【作者】 鄂亚军；

【导师】 贺能树；

【作者基本信息】 天津医科大学 ， 影像医学与核医学， 2009， 博士

【摘要】 目的：评价雷帕霉素、紫杉醇等抗增殖药物对血管成形术(percutaneous transluminalangioplasty，PTA)后新生内膜增生及血管重塑的影响；评价雷帕霉素、紫杉醇对PTA后血管内皮化及血管壁凝血相关因子的影响；比较不同给药方式下紫杉醇对PTA后新生内膜增生及血管壁凝血相关因子的影响。材料与方法：1．建立大鼠颈总动脉损伤模型。2．采取外膜给药(以pluonic F-127为药物释放载体)，测定大鼠颈总动脉损伤后15天、30天新生内膜、中膜厚度与面积，分别计算内膜与中膜面积比、狭窄率、内弹力膜及外弹力膜围绕面积等指标，与对照组进行比较，观察并定量分析在抗增殖药物影响下血管壁大体形态学变化。3．采取外膜给药，以CD31标记血管内皮细胞检测抗增殖药物对血管内皮化的影响；通过免疫组化及原位杂交评估抗增殖药物对血管壁组织因子(tissue factor，TF)、组织型纤溶酶原激活剂(tissue type plasminogen activator，t-PA)及纤溶酶原激活物抑制剂1(inhibitors of plasminogen activator-1，PAI-1)表达的影响。4．采取腔内给药，观察不同浓度紫杉醇局部瞬时给药对损伤后新生内膜增生、血管内皮化的影响及其血栓形成倾向，与外膜给药进行比较。结果：1．外膜给药，损伤后15天、30天，雷帕霉素(300μg／100μl组及600μg／100μl组)及紫杉醇(20μg／100μl及40μg／100μl)实验组新生内膜厚度及面积、内膜与中膜面积比及狭窄率均低于对照组(P＜0．01)；对血管重塑的影响，损伤后30天两种抗增殖药物组均出现内、外弹力膜围绕面积的缩小(P＜0．05)；两种抗增殖药物高低两种浓度对血管壁的影响无统计学差别。2．外膜给药，损伤后15天、30天，雷帕霉素及紫杉醇实验组不同程度阻碍损伤后血管内皮化，内膜游离缘不光整并且出现异常增大变形的内皮细胞；与对照组相比，雷帕霉素及紫杉醇实验组新生内膜中TF mRNA及TF的表达高于对照组(P＜0．05)，TF在新生内膜具有向心性分布的特点，即主要集中于新生内膜游离缘，损伤后30天尤为明显；与对照组相比，雷帕霉素及紫杉醇实验组新生内膜PAI-1 mRNA及PAI-1的表达高于对照组(P＜0．05)；雷帕霉素与紫杉醇实验组新生内膜t-PA蛋白的表达低于对照组(P＜0．05)。3．腔内瞬时给药：血管损伤后15天及30天，紫杉醇高浓度(180μg／30μl)2分钟、10分钟组及低浓度(90μg／30μl)10分钟组新生内膜厚度、面积、内膜与中膜面积比及狭窄率等均低于对照组(P＜0．05)；内、外弹力膜围绕面积测定显示，实验组与对照组之间无统计学差异；与对照组相比较，实验组TF mRNA及TF蛋白在新生内膜中的表达与对照组没有统计学差别(P＞0．05)；实验组对新生内膜中PAI-1mRNA的影响与对照组相比没有统计学差别(P＞0．05)；对于PAI-1蛋白在新生内膜中的表达，高浓度10分钟组高于对照组(P＜0．05)，而其他3组(高浓度2分钟、低浓度2分钟及10分钟组)与对照组相比没有统计学差别(P＞0．05)；对新生内膜中t-PA蛋白表达的影响，实验组除了高浓度2分钟组低于对照组(P＜0．05)以外，其余各组虽然在数值上低于对照组，但无统计学意义。结论：1．雷帕霉素与紫杉醇外膜给药缓慢持续释放有效的抑制PTA后新生内膜增生。2．雷帕霉素与紫杉醇外膜给药缓慢持续释放具有一定的负性重塑效应(本实验中损伤后30天)，此效应对于以支架作为释放载体来说意义不大，但对于非支架释放载体以及未来新治疗方式的研发具有一定参考意义。3．雷帕霉素与紫杉醇外膜给药缓慢持续释放在抑制PTA后新生内膜增殖的同时阻碍内皮化，再生或残存内皮细胞发生形态改变。4．雷帕霉素与紫杉醇外膜给药缓慢持续释放诱导损伤后血管内膜TF在mRNA及蛋白水平的表达，并且TF蛋白有沿内膜腔面游离缘分布的特点。5．雷帕霉素与紫杉醇外膜给药缓慢持续释放诱导血管内膜PAI-1在mRNA及蛋白水平的表达，而相应t-PA在蛋白水平的表达有下降趋势。6．紫杉醇局部瞬时用药(180μg／30μl 2分钟及10分钟组；90μg／30μl 10分钟组)可以有效的抑制新生内膜增生，而且这种效应具有持续性。7．紫杉醇局部瞬时用药(180μg／30μl 2分钟组及90μg／30μl 10分钟组)在有效抑制内膜增生的同时不增加TF及PAI-1的表达。更多还原

【Abstract】 Objective①to evaluate the effect of sirolimus and paclitaxel on neointimal hyperplasia andvessel wall remodeling after PTA.②to investigate the effect of sirolimus andpaclitaxel on endothelialization and related coagulation factors in the vessel after PTA.③to compare the effect of different ways of administration of paclitaxel onrendothelialization and related coagulation factors in the vessel after PTA.Materials and methods1.Establishment of experimental common carotid artery injury model in the rat.2.Adventitial administration (Pluronic gel containing sirolimus or paclitaxel wasapplied to the exposed adventitial surface of the injured carotid artery)was applied.The neointimal thickness and area,medial thickness and area,the ratio of intimal areato medial area,stenotic ratio,internal elastic lamina area,and external elastic laminaarea were measured or calculated 15 and 30 days after injury respectively,which werecompared to those of controls,in order to evaluate the morphological changes of thevessel wall under the influence of the anti-proliferative agents.3.Adventitial administration was applied.CD31 antibody was used to label theendothelial cells in order to assess the effect of anti-proliferative agents onrendothelialization.The influence of anti-proliferative agents on the expression of TF,PAI-1 and t-PA of the vessel wall was observed by immunohistochemistry (IHC)andin situ hybridization (ISH).4.Local intra-arterial administration (an admixture of paclitaxel to contrast medium)was applied,and the effects of short-term delivery of paclitaxel on neointimalhyperplasia,vessel wall remodeling and related coagulation factors in the vessel wereevaluated.Relults1.Adventitial administration:15 and 30 days after injury,the neointimal thicknessand area,the ratio of intimal area to medial area and stenotic ratio were significantlydecreased in sirolimus (300μg/100μl,600μg/100μl)and paclitaxel (20μg/100μl,40μg/100μl)groups compared to control group (P＜0.01);Compared to controlgroup,the decrease of internal and external elastic lamina area could be seen (P＜0.05)30 days after injury;No significant difference of the effects of sirolimus andpaclitaxel on the vessel of could be seen between two concentrations respectively.2.Adventitial administration:15 and 30 days after injury,rendothelization of thevessel wall in sirolimus and paclitaxel experimental groups was delayed anduncompleted,and the inside surface of intima was uneven with enlarged and deformed endothelial cells diffusely distributed;Compared to control group,theexpression of TF mRNA and TF in neointima of sirolimus and paclitaxel groups wasincreased significantly (P＜0.05),and the expression of TF mainly located at theinside surface of intima,especially 30 days after injury;The expression of PAI-1mRNA and PAI-1 was increased significantly (P＜0.05),but the expression of t-PAwas decreased significantly (P＜0.05)in neointima of sirolimus and paclitaxel groupscompared to controls.3.Local intra-arterial administration:15 and 30 days after injury,the neointimalthickness and area,the ratio of intimal area to medial area and stenotic ratio weresignificantly decreased in paclitaxel high concentration (180μg/30μl)2 min,10mingroup and low concentration (180μg/30μl)10 min group(P＜0.05);No significantdifference of the internal and external elastic lamina area could be seen betweenpaclitaxel experimental groups and controls;Compared to controls,no obviouslysignificant changes for the expression of TF mRNA and TF exists in the neointima ofpaclitaxel experimental groups;there was no significant difference for the expressionof PAI-lmRNA in the intima between experimental groups and controls;Theexpression of PAI-1 in the intima of high concentration 10 min group was higher thancontrol group (P＜0.05),but no statistical difference was found between other 3groups(high concentration 2min,low concentration 2min and 10min groups)andcontrols;The expression t-PA in neointima of high concentration 2 min groupdecreased significantly than control group (P＜0.05),and for other 3 groups,Theexpression t-PA in neointima seemed to be lower than control group,but no statisticalsignificance could be seen.Conclusion1.A sustained release of sirolimus and paclitaxel with adventitial administrationcould inhibit the neointimal hyperplasia effectively after PTA.2.There was a negative remodeling effect after PTA (30 days after injury in theexperiment)for sustained release of sirolimus and paclitaxel with adventitialadministration.Although the negative remodeling effect seemed to have no effects forstent-based local drug delivery,but for non-stent-based local drug delivery or for thenew therapy of restenosis prevention,it had practical significance.3.A sustained release of sirolimus and paclitaxel with adventitial administrationdelayed rendothelization and caused the remained or regenerative cell deformed orenlarged after PTA.4.A sustained release of sirolimus and paclitaxel with adventitial administrationinduced high expression of TF mRNA and TF in intima.5.A sustained release of sirolimus and paclitaxel with adventitial administrationcaused high expression of PAI-1mRNA and PAI-1 in intima,however the expressionof t-PA in intima had a decreasing tendency. 6.A short-term release of paclitaxel with local intra-arterial administration(180μg/30μl 2 min and 10 min,90μg/30μl 10 min group in the experiment)couldeffectively suppress the neointimal hyperplasia in the long term (30 days in theexperiment).7.A short-term release of paclitaxel with local intra-arterial administration(180μg/30μl 2 min and 90μg/30μl 10min group in the experiment)could effectivelysurpress the neointimal hyperplasia,meanwhile,have no influence on the expressionof TF and PAI-1 in intima.更多还原