肝素化bFGF支架在心肌损伤修复中的实验研究
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摘要
目的：冠状动脉粥样硬化性心脏病(Coronary artery disease, CAD)严重威胁着人类的健康,冠状动脉搭桥术和经皮冠脉介入治疗挽救了大量冠心病患者的生命。但是,约12%的患者因弥漫性冠脉血管病变、搭桥远端靶血管直径偏小以及二次手术缺乏桥血管等原因不适合上述治疗。最近,我们发明了一种新的心肌再血管化的方法：心肌钻孔结合肝素化碱性成纤维细胞生长因子(Basic fibroblast growth factor, bFGF)缓释支架植入。前期的实验已经证实了这种新方法的可行性,本文旨在揭示其在抗心肌缺血性损伤中的作用。
方法：利用小型猪急性心梗模型,设立对照组和治疗组。对照组不接受任何治疗,治疗组于梗死区内钻两个直径为3.5mm的穿心肌孔道并植入两枚支架。
(1)采用MRI扫描及生理记录仪评价心肌重构、心功能相关参数变化；
(2)采用RT-PCR、免疫组化方法观察心肌纤维化,细胞凋亡及心肌干细胞增殖、分化情况,以揭示该方法的作用机制；
(3)于细胞水平测量每个孔道的有效治疗半径,为确定孔道间距提供指导性意见。
结果：(1)术后6周,治疗组左室舒张末容积(Left ventricular end-diastolic volume, LVEDV)、左室收缩末容积(Left ventricular end-systolic volume, LVESV)明显低于对照组(P<0.05)；缺血区室壁厚度明显厚于对照组(P<0.001)；左室射血分数(Left ventricular ejection fraction, LVEF)明显高于对照组(P<0.001),左室内压最大上升速率(The maximum velocity of LV contraction, dP/dtmax)和左室内压最大下降速率(Themaximum velocity of LV diastole, dP/dtmin)亦明显优于对照组(P<0.001)。
(2)术后6周,治疗组血管内皮细胞生长因子(Vascular endothelial growth factor, VEGF)、血管性血友病因子(Von Willebrand Factor, vWF)、转化生长因子β3(Transforming growth factor-β3, TGF-β3)、白介素1β(Interleukin-1β, IL-1β)、基质细胞衍生因子-1(stromal cell-derived factor-1, SDF-1)和CXC族细胞因子受体4(CXC chemokine receptor4,CXCR4) mRNA表达较对照组上调(P<0.05)。vWF染色发现治疗组新生血管密度较对照组显著增加(P<0.001)。TUNEL染色结果显示治疗组细胞凋亡明显少于对照组(P<0.001)。5-溴脱氧尿嘧啶核苷(5-bromo-2-deoxyuridine, BrdU)染色定量分析显示治疗组新生心肌明显多于对照组(P<0.001)。c-kit染色有相似的结果(P<0.001)。同时,Masson三色法染色证实治疗组存活心肌面积明显高于对照组(P<0.001)。
(3)心肌打孔结合支架植入治疗的效果与距孔道中心距离呈明显的负相关。促血管再生的有效范围为距孔道中心10 mm,增加心肌存活的有效范围为5-7.5 mm,凋亡则在7.5-10mm之间。
结论：(1)心肌钻孔结合肝素化bFGF缓释支架植入可以明显抑制心室重构,改善心功能。
(2)该方法通过减轻心梗后纤维化,抑制心肌细胞凋亡,促进心肌干细胞增殖、向心肌细胞分化,发挥心肌缺血性损伤修复作用。
(3)每个孔道的有效治疗半径为5-7.5mm。因此建议孔道间距为10mm。
Objective:Coronary artery disease remains the leading cause of morbidity and mortality in the world. Approximately 12% of patients with coronary artery disease, because of unfavorable characteristics such as diffuse coronary atherosclerosis, small distal vessels or lack of suitable bridge vessels, are not amenable to coronary artery bypass grafting or percutaneous coronary intervention. Recently, we have developed a novel method:transmyocardial drilling revascularization combined with heparinized basic fibroblast growth factor (bFGF)-incorporating stent implantation. In previous studies, the feasibility of this method has been confirmed. The objective of this study is to investigate the effects of the method against ischemic myocardial injury.
Methods:The miniswine model of acute myocardial ischemia was used in present study. The animals were divided into control group (without any treatment) and treatment group. In treatment group, two channels with 3.5 mm in diameter were established in ischemic region, followed by implantation of two stents.
(1) Myocardial remodeling and left ventricular function related parameters were determined by MRI scan and physiological recorder.
(2) Myocardial fibrosis, cell apoptosis, proliferation and differentiation of cardiac stem cell were evaluated by RT-PCR and immunohistochemical analysis to explore the underlying mechanisms.
(3) The effective radius of every channel was determined at cell level, to provide a guide for selection of pitch of channels
Results:
(1) Six weeks after operation, left ventricular end-diastolic volume (LVEDV) and LV end-systolic volume (LVESV) were significantly decreased (P<0.05), the wall thick of ischemic region increased significantly (P<0.001), LV ejection fraction (LVEF) was significantly promoted (P<0.001), and the maximum velocity of LV contraction (dP/dtmax) and the maximum velocity of LV diastole (dP/dtmin) were significantly improved (P<0.001) in treatment group, compared with control group.
(2) Six weeks after therapy, there was up-regulative expressions of vascular endothelial growth factor (VEGF), von Willebrand Factor (vWF), transforming growth factor-β3 (TGF-β3), Interleukin-1β(IL-1β), stromal cell-derived factor-1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) mRNA in treatment group (P<0.05), compared with control group. vWF staining analysis showed an enhanced angiogenesis in treatment group relative to control group (P<0.001). It was demonstrated by TUNEL that cell opoptosis was significantly inhibited in treatment group compared with control group (P<0.001). Quantitative analysis of 5-bromo-2-deoxyuridine (BrdU) stained positive region revealed more neonatal myocytes in treatment group than the control (P<0.001). There were similar results in c-kit staining analysis (P<0.001). Masson trichrome staining analysis demonstrated that myocardial viability was significantly improved in treatment group compared with control group (P<0.001).
(3) There was a significant negative correlationship between the effects of transmyocardial drilling revascularization combined with stent implantation and the distance away from the center of channels. Although the effective radius of angiogenesis was more than 10 mm, the result of myocardial viability analysis was 5 mm, and cell apoptosis analysis yielded an effective radius of 7.5 mm.
Conclusions:
(1) Transmyocardial drilling revascularization combined with heparinized bFGF-incorporating stent implantation may reverse ventricular remodeling, and promote left ventricular function.
(2) This method may attenuate myocardial fibrosis, inhibit cell apoptosis, and promote proliferation of cardiac stem cells and differentiation to myocytes, consequently provide protective effects against ischemic injury.
(3) The effective radius of every channel was 5-7.5 mm, it is therefore suggested that the pitch between two channels should be 10 mm.
方法：利用小型猪急性心梗模型,设立对照组和治疗组。对照组不接受任何治疗,治疗组于梗死区内钻两个直径为3.5mm的穿心肌孔道并植入两枚支架。
(1)采用MRI扫描及生理记录仪评价心肌重构、心功能相关参数变化；
(2)采用RT-PCR、免疫组化方法观察心肌纤维化,细胞凋亡及心肌干细胞增殖、分化情况,以揭示该方法的作用机制；
(3)于细胞水平测量每个孔道的有效治疗半径,为确定孔道间距提供指导性意见。
结果：(1)术后6周,治疗组左室舒张末容积(Left ventricular end-diastolic volume, LVEDV)、左室收缩末容积(Left ventricular end-systolic volume, LVESV)明显低于对照组(P<0.05)；缺血区室壁厚度明显厚于对照组(P<0.001)；左室射血分数(Left ventricular ejection fraction, LVEF)明显高于对照组(P<0.001),左室内压最大上升速率(The maximum velocity of LV contraction, dP/dtmax)和左室内压最大下降速率(Themaximum velocity of LV diastole, dP/dtmin)亦明显优于对照组(P<0.001)。
(2)术后6周,治疗组血管内皮细胞生长因子(Vascular endothelial growth factor, VEGF)、血管性血友病因子(Von Willebrand Factor, vWF)、转化生长因子β3(Transforming growth factor-β3, TGF-β3)、白介素1β(Interleukin-1β, IL-1β)、基质细胞衍生因子-1(stromal cell-derived factor-1, SDF-1)和CXC族细胞因子受体4(CXC chemokine receptor4,CXCR4) mRNA表达较对照组上调(P<0.05)。vWF染色发现治疗组新生血管密度较对照组显著增加(P<0.001)。TUNEL染色结果显示治疗组细胞凋亡明显少于对照组(P<0.001)。5-溴脱氧尿嘧啶核苷(5-bromo-2-deoxyuridine, BrdU)染色定量分析显示治疗组新生心肌明显多于对照组(P<0.001)。c-kit染色有相似的结果(P<0.001)。同时,Masson三色法染色证实治疗组存活心肌面积明显高于对照组(P<0.001)。
(3)心肌打孔结合支架植入治疗的效果与距孔道中心距离呈明显的负相关。促血管再生的有效范围为距孔道中心10 mm,增加心肌存活的有效范围为5-7.5 mm,凋亡则在7.5-10mm之间。
结论：(1)心肌钻孔结合肝素化bFGF缓释支架植入可以明显抑制心室重构,改善心功能。
(2)该方法通过减轻心梗后纤维化,抑制心肌细胞凋亡,促进心肌干细胞增殖、向心肌细胞分化,发挥心肌缺血性损伤修复作用。
(3)每个孔道的有效治疗半径为5-7.5mm。因此建议孔道间距为10mm。
Objective:Coronary artery disease remains the leading cause of morbidity and mortality in the world. Approximately 12% of patients with coronary artery disease, because of unfavorable characteristics such as diffuse coronary atherosclerosis, small distal vessels or lack of suitable bridge vessels, are not amenable to coronary artery bypass grafting or percutaneous coronary intervention. Recently, we have developed a novel method:transmyocardial drilling revascularization combined with heparinized basic fibroblast growth factor (bFGF)-incorporating stent implantation. In previous studies, the feasibility of this method has been confirmed. The objective of this study is to investigate the effects of the method against ischemic myocardial injury.
Methods:The miniswine model of acute myocardial ischemia was used in present study. The animals were divided into control group (without any treatment) and treatment group. In treatment group, two channels with 3.5 mm in diameter were established in ischemic region, followed by implantation of two stents.
(1) Myocardial remodeling and left ventricular function related parameters were determined by MRI scan and physiological recorder.
(2) Myocardial fibrosis, cell apoptosis, proliferation and differentiation of cardiac stem cell were evaluated by RT-PCR and immunohistochemical analysis to explore the underlying mechanisms.
(3) The effective radius of every channel was determined at cell level, to provide a guide for selection of pitch of channels
Results:
(1) Six weeks after operation, left ventricular end-diastolic volume (LVEDV) and LV end-systolic volume (LVESV) were significantly decreased (P<0.05), the wall thick of ischemic region increased significantly (P<0.001), LV ejection fraction (LVEF) was significantly promoted (P<0.001), and the maximum velocity of LV contraction (dP/dtmax) and the maximum velocity of LV diastole (dP/dtmin) were significantly improved (P<0.001) in treatment group, compared with control group.
(2) Six weeks after therapy, there was up-regulative expressions of vascular endothelial growth factor (VEGF), von Willebrand Factor (vWF), transforming growth factor-β3 (TGF-β3), Interleukin-1β(IL-1β), stromal cell-derived factor-1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) mRNA in treatment group (P<0.05), compared with control group. vWF staining analysis showed an enhanced angiogenesis in treatment group relative to control group (P<0.001). It was demonstrated by TUNEL that cell opoptosis was significantly inhibited in treatment group compared with control group (P<0.001). Quantitative analysis of 5-bromo-2-deoxyuridine (BrdU) stained positive region revealed more neonatal myocytes in treatment group than the control (P<0.001). There were similar results in c-kit staining analysis (P<0.001). Masson trichrome staining analysis demonstrated that myocardial viability was significantly improved in treatment group compared with control group (P<0.001).
(3) There was a significant negative correlationship between the effects of transmyocardial drilling revascularization combined with stent implantation and the distance away from the center of channels. Although the effective radius of angiogenesis was more than 10 mm, the result of myocardial viability analysis was 5 mm, and cell apoptosis analysis yielded an effective radius of 7.5 mm.
Conclusions:
(1) Transmyocardial drilling revascularization combined with heparinized bFGF-incorporating stent implantation may reverse ventricular remodeling, and promote left ventricular function.
(2) This method may attenuate myocardial fibrosis, inhibit cell apoptosis, and promote proliferation of cardiac stem cells and differentiation to myocytes, consequently provide protective effects against ischemic injury.
(3) The effective radius of every channel was 5-7.5 mm, it is therefore suggested that the pitch between two channels should be 10 mm.
引文
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