人脐血单个核细胞移植对急性心肌梗死大鼠心功能及左室重构的影响

Human umbilical cord blood mononuclear cell transplantation effects on rat cardiac function and left ventricular remodeling following acute myocardial infarction

背景:以往心肌梗死后心肌修复实验多集中于骨髓干细胞,甚少报道涉及富含造血干细胞、间充质干细胞及内皮祖细胞的脐血干细胞,而以其作为种子细胞的可行性及安全性尚未确定。目的:探讨人脐血单个核细胞移植对急性心肌梗死大鼠心功能和左室重构的影响。设计、时间及地点:随机对照动物实验,于2005-11/2007-09在中山大学动物试验中心完成。材料:脐血来源于足月分娩的健康产妇,由中山大学附属第一医院产科提供。Wistar雄性大鼠45只,随机分为假手术组、模型组、细胞移植组,15只/组。方法:采用羟乙基淀粉沉淀联合ficoll-paque密度梯度离心法体外分离培养人脐血单个核细胞,移植前细胞行BrdU标记。模型组、细胞移植组大鼠结扎冠状动脉左前降支建立急性心肌梗死模型,造模成功后,细胞移植组在梗死区周边注射脐血单个核细胞悬液,模型组注射DMEM培养基,培养4周。假手术组仅开胸并在左前降支下过线。主要观察指标:通过超声心动图评价心脏结构,以左心导管检测血流动力学改变,采用苏木精-伊红染色、Masson染色和BrdU免疫组化染色观察心肌组织病理学变化、心肌胶原含量变化及移植细胞存活情况。结果:与模型组比较,细胞移植组左室壁厚度增加,室壁活动度明显改善,左室舒张末期内径缩小,球形指数增大,左室舒张末压显著下降,左室内压最大上升/下降速率明显增快,差异均有显著性意义(P<0.05)。假手术组心肌细胞间仅见少量胶原纤维,呈条索状与心肌纤维平行排列;与模型组比较,细胞移植组心肌细胞肿胀程度及排列情况均有所改善,细胞移植组胶原容积分数明显降低(t=3.81,P=0.001)。模型组梗死区及其周边和假手术组均未检测到BrdU阳性细胞,细胞移植组胞核呈BrdU阳性的细胞散在分布于梗死区,部分阳性细胞参与到血管壁的组成。结论:在未使用免疫抑制剂的条件下,人脐血单个核细胞可成功移植到大鼠心肌梗死区,且短期内具有改善心功能、抑制梗死后心肌重构的作用。

BACKGROUND： Previous studies on myodardial repair are focus on bone marrow stem cells following myocardial infarction. Few studies address umbilical cord blood stem cells containing hemopoietic stem cells, mesenchymal stem cells and endothelial progenitor cells. The feasibility and safety of umbilical cord blood stem cells as seed cells remain unclear. OBJECTIVE： To evaluate the effects of human umbilical cord blood mononuclear cell transplantation on cardiac function and left ventricular remodeling in rats after myocardial infarction. DESIGN, TIME AND SETTING： The randomized, controlled, animal experiment was performed at the Animal Experimental Center of Sun Yat-sen University from November 2005 to September 2007. MATERIALS： Umbilical cord blood collected from healthy term-delivery puerperants was obtained from Department of Obstetrics, First Affiliated Hospital, Sun Yat-sen University. Totally 45 male Wistar rats were randomly assigned into a sham operation, model, and cell transplantation groups, with 15 in each group. METHODS： Human umbilical cord blood mononuclear cells were harvested and incubated by hydroxyethyl starch and Ficoll-paque density gradient centrifugation. Before transplantation, cells were labeled. Rats in the model and cell transplantation groups were used to establish models of acute myocardial infarction by ligating the left anterior descending coronary artery. Following successful model establishment, umbilical cord blood mononuclear cell suspension was injected into infarcted regions of rats in the cell transplantation group. Rats in the model group were injected with DMEM for 4 weeks. Rats in the sham operation group received ligation of the left anterior descending artery. MAIN OUTCOME MEASURES： Cardiac structure was assessed by echocardiography. Changes in hemodynamics were detected by the left cardiac catheter. Pathological changes, collagen contents and transplanted cell survival were observed using hematoxylin-eosin staining, Masson staining and BrdU immunohistochemistry. RESULTS： Compared with the model group, wall thicking index of the left ventricle was increased, and activity was significantly improved, left ventricular end-diastolic diameter was decreased, sphericity index was increased, left ventricular end-diastolic pressure was significantly decreased, maximal rate of ventricular pressure （＋dp/dtmax） was significantly increased in the cell transplantation group （P 〈 0.05）. A few trabs-shaped collagen fiber was found in cardiomyocytes in the sham operation group. Compared with the model group, cardiomyocyte swelling and arrangement were improved, and collagen volume fraction was significantly decreased in the cell transplantation group （t=3.81, P=0.001 ）. BrdU-positive cells were not detected around the infarcted regions in the model group and in the sham operation group. BrdU-positive cells were distributed surrounding the infarcted region in the cell transplantation group, and some positive cells participated in the composition of vessel wall. CONCLUSION： Without using immunosuppressor, human umbilical cord blood mononuclear cells can be successfully transplanted into rat myocardial infarction, which can improve cardiac function, and inhibit myocardial reconstruction following infarction.