Orthotopic transplantation of human amniotic mesenchymal stem cells for treatment of cerebral infarction in rats

doi:10.3969/j.issn.2095-4344.2017.09.019

Abstract

Abstract:

BACKGROUND: Preliminary experimental study found that the human amniotic mesenchymal stem cells (hAMSCs) transplantation can improve nerve injury symptoms of rats with cerebral infarction.
OBJECTIVE: To observe the survival, colonization and differentiation of hAMSCs in the infarct area of cerebral infarction rats.
METHODS: Sixty Sprague-Dawley rats were randomly assigned into hAMSCs transplantation, model or sham operation groups (n=20/group). Animal models of middle cerebral artery occlusion were produced in the model and transplantation groups by Zea-Longa method. One day after modeling, rats in the hAMSCs transplantation group were given in situ transplantation of 10 μL of hAMSCs (2×10⁶) into the damaged striatum and cortex, while those in the model and sham operation group were given the same volume of PBS. Within 1 week after transplantation, rat neurological defects were assessed and changes in their body mass were continuously monitored. Two weeks after transplantation, TTC staining was used to observe cerebral infarct size, hematoxylin-eosin staining was used for pathological observation of brain tissues, and immunofluorescent staining was used to detect expression of neuron-specific nuclear protein.
RESULTS AND CONCLUSION: With time, weight loss was increased while neurologic deficit scores were gradually reduced in the hAMSCs and model groups. Compared with the model group, the weight loss and neurologic deficit scores were lower in the hAMSCs group,; however, there was a significant difference in the neurologic deficit scores but not in the weight loss between the two groups. Additionally, the hAMSCs significantly reduced infarct size, attenuated pathologic injury, and decreased the number of inflammatory cells. Immunofluorescence staining showed that the hAMSCs were observed at 1 week after transplantation under inverted luorescence microscope, and gradually differentiated into nerve cells at 2 weeks after transplantation. In conclusion, transplanted hAMSCs may migrate to and survive in the cerebral infarct region, and differentiate into nerve cells in situ in rats with cerebral infarction.

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: Stem Cells, Stem Cell Transplantation, Infarction, Middle Cerebral Artery, Tissue Engineering

CLC Number:

R394.2

Wang Yu-ying, Su Xu, Liu Bo, Liu Juan, Wan Xue. Orthotopic transplantation of human amniotic mesenchymal stem cells for treatment of cerebral infarction in rats[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(9): 1414-1419.

Figures/Tables 3

2.1 hAMSCs细胞的生长特性原代hAMSCs培养48 h后可见细胞形态呈梭形、多角形、星形等；培养4 d左右，贴壁细胞融合达80%左右进行传代，培养的第3代细胞以梭形细胞为主，单层放射状排列或漩涡状生长，见图1。 2.2 hAMSCs细胞的表型特征用流式细胞仪鉴定第3代hAMSCs细胞表型，结果显示CD44、CD29、CD73阳性表达率分别为97.33%、94.11%、62.67%，低表达或不表达CD34、CD45、HLA-DR，说明分离的hAMSCs纯度较高，见图2。 2.3 实验动物数量分析模型组死亡6只，造模24 h内死亡3只；实验组死亡2只，均为造模24 h内死亡，主要与麻醉和动物模型制备手术有关。模型组移植后1，2周各处死7只大鼠，其中3只做TTC染色，剩余4只做组织病理学及免疫荧光染色；实验组移植后1，2周各处死9只大鼠，4只用于TTC染色，5只用于组织病理学相关检测。 2.4 hAMSCs细胞移植对脑梗死大鼠体质量的影响 hAMSCs移植治疗前后，监测各组大鼠体质量情况，结果显示，假手术组体质量基本呈持续上升趋势；模型组较假手术组大鼠体质量下降明显，尤其在移植4 d后，差异显著(P < 0.05)；实验组较模型组体质量有所改善，但没有显著性差异(P > 0.05)，见表1。 2.5 hAMSCs细胞移植对脑梗死大鼠神经功能缺损评分的影响 hAMSCs移植前后，对各组大鼠进行神经功能缺损评分，结果显示，模型组造模后及移植后2 d的神经功能缺损评分高于假手术组(P < 0.05)；随着时间推移模型组和实验组神经功能缺损评分呈逐渐降低趋势，虽然实验组各时间点评分较模型组略低，但两组之间差异无显著性意义(P > 0.05)，见表2。 2.6 TTC染色观察大鼠脑梗死面积 TTC染色观察大鼠脑梗死面积，结果显示，模型组大鼠大脑皮质出现局灶性缺血坏死且范围较大；与模型组比较，实验组缺血灶面积明显缩小，见图3。 2.7 脑组织组织病理学变化苏木精-伊红染色结果显示，假手术组神经细胞形态正常，未见间质水肿及囊腔形成；模型组脑组织内可见淡染边界处有清楚的镂空筛网状软化病灶，神经细胞变性、坏死，胞体皱缩，核固缩、碎裂、溶解，神经纤维疏松，间质水肿明显，局灶周围有大量炎性细胞浸润；实验组炎性细胞浸润明显减轻，软化病灶面积明显缩小，组织结构、层次较清晰，见图4。 2.8 免疫荧光双染色结果实验组hAMSCs移植后1，2周，脑组织内均可见MAB1281标记的阳性细胞(红色荧光标记细胞核)，说明经原位移植的hAMSCs能定植到脑组织内并存活；hAMSCs移植后1周，无绿色荧光阳性细胞存在，说明移植的hAMSCs不表达特异性蛋白NeuN；hAMSCs移植后2周，NeuN标记的阳性细胞有表达，且少部分细胞与MAB1281阳性细胞重叠，说明移植的hAMSCs可能向神经样细胞分化，并表达神经特异性蛋白NeuN，见图5。"

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