重组人粒细胞集落刺激因子动员急性心肌缺血大鼠自体骨髓干细胞归巢缺血心肌的短期安全性评价

Short-term safety in mobilizing autologus bone marrow stem cells homing to ischemic myocardium using recombinant human granulocyte colony-stimulating factor in acute myocardial ischemia rats

背景：骨髓中存在多潜能干细胞,利用其多向分化和归巢的能力,已成为近年来多种疾病治疗的研究方向。目的：观察重组人粒细胞集落刺激因子动员急性心肌梗死大鼠骨髓干细胞归巢于缺血心肌的高度选择性,并评价其短期安全性。设计、时间及地点：随机对照动物实验,于2006-09/2007-04在吉林大学基础医学院病理生理实验室完成。材料：选取清洁级SD大鼠82只,雌雄各半,体质量250-300g,62只大鼠结扎冠状动脉前降支制成急性心肌梗死模型,20只作为假手术组。方法：造模成功41只,分为模型组（n=20）和动员组（n=21）。模型组：皮下注射生理盐水2mL/d,连续7d;动员组：皮下注射生理盐水稀释至浓度2.5mg/L重组人粒细胞集落刺激因子15μg/（kg·d）,连续7d。假手术组：在相应冠状动脉结扎部位穿线,不结扎,其余操作步骤相同,皮下注射生理盐水2mL/d,连续7d。主要观察指标：1周后计数3组大鼠外周血白细胞及单个核细胞百分比,4周后比较3组大鼠在体心功能。并取大鼠心脏、肺脏、肝脏、骨骼肌组织制切片行苏木精-伊红染色及CD34免疫组织化学染色观察心脏组织病理改变,毛细血管密度和CD34＋细胞归巢情况。结果：①造模1周后动员组大鼠的外周血白细胞及单个核细胞百分比较动员前明显增加（P〈0.05）,且动员组大鼠的外周血白细胞计数及微核细胞率明显高于模型组（P〈0.05）。②造模1周时,动员组较模型组大鼠心肌梗死区可见较多CD34＋细胞（P〈0.05）;4周时,动员组和模型组均未见CD34＋细胞。各组大鼠肝脏、肺脏及骨骼肌各个时段则均无明显差异。造模后4周,动员组心功能各项指标均优于模型组（P〈0.05）,梗死面积明显小于模型组（P〈0.05）,毛细血管密度明显高于模型组（P〈0.05）。结论：重组人粒细胞集落刺激因子动员心肌梗死后大鼠自体骨髓干细胞到外周血循环并归巢于梗死心肌,并可分化成心肌样细胞及毛细血管内皮细胞,减少心肌梗死范围,改善心功能。短期观察重组人粒细胞集落刺激因子对肺脏、肝脏、骨骼肌无明显组织学影响。

BACKGROUND： Multipotential stem cells exist in bone marrow. With the abilities to multiple direction and homing, these stem cells have become the studying direction for the research of various disease treatments. OBJECTIVE： To observe the therapeutic efficacy of bone marrow stem cell mobilization with recombinant human granulocyte colony-stimulating factor （rhG-CSF） in rats with acute myocardial infarction, and to certificate the selectivity in stem cells homing ischemic myocardium, and to evaluate short-term security. DESIGN, TIME AND SETTING： The randomized, controlled animal study was performed at the Laboratory of Pathology and Physiology, Basic Medical College, Jinlin University from September 2006 to April 2007. MATERIALS： A total of 82 clean Sprague Dawley rats, of both gender, weighing 250-300 g, were selected. Of them, 62 rats were made into acute myocardial infarction models by ligating anterior descending coronary artery, and 20 rats served as the sham operation group. METHODS： A total of 41 successful rat models were divided into a model group （n=20） and a mobilization group （n =21 ）. Rats in the model group underwent subcutaneously saline （2 mL/d） for 7 days. Rats in the mobilization group underwent subcutaneously rhG-CSF 15μg/（kg·d） and saline （2.5 mg/L） for 7 days. Rats in the sham operation group received braid at the coronary artery ligation site, no deligatin, and other procedures were similar, subcutaneous injection of saline （2 mL/d）, for 7 days. MAIN OUTCOME MEASURES： One week later, percentage of rat peripheral blood leucocytes and mononuclear cells was counted in each group. Heart function was compared among groups 4 weeks later. Rat heart, lung, liver and skeletal muscle tissues were obtained, sectioned, and subjected to hematoxylin-eosin staining and CD34 immunohistochemistry to observe pathological changes in heart tissues, capillary density and CD34^＋ cell homing. RESULTS： One week later, in the mobilization group, the peripheral blood leukocytes and mononuclear cells increased obviously compared with that before mobilization （P 〈 0.05）. Rat peripheral blood leukocyte counting and micronucleated cell rate were significantly greater in the mobilization group than in the model group （P〈 0.05）. At week 1, there were a great number of monocytes infiltrating with CD34 expression in the rat myocardial infracted zone in the mobilization group compared with the model group （P 〈 0.05）. At week 4, there was no CD34 expression in both mobilization group and model group. No significant differences were found in the rat liver, lung and skeletal muscle at various time points. Four weeks after model establishment, various indexes of heart function were better （P 〈 0.05）, and infarcted area was less （P 〈 0.05）, and capillary density was significantly higher （P 〈 0.05） in the mobilization group compared with the model group. CONCLUSION： The rhG-CSF can mobilize bone marrow stem cells to home to infracted zones and differentiate to cardiomyocyte-like cells and capillary endothelium cells in rats following myocardial infarction. Thus, myocardial infarcted range was reduced and heart function was improved. Short-term observation of rhG-CSF has no significant histology effects on the lung liver and skeletal muscle.