经冠状动脉内注射体外培养骨髓间充质干细胞的安全性

Safety of intra-coronary artery injection of in vitro cultured bone marrow mesenchymal stem cells

目的:经冠状动脉内移植可以使移植细胞均匀的分布在移植血管相关心肌组织内,同时避免了心肌内注射引起的心肌损伤,是目前很多临床试验采用的移植途径。然而部分学者指出冠状动脉内注射移植可能会导致心肌梗死等并发症。观察经介入途径将骨髓间充质干细胞注射到心肌梗死猪冠状动脉内的可行性及安全性。方法:实验于2005-06/2006-03在中国协和医科大学阜外心血管病医院、卫生部心血管病再生医学重点实验室完成。①实验材料:中华小型猪由中国农业大学实验动物基地提供,雌性,6个月龄。实验过程中对动物处置符合动物伦理学标准。②实验方法:采用密度梯度离心法分离猪骨髓单个核细胞获取骨髓间充质干细胞,并采用超顺磁性氧化铁颗粒和荧光细胞膜标记物CM-DiI进行双重标记。阻断冠状动脉前降支中下1/3处90min后开放制作猪心肌梗死模型。通过导管将骨髓间充质干细胞注射到心肌梗死模型动物的冠状动脉内,观察心电图及肌钙蛋白I在细胞移植后不同时期的变化,应用心脏磁共振在体示踪移植细胞在心脏内的分布,评价心脏形态和功能在细胞移植前后的变化,病理检查分析移植细胞在心肌组织内的分布以及体内非靶器官的再分布情况及组织形态改变。结果:①冠状动脉内移植骨髓间充质干细胞当时及24h后,心电图未见ST-T明显改变,肌钙蛋白未见明显升高。细胞移植后当时及24h后左室舒张末期容积、左室收缩末期容积未见明显增大,左心室射血分数未见降低。移植后2周心脏功能明显改善,左心室容积明显缩小,左心室射血分数明显升高。②细胞移植前、移植后24h、移植后2d,反映心肌损伤的肌钙蛋白I均未出现明显升高,细胞移植过程中心电图未出现明显的变化。③病理检查发现移植细胞在心肌组织内均匀分布,2周后可见移植细胞在梗死区域存活,移植细胞存在向肺、肝、肾等非靶器官的再分布,移植细胞对非靶器官的组织形态无明显影响。结论:实验证明经冠状动脉内移植的骨髓间充质干细胞能够均匀的分布到相关心肌组织内,不会造成梗死心肌组织的功能损伤,并可以明显改善梗死心肌功能,对非靶器官组织形态无明显影响,经冠状动脉内移植骨髓间充质干细胞治疗猪急性心肌梗死是安全可行的。

AIM： Transplanted cells via coronary artery can migrate into the corresponding myocardium and avoid myocardial injury due to myocardial injection. It is a common used transplanted approach presently in clinical tests. However, a few scholars believed that transplantation via coronary artery could induce complications of myocardial infarction. This study aimed to investigate the feasibility and safety of injecting BMSCs into the coronary artery of myocardial infarction pigs. METHODS： Experiments were performed at the Fuwai Cardiovascular Disease Hospital of Peking Union Medical College from June 2005 to March 2006. Female Chinese minipig aged 6 months were provided by Experimental Animal Center of China Agricultural University. The protocol was in accordance with animal ethical standards. BMSCs were harvested from bone marrow mononuclear cells of pigs by density gradient centrifugation, and then labeled with superparamagnetic iron oxide （SPIO） granules and fluorescent cell membrane marker CM-DiI. Pig model of myocardial infarction was made by ligating 1/3 anterior descending coronary artery for 90 minutes. BMSCs were injected into the coronary artery of myocardial infarction models. Electrocardiogram were recorded and plasma concentration of troponin 1 were measured before and after the transplantation. Cardiac magnetic resonance imaging （MRI） was used to trace the transplanted cells and evaluate the morphology and function of the heart before and after transplantation. Pathological examination was performed to analyze distribution of transplanted cells, redistribution of non-targeted organs and changes in tissue morphous. RESULTS： No significant changes in ST-T or an increase in troponin I concentration were found by electrocardiogram immediately and 24 hours after transplantation. Left ventricular end diastolic volume （LVEDV） and left ventricular end systolic volume （LVESV） were not increased, and left ventricular ejection fraction （LVEF） was not decreased immediately and 24 hours after transplantation. Cardiac function was not improved, left ventricular volume （LVV） was significantly shortened, and LVEF was significantly increased 2 weeks after transplantation. No significant increase in troponin I concentration or significant changes in electrocardiogram were detected before, 24 hours and 48 hours after transplantation. Pathological examination demonstrated that transplanted cells were evenly distributed in myocardial tissues, and survived in the infarcted region two weeks later. The transplanted cells had redistribution to the non-targeted organs, but the structure of these organs remained normal. CONCLUSION： BMSCs can migrate into the corresponding tissues by coronary artery transplantation and cannot induce functional injury of infarcted myocardium, resulting in significant improvement of myocardial function and morphous of non-targeted organs remains normal. It is safe and feasible to treat pig with acute myocardial infarction by BMSCs transplantation via the coronary artery.