巨噬细胞吞噬骨髓间充质干细胞后对梗死心肌修复作用及可能机制的研究

目的研究巨噬细胞吞噬骨髓间充质干细胞(MSCs)后对梗死心肌的修复作用以及可能的机制。方法体外分离培养MSCs,经流式细胞仪分离纯化及进一步缺氧诱导凋亡后与脂多糖激活的巨噬细胞共培养48h,随后获得吞噬了MSCs的巨噬细胞(pMΦ)。将磷酸盐缓冲液(阴性对照组)、MSCs(阳性对照组)、pMΦ(实验1组)、巨噬细胞炎性蛋白(MIP)-1α+pMΦ(实验2组)经尾静脉注射入急性心肌梗死(AMI)后BALB/C小鼠体内。应用磁共振及多光谱分析仪分析移植后细胞在梗死区的定植及强度,小鼠心脏超声分析移植细胞对小鼠梗死心肌的修复作用,流式细胞技术及弹力纤维染色分析移植细胞修复小鼠梗死心肌的可能机制。结果多光谱分析显示,与阴性对照组相比,阳性对照组、实验1组和实验2组的细胞均可定植于梗死心肌局部,实验1组的相对荧光强度显著高于阳性对照组(P<0.001),但显著低于实验2组(P<0.001)。AMI后两周小鼠超声心动图显示,阳性对照组、实验1组和实验2组治疗后的射血分数均较阴性对照组有显著改善,其中实验2组的改善最为明显,其次为实验1组,两两比较差异均有统计学意义(P值均<0.001)。弹力纤维染色显示,与阴性对照组比较,阳性对照组、实验1组和实验2组细胞移植治疗后小鼠的心肌梗死面积均显著缩小(P<0.001),实验1组的心肌梗死面积占左心室面积比例显著低于阳性对照组(P<0.001),但仍显著高于实验2组(P<0.001);实验1组的梗死区弹性纤维占梗死区瘢痕的比例显著高于阳性对照组(P<0.001),但与实验2组的差异无统计学意义(P=0.32)。经胰酶消化及流式细胞仪检测结果显示,与阴性对照组比较,阳性对照组、实验1组和实验2组梗死区附近及外周血中修复性的巨噬细胞显著增多(P值均<0.001),阳性对照组、实验1组、实验2组的CD86阳性巨噬细胞比例依次增多,组间差异均有统计学意义(P值均<0.001)。结论 AMI后移植的pMΦ可有效定植于梗死心肌并显著改善梗死后的心脏功能,其机制与促进弹力纤维增生及促使更多修复性巨噬细胞的动员与募集有关,MIP-1α可增强这种作用。

Effect of macrophages phagocytizing mesenchymal stem cells on the repair of infarcted myocardium and possible mechanisms

Objective To study the effect of macrophages phagocytizing mesenchymal stem cells(MSCs) on the repair of infarcted myocardium and the possible mechanisms.Methods MSCs were isolated and cultured in vitro.After purified by flow cytometry and hypoxia-induced apoptosis,the dead MSCs were co-cultured with lipopolysac-charide(LPS)-activated macrophages for 48 h.Then the macrophages(pMΦ) which had phagocytized MSCs were obtained.Phosphate buffer saline(negative control group),MSCs(positive control group),pMΦ(Group 1),and macrophage inflammatory protein-1α(MIP-1α) + pMΦ(Group 2) were injected into acute myocardial infarction(AMI) BALB/C mice through caudal vein,respectively.MRI and multi-spectrometer were used to identify the colonization intensity of the transplanted cells in the infarct area.Mice cardiac ultrasound was used to analyze the effect of transplanted cells on the repair of infarcted myocardium.The possible repair mechanisms were speculated by flow cytometry and elastic fiber staining.Results All the cells except for the ones in negative control group were successfully implanted in infarted myocardium.The relative fluorescence intensity in Group 1 was significantly higher than that in positive control group(P<0.001),and lower than that in Group 2(P<0.001).Echocardiography showed that,compared with negative control group,ejection fraction in Group 1 and Group 2 as well as positive control group was significantly improved two weeks after AMI.The improvement was most obvious in Group 2,followed by Group 1.Pairwise comparisons were statistically significant(P<0.001).Elastic fiber staining showed that myocardial infarct size was significantly decreased in positive control group,Group 1 and Group 2 compared to negative control group(P<0.001).The percentage of infarction size in left ventricular area in Group 1 was significantly lower than that in positive control group(P<0.001),and higher than that in Group 2(P<0.001).The percentage of elastic fibers in infarct scar area in Group 1 was significantly higher than that in positive control group(P <0.001),but there was no significant difference between Group 1 and Group 2(P=0.32).Compared with negative control group,CD86-positive macrophages in the infarct zone and peripheral blood significantly increased in positive control group,Group 1 and Group 2(P<0.001);the proportions of CD86-positive macrophages in the three groups increased gradually in turn(P<0.001).Conclusion pMΦ can effectively colonize in the infarcted myocardium after transplanted into AMI mice and significantly improve cardiac function.The mechanisms may be associated with the promotion of elastic fiber proliferation and mobilization of more reparative macrophages.MIP-1α can enhance this action.