急性心肌梗死后心肌细胞增殖和再生的初步实验研究

Myocardial proliferation/regeneration in rats with experimental acute myocardial infarction

目的 评估急性心肌梗死后,不同缺血时段心脏不同部位心肌细胞增殖变化,探讨增殖发生时间和部位、分布特点及增殖细胞是否与心脏干细胞有关联及其在修复损伤心肌中的可能作用.方法 成年SD大鼠64只按随机数字表法分为2组,实验组（n=44）开胸结扎冠状动脉前降支制作急性心肌梗死模型,对照组（n=20）开胸置缝线而不结扎.于术后第3、5、7、14和60 d处死大鼠（第7和14天两组各9只,其余各组均为5只）,采集左心室梗死边缘区及右心室非梗死区心肌组织.处死大鼠前12和24 h分别腹腔注射50 mg/kg5-溴-2-脱氧尿苷（Brdu）.HE染色观察心肌组织形态学和病理学变化,用免疫组化方法检测Brdu阳性心肌细胞数量;磷钨酸苏木素（PTAH）染色检测新生细胞的横纹结构;α-横纹肌肌动蛋白抗体检测判别增殖的细胞是否为心肌细胞;免疫荧光法测定Brdu和c-kit阳性的心肌细胞.结果 实验组大鼠急性心肌梗死后第5天梗死边缘区和非梗死区Brdu阳性心肌细胞明显多于对照组（P＜0.01）;第7天梗死边缘区Brdu阳性心肌细胞达高峰,为对照组的5.7倍[（1.26±0.15）％比（0.22±0.06）％,P＜O.01],非梗死区为对照组的4.2倍[（0.75±0.12）％比（0.18±0.07）％,P＜0.01];第60天两组差异无统计学意义.实验组第7天梗死边缘区的Brdu阳性细胞数量为右心室非梗死区的1.7倍[（1.26±0.15）％比（0.75±0.12）％,P＜0.01],第14天为1.4倍[（0.77±0.09）％比（0.54±0.11）％,P＜0.01].PTAH染色显示部分增殖的新生细胞有心肌横纹结构.免疫荧光检测显示部分Brdu阳性细胞心肌肌动蛋白标记阳性;梗死边缘区部分Brdu阳性细胞c-kit标记亦阳性,这些心肌细胞核较小且圆.结论 急性心肌梗死后,成年大鼠新生心肌细胞明显增多,梗死边缘区及右心室非梗死区均显著高于对照组,心肌细胞增殖和（或）再生现象呈时间依赖性.部分新生的心肌细胞具有心脏干细胞特性.本研究表明急性心肌缺血损伤后心肌细胞似有激活增殖和（或）再生的现象.

Objective To investigate the myocardial proliferation/regeneration capacities at different time points and at different parts of the heart post acute myocardial infarction （AMI） in rats. Methods A total of 64 adult Sprague-Dawley （SD） rats were randomly divided into AMI group （left anterior descending coronary ligation, n = 44 ） and sham-operated group （ n = 20 ）. Rats were sacrificed on day 3,5,7,14 and 60 respectively post operation （ n = 5 - 9 at each time point） and ventricular tissues were harvested. 5-Bromo-2-deoxyUridine （Brdu, 50 mg/kg） was injected intraperitoneally at 12 and 24 hours before sacrifice. Morphological and pathological changes of the myocardium were observed after HE staining. Brdu-positive and c-kit and Brdu double-positive cardiomyocytes were analyzed post immunohistocbemistry and immunofluorescence staining. Striated structure of new cells was detected by PTAH staining. Alpha-sarcomeric actin antibody was used to identify new cardiomyocytes. Result Brdu- positive cardiomyocytes at border zone and non-ischemic zone were significantly increased at 5 days post AMI and peaked at 7th day post operation （Border zone,AMI. 1.26% ＋0. 15% vs. Sham： 0.22% ＋0.06% ,P 〈0.01; right ventricle,AMI： 0.75% ＋ 0. 12% vs. Sham： O. 18% ＋0.07% ,P 〈0.01 ）. There was nosignificant difference between the two groups on the 60th post-operation day. Brdu-positive cells were 1.7-fold higher in infarct border zone than in the right ventricular area of AMI rats on the 7th post operation day （ 1.26% -＋0. 15% ,vs. 0. 75% ＋0. 12% ,P 〈0. 01 ） and was 1. d-fold higher on the 14th post operation day （0. 77% -＋ 0. 09%, vs. 0. 54% -＋ 0. 11% ,P 〈 0. 01 ）. PTAH staining evidenced myocardial striated structure inside the new cells. Immunofluorescent assay showed that parts of Brdu positive ceils were myocardial actin positive, and the c-kit and Brdu double-positive myocardial cells were also observed. Most nuclei of tehse new cardiomyocytes were small and round-shaped. Conclusions Myocardial proliferation/regeneration increased significantly after AMI in rats, especially around the infarct border zone. The myocardial proliferation/ regeneration was time-dependent. Parts of the new cardiomyocytes had some characteristics of cardiac stem ceils. This study suggests that myocardial proliferation/regeneration may be activated after acute myocardial injury.