氨氯地平对糖尿病大鼠心肌梗死后骨髓内皮祖细胞动员及血管新生的改善作用

Amlodipine Improves Endothelial Progenitor Cell Mobilization and Neo-vascularization in Experimental Diabetic Rats After Myocardial Infarction

目的：观察氨氯地平对糖尿病大鼠心肌梗死（心梗）后骨髓内皮祖细胞（EPC）动员和血管新生障碍的改善作用以及对心功能的影响,并探讨其可能的分子机制。方法：180-200g SPF级雄性Sprague-Dawley大鼠60只随机分为两部分：糖尿病大鼠（n=40）给予高脂饲料饲养四周后,腹腔注射30 mg/kg链脲佐菌素;非糖尿病大鼠（n=20）为正常饮食饲养。40只糖尿病大鼠结扎冠状动脉左前降支造成急性心梗模型。术后将大鼠随机分成对照组（n=20）,每日予以0.5%羧甲基纤维素钠溶剂1ml灌胃,治疗组（n=20）每日予以氨氯地平2 mg/kg灌胃,继续高脂喂养四周。流式细胞术检测术前及术后不同时间点（第1、3、5、7、14和28 d）外周血CD45-/low＋/CD133＋/KDR＋早期EPC数量,酶联免疫吸附法检测血浆血管内皮生长因子（VEGF）水平。CD31免疫荧光染色法评估心梗周围区血管新生情况。超声心动图评估心功能。免疫印迹法测定骨髓细胞中EPC动员相关信号通路蛋白的表达。结果：外周血CD45-/low＋/CD133＋/KDR＋EPC水平术后峰值治疗组（第7 d,112±30/106单核细胞）较对照组（第5d,55±10/106单核细胞）升高;血浆VEGF水平在心梗后峰值治疗组[第7 d,（5.63±1.33）ng/L]较对照组[第5 d,（3.68±0.98）ng/L]升高;骨髓细胞蛋白激酶B与内皮型一氧化氮合酶的活化水平及基质金属蛋白酶-9的表达增加;心梗周围区新生毛细血管密度治疗组大鼠较对照组显著增加,左心室射血分数及左心室短轴缩短率明显提高。上述比较差异均有统计学意义（P〈0.05-0.01）。结论：氨氯地平治疗改善糖尿病大鼠缺血诱导的骨髓EPC动员障碍,缺血区血管新生以及心梗后心功能。这种作用可能通过改善VEGF/内皮型一氧化氮合酶信号通路活化而介导。

Objective： To observe the effect of amlodipine on bone marrow endothelial progenitor cell （EPC） mobilization, neo-vascularization and cardiac function in diabetic rats after myocardial infarction （MI） with the possible mechanisms. Methods： A total of 60 male SD rats were divided into 2 groups. Normal group, n=20. Diabetic group, n=40, the rats were fed with high fat diet （HFD） for 4 weeks and then received streptozotocin followed by left anterior descending coronary artery ligation to establish MI model, those rats were further divided into 2 sub-groups：Control group, the rats received sodium carboxymethylcellulose 1 ml/day with HFD and Treatment group, the rats received amlodipine 2 mg/kg/day with HFD, n=20 in each sub-group, all animals were treated for 4 weeks. The EPC level in peripheral blood CD45-/low＋/CD133＋/KDR＋ at before and 1, 3, 5, 7, 14, 28 days after operation were examined by lfow cytometry, plasma vascular endothelial growth factor （VEGF） level was measured by ELISA, capillary density in MI area was determined by CD31 staining, EPC related protein expressions were detected by western blot analysis and the cardiac function was evaluated by echocardiography. Results： EPC in CD45-/low＋/CD133＋/KDR＋in Treatment group at 7 days after operation was increased than Control group at 5 days after operation （112 ± 30/106） vs （55 ± 10/106）, plasma VEGF in Treatment group was higher than Control group （5.63 ± 1.33） ng/L vs （3.68 ± 0.98） ng/L; Treatment group presented increased expressions of protein kinase B, endothelial nitric oxide synthase （eNOS） and matrix metallopeptidase-9, increased capillary density in MI area, higher LVEF and left ventricular fractional shorting, all P〈0.05-0.01. Conclusion： Amlodipine improves EPC mobilization, neo-vascularization and cardiac function in diabetic-MI rats, it may be related to VEGF/eNOS cascade activation.