Effects of intravenous transplantation of human umbilical cord blood mesenchymal stem cells on conversion of intra-and extra-cardiac monocyte-macrophages M1/M2 subtypes in mice with acute myocardial infarction

doi:10.3969/j.issn.2095-4344.0643

Abstract

Abstract:

BACKGROUND: Studies have shown that transplantation of human umbilical cord blood mesenchyme stem cells (hUCB-MSCs) may reduce inflammation, improve cardiac function and participate in ischemic myocardial protection. However, there is still no final conclusion on the anti-inflammatory effect of hUC-MSCs.
OBJECTIVE: To investigate the effects of intravenous transplantation of hUCB-MSCs on the conversion of intra- and extra-cardiac monocyte-macrophages subtypes LY-6C hi (M1)/LY-6 low (M2) and cardiac protection in mice with acute myocardial infarction (AMI).
METHODS: Balb/C mice with AMI, aged 7-8 weeks, were randomly divided into experimental group (n=7) and control group (n=8). At 1 week after AMI, the experimental group was injected with 0.2 mL of saline containing 1×10⁶ hUCB-MSCs via tail vein, and the control group was given 0.2 mL of saline with no cells. At 4 weeks after treatment, the M1/M2 ratio in the peripheral blood and spleen was measured by flow cytometry. Cardiac function was measured by echocardiography. Pathological changes of the myocardium, collagen sediment, apoptosis in myocardial cells, and myocardial macrophage M1/M2 cell count were detected by hematoxylin-eosin staining, Masson staining, TUNEL staining and immunofluorescence analysis, respectively.
RESULTS AND CONCLUSION: Compared with the control group, monocyte-macrophages M1/M2 ratio in the peripheral blood, spleen and peri-infarcted myocardial tissue was lower in the experimental group (P < 0.05). Compared with the control group, cardiac functions were significantly improved in the experimental group presenting with lower left ventricular end-systolic diameter, left ventricular end-diastolic diameter as well as higher left ventricular ejection fraction (P < 0.05). Compared with the control group, the inflammatory cell count in the infarction area and surrounding tissue was significantly lower in the experimental group (P < 0.05). Compared with the control group, the amount of collagen sediment was significantly less in the experimental group (P < 0.05). Compared with the control group, the apoptosis index was lower in the experimental group, but there was no significant difference between the two groups (P > 0.05). These experimental findings verify that the intravenous transplantation of hUCB-MSCs can improve cardiac function of AMI mice by regulating systemic inflammatory responses and focal infarct lesions, which is one of the mechanisms by which hUCB-MSCs transplantation for myocardial infarction produces myocardial protection.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Myocardial Infarction, Cord Blood Stem Cell Transplantation, Injections, Intravenous, Mononuclear Phagocyte System, Tissue Engineering

CLC Number:

R394.2

Peng Yi, Chen Bing-quan, Zhao Ji-ling, Peng Zhi-yong, Xu Wei-fang, Yu Guo-long. Effects of intravenous transplantation of human umbilical cord blood mesenchymal stem cells on conversion of intra-and extra-cardiac monocyte-macrophages M1/M2 subtypes in mice with acute myocardial infarction[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(33): 5327-5332.

Figures/Tables 2

2.1 hUCB-MSCs的形态冷冻复苏后显微镜下观察hUCB-MSCs状况良好，贴壁牢固，形态呈长梭形，呈极性排列，少量未贴壁细胞呈圆形漂浮，培养基透亮度佳(图1)。 2.2 hUCB-MSCs经小鼠尾缘静脉注射及小鼠实验期间内生存状况细胞移植后每日观察小鼠存活情况，实验组小鼠全体存活致实验结束，对照组1只小鼠于治疗后25 d死亡，解剖发现该鼠出现左室前壁大面积梗死，其左室明显扩大，室壁变薄。所有小鼠实验结束解剖肝、肺、肾、脑等实质器官，均未引起新生明显实质性病变占位。 2.3 两组外周血及脾脏单核细胞M1/M2亚型比较治疗4周后，实验组外周血和脾脏单核细胞亚型M1/M2比值显著低于对照组，差异有显著性意义(外周血1.636±0.147，2.866±0.383；脾脏1.808±0.082，5.040±0.483，n=7，P均< 0.05)，见图2。 2.4 两组间免疫荧光巨噬细胞计数及M1/M2比值比较如图3及表1所示，治疗4周后，实验组心肌组织内巨噬细胞总数、M1细胞数、M1/M2比值均低于对照组(P < 0.05)，但M2型细胞数差异无显著性意义(P > 0.05)。 2.5 两组间梗死区域残存心肌面积比例及心肌纤维化比较如图4所示，两组小鼠心脏横截面切片经Masson染色，治疗4周后，实验组残存心肌面积较对照组显著增多，梗死区胶原纤维数量显著减少。 2.6 两组间缺血梗死区病理学检测及白细胞计数比较由图5(箭头所示为白细胞)所示，治疗后4周，缺血梗死区苏木精-伊红染色病理学检测，显微镜下均有心肌梗死区域及边缘处心肌水肿，胞质疏松，部分肌纤维及核溶解消失，心肌细胞排列紊乱，单核淋巴细胞浸润，梗死边缘区心肌细胞代偿性肥大。与对照组比较，实验组残存心肌细胞数量较多，单核淋巴细胞浸润减少。实验组心肌组织内浸润白细胞计数为(15.93±0.88)/高倍视野，明显低于对照组(27.27±0.52)/高倍视野，差异有显著性意义(n=5，P < 0.05)。 2.7 两组小鼠心肌凋亡指数比较 TUNEL法检测结果显示正常心肌细胞核呈蓝色，而凋亡阳性心肌细胞核呈棕褐色(图6，箭头标记为凋亡细胞核)，实验组治疗后4周心肌细胞凋亡指数低于对照组，但差异无显著性意义[(30.55±3.97)%，(32.41±2.27)%，n=5，P=0.705]。 2.8 两组小鼠心脏重构与心功能比较与对照组比较，治疗后4周实验组左室射血分数显著高于对照组[(40.00±2.52)%，(30.00±2.27)%，n=7]，实验组左心室收缩末期内径[(2.91±0.08) mm，(4.16±0.31) mm，n=7]以及左心室舒张末期内径[(3.47±0.14) mm，(4.72±0.42) mm，n=7]均低于对照组，差异均有显著性意义(P < 0.05)。"

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