目的观察心肌梗死后应用粒细胞-巨噬细胞集落刺激因子(GM-CSF)动员骨髓干细胞能否促进心肌细胞的再生、血管的新生。方法结扎大鼠的冠状动脉前降支制成心肌梗死模型,实验组在心肌梗死后皮下注射GM-CSF50μg·kg^-1·d^-1,连续7d。用流式细胞仪检测骨髓和外周血中CD34^+干细胞的含量,通过Ⅷ因子抗体、Ki67抗体免疫组化染色结合磷钨酸-苏木精(PTAH)染色,判定血管新生、心肌再生,测定心肌梗死的面积。结果心肌梗死后第3天,实验组的CD34^+干细胞在外周血和骨髓中开始上升;第7天(外周血0.350%±0.026%,骨髓2.250%±0.140%)和第14天(外周血0.260%±0.022%,骨髓2.060%±0.110%)时均明显高于对照组(外周血0.170%d-O.015%,骨髓1.240%±0.064%)(P〈0.01),第28天时回落,与对照组比较差异无统计学意义(P〉0.05)。实验组瘢痕区内毛细血管数(313±10)、Ki67阳性细胞数(275±5)明显多于对照组(264±10,207±10)(均P〈0.05),并且与外周血和骨髓中的干细胞数呈正相关关系(r=0.961,P=0.019,r=0.975,P=0.005)。但这些Ki67阳性细胞核仅与部分淋巴、间质细胞、血管内皮细胞重叠,未见Ki67阳性的细胞核与心肌细胞重叠,实验组心肌梗死面积也无明显减少(P〉0.05)。结论GM-CSF能有效地动员骨髓CO34^+干细胞,促进梗死心肌的血管新生,但并不能促进心肌细胞的再生。
Objective To test the potential of bone marrow stem cells mobilized by granulocyte macrophage colony stimulating factor (GM-CSF) to promote regeneration of myoeyte and neovascularization in a rat model of myocardial infarction. Methods A myocardial infarction model was created by ligation of the left anterior descending coronary artery in adult Sprague-Dawley rats. Then the infracted rats were randomly assigned to receive GM-CSF injection subcutaneously 50 μg·kg^-1·day^-1 as experimental group or receive saline injection as control group. Enumeration of CD34 stem cells in peripheral blood and bone marrow was performed by flow cytometric analysis. Histologic and immunohistological staining such as Factor VIH, Ki67 and PTAH staining (phosphotungstic acid hematoxylin) was performed to assess angiogenesis and myogenesis, calculate myocardial infarction size in the heart. Results The CD34^+ stem cells in the blood and bone marrow increased from Day 3, peaked between Day 7 (0. 350% ± 0. 026% , 2. 250% ± 0. 140% ) and Day 14 ( 0. 260% ± 0. 022% , 2. 060%± 0. 110% ) in experimental group compared with control group (0. 170% + 0. 015% , 1. 240% ± 0. 064% ) ( P 〈 0. 01 ) , declined on Day 28 and no difference versus control group( P 〉 0.05 ). Immunohistological staining showed apparent neovasculature formation and much more Ki67 expression in the infracted regions in experimental group (313 ± 10,275 ± 5) compared with control group (264 ± 10,207 ± 10) (P 〈 0. 05 ). The Ki67 positive ceils in the experimental hearts were significantly positively correlated with CD34^+ stem cells in the blood and bone marrow (r = 0. 961 ,P = 0. 019, r =0. 975 ,P =0. 005). Ki67 and PTAH double staining showed that the Ki67 positive nucleoli overlay the lvmnhocvtes, fibroblasts and endothelial cells but not mvocvtes. No significant decrease of infracted size occurred in experimental group (P〉0.05). Conclusions Bone marrow and peripheral blood stem cells could be mobilize