于其它两种抗体裱衬;EPCs在用CD133裱衬的Chamber上受到剪应力作用,NO表达量都显著增加,一定剪应力作用可能会诱导EPCs细胞向EC细胞定向分化。④动物实验证明苦参总碱能有效地抑制内膜增生,当浓度是100μg/ml时,苦参总碱对EPCs有促增殖作用,细胞凋亡率为(1.87±1.682)%,且不会减少EPCs对特异性基质的粘附能力;同时NO分泌量在该浓度的苦参总碱作用60h达到峰值。⑤体外结果显示,CD133抗体涂层对EPCs的捕获能力强于CD34抗体涂层。总之,本文通过研究EPCs对促粘附基质的特异性结合,流场对EPCs与基质特异性结合后其生物学性能的影响,抑制内膜增生药物对EPCs数量和功能的影响,以及抗体涂层支架体外捕获EPCs能力的检测,为血管内支架内皮化早日实现提供了新的思路,也为人工心瓣膜、人工血管的内皮化研究提供了新的参考。更多还原

【Abstract】 Percutaneous coronary interventional (PCI)as the main interventional therapies of coronary artery disease has induced the restenosis of blood vessel, which has restricted its development. The planting of stent in blood vessel can decrease the restenosis incidence rate, but that also brings the in-stent restenosis (ISR). Now the emergence of drug-eluting stents has reduced the incidence of ISR. The drug coated on stents can inhibit or breakdown DNA synthesis in cell, which can inhibit the neoinerma hyperplasy and affect the cell proliferation. Studies shows that rapid endothelium of stents may be an available method to inhibit ISR, which can make injuried vessel wall reendothelialization quickly. However, the adhesion, proliferation and resource of cells planted on the stent are the unsolved questions in the endothelialization process of stents. Recent studies show that endothelial progenitor cells (EPCs) are a cell population that has the capacity to circulate, proliferate, and differentiate into mature endothelial cells, and EPCs has been identified as a key factor for re-endothelialization.The EPCs capture stents have been developed using immobilized antibodies targeted at EPCs surface antigens. Antibody-coated stent planted into vessel to capture EPCs in circulating blood and to keep them on stent and to make stent endotheliazation depends on the adhesion, proliferation of EPC on antibody coating.Now the studies on stent surface endothelialum focus on the resource of cell, the time of endothelium and the anti-restenosis capability. In this study, EPCs from SD rat were isolated by density gradient centrifugation with Percoll solution. And immunofluorescence, immunocytochemistry and flow cytometry were used to identify EPCs. EPCs adhesion force on different substrates was detected by use of single micropipette aspiration technique. The effect of shear stress on adhesion of EPCs to substrates (anti-VEGFR2, anti-CD34 and anti-CD133) by parallel plate flow Chamber technique at multi-cell level. The proliferation of EPCs treated with sophorcarpidine solution was assayed with MTT assay, and the cell cycle was detected with flow cytometer (FCM). At last, the EPCs capture ability was valued.Results showed that the high purity EPCs was obtained by density gradient centrifugation with Percoll solution, and EPCs were VEGFR2+CD34+CD133+ cells. The adhesion force of EPCs and endothelial cells on different substrates depended on concentration of gelatin, anti-VEGFR2, anti-CD34 and anti-CD133. The adhesion characters of EPCs on the four substrate materials were better than that of ECs. And the same time, the adhesion force of EPCs to antibody CD133 was the strongest among the four substrate materials. The study on the effect of shear stress on EPCs showed that the proliferation rate and reservation rate on anti-CD133 coating have great difference to compare with other substrates (anti-VEGFR2 and anti-CD34). And the NO expression of EPCs was increased with the treatment of shear stress. Sophorcarpidine increased EPCs adhesive activity at concentration 100μg/ml. With the increase of Sophorcarpidine concentration, the apoptosis rate decreased with significant difference (P<0.05). However, cytotoxicity was seen at higher sophorcarpidine concentration (150μg/ml). The minimum apoptosis rate was (1.87±1.682) % at concentration 100μg/ml. The ability of capturing EPCs of CD133 antibody coating was stronger than that of CD34 antibody coating.In a word, these findings in this study may offer the researchers the inspiration to realize the endothelialization of stent surface, and be the reference for the research of endothelialization of artificial heart valve and artificial vascular.更多还原