Transplantation of umbilical cord blood mesenchymal stem cells at two states: comparison of neurological function recovery in cerebral infarction rats

doi:10.3969/j.issn.2095-4344.0470

Abstract

Abstract:

BACKGROUND: Human umbilical cord blood mesenchymal stem cells (hUC-MSCs) can repair the injury of nerve cells caused by ischemia and hypoxia, but which state of cells has a better therapeutic efficacy, primary isolation or induced differentiation is not yet known.
OBJECTIVE: To compare the therapeutic effects of hUC-MSCs primary cultured and differentiated in a rat model of cerebral infarction.
METHODS: After full-term delivery, fetal umbilical cord blood samples were obtained using quadruple bags by means of density gradient centrifugation. hUC-MSCS were induced in the medium containing basic fibroblast growth factor. Sixty rats were equivalently randomized into four groups: sham, model, primary culture and induced differentiation groups. Animal models of cerebral infarction were made in the rats in the latter three groups. Model rats in the primary culture and induced differentiation groups were subjected to tail vein transplantation of hUC-MSCs that were primary cultured or induced to differentiate in vitro at 7 days after modeling.
RESULTS AND CONCLUSION: A marked improvement in balance, walking, spatial orientation, and learning and memory abilities was found in the rats after transplantation of hUC-MSCs that were primary cultured or induced to differentiate. Moreover, compared with the primary culture group, a significant improvement was found in the induced differentiation group, including improved pathological injury of the brain, higher expression of CD34 and Ki-67, lower expression of glial fibrillary acidic protein, lower expression of interleukin 1β and tumor necrosis factor β. Compared with the primary culture group, similar infarction size and expression of interleukin-6 were also found in the induced differentiation group. These findings indicate that hUC-MSCs with induced differentiation exhibit better therapeutic outcomes in the recovery of neurological function of cerebral infarction rats.

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: Cord Blood Stem Cell Transplantation, Infarction, Middle Cerebral Artery, Tissue Engineering

CLC Number:

R394.

Han Xiao-gai, Li Jian-bin, Shan Hong, Bie Li-li, Wang Jiao-jie, Liu Ying, Qi Zheng. Transplantation of umbilical cord blood mesenchymal stem cells at two states: comparison of neurological function recovery in cerebral infarction rats[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(9): 1395-1401.

Figures/Tables 3

2.1 脐血间充质干细胞诱导培养后鉴定细胞培养1周后，大多数细胞形态发生明显变化，细胞核大，收缩呈典型的核周体形态(图1A)，2周后转变为类神经元形态，出现双极及复杂的多极细胞(图1B)，3周后突起末端出现一、二级分支，类似于树突形成纤维网状(图1C，D)。脐血间充质干细胞形成小的克隆团，细胞可见核仁，胞浆丰富，细胞呈纤维样形态，贴壁的细胞呈圆形和长梭形，培养细胞生长可达75%-90%融合，有的可呈克隆团样生长。原代细胞呈神经标志物Nestin、神经元特异烯醇化酶、神经胶质纤维酸性蛋白mRNA低表达。经诱导分化后呈现Nestin、神经元特异烯醇化酶、神经胶质纤维酸性蛋白mRNA高表达，2种细胞的表达水平差异有显著性(P < 0.05，图1)。 2.2 实验动物数量分析各组大鼠均出现由于伤口感染、营养不良等问题导致的死亡，各组均补齐大鼠数量。 2.3 动物模型的鉴定结果 MRI结果显示正常大鼠脑实质未见明显异常信号。而脑梗死大鼠右侧额叶可见条片状T1略低T2高信号影，右侧顶叶白质可见班点状T1等T2高信号影，中线结构居中，脑室系统略扩张，表明大鼠脑梗死缺血灶形成，造模成功(图2)。 2.4 行为学结果 2.4.1 平衡木和平衡木行走实验细胞移植前大鼠在平衡木上站立不稳，晃动，跌落，细胞移植后诱导分化细胞治疗组大鼠在平衡木上的时间增加，从第7天的3 s逐渐增加到第60天的50 s，从细胞移植后第14天开始，诱导分化细胞治疗组大鼠在平衡木上的时间比原代细胞治疗组明显增加。各组大鼠平衡木实验结果差异有显著性意义(F=40.13，P < 0.01)，其中脑梗死组大鼠平衡时间明显低于假手术组(P < 0.05)；采用原代细胞和诱导细胞治疗后，平衡功能得到改善(P < 0.05)；且诱导分化细胞治疗组平衡功能比原代细胞治疗组显著(P < 0.05；图3A)。各组平衡木行走实验结果差异有显著性意义(F=35.56，P < 0.01)，其中脑梗死组大鼠平衡木行走花费时间明显长于假手术组(P < 0.05)；采用原代细胞和诱导细胞治疗后，行走能力得到改善(P < 0.05)；且诱导分化细胞治疗组改善比原代细胞治疗组显著(P < 0.05；图3B)。 2.4.2 水迷宫实验定位巡航试验10-14 d时，各组大鼠寻找到平台的潜伏期逐渐缩短，且诱导分化细胞治疗组大鼠潜伏期小于原代细胞治疗组(P < 0.05)。54-58 d时的结果与早期相一致，说明诱导分化细胞治疗能够明显改善大鼠的学习能力(图4A，B)。空间探索试验14，60 d时，各组大鼠在目标象限探索的时间百分比差异有显著性意义(F=26.30，P < 0.01；F=10.06，P < 0.01)(图4C)。其中14，60 d时脑梗死组、原代细胞治疗组和诱导分化细胞治疗组大鼠在目标象限探索的时间百分比明显少于假手术组(F=6.50，P < 0.05； F =12.39，P < 0.05)；且3组大鼠在目标象限探索的时间百分比在14 d时接近(P > 0.05)，60 d时诱导分化细胞治疗组大鼠在目标象限探索的时间百分比长于原代细胞治疗组 (P < 0.05；图4D)。 2.4.3 神经功能评分细胞移植7 d时，与脑梗死组相比，原代细胞治疗组和诱导分化细胞治疗组大鼠NSS评分较低(P < 0.05)，且细胞移植14，60 d时诱导分化细胞治疗组大鼠NSS评分低于原代细胞治疗组(P < 0.05；表1)。 2.5 梗死体积造模14 d后，TTC染色结果显示，原代细胞治疗组和诱导分化细胞治疗组脑梗死体积接近(P > 0.05；图5)。 2.6 神经元生存情况细胞移植后60 d，Nissl染色发现原代细胞治疗组细胞空泡细胞，细胞稀疏，失去完整性，细胞质皱缩，可见椭圆形或三角形神经核，细胞肿胀，完整饱满细胞少，而诱导分化细胞治疗组细胞形态规则，排列整齐，胞浆饱满，染色正常，偶见空泡细胞，周围无水肿。原代细胞治疗组Nissl阳性细胞数量明显少于诱导分化细胞治疗组(P < 0.05；图6)。 2.7 CD34、神经胶质纤维酸性蛋白和ki-67阳性细胞数量 CD34是血管新生标志物[13]，ki-67是细胞增殖标志物[14]。免疫组化检测结果显示，细胞移植后14 d，CD34、神经胶质纤维酸性蛋白和ki-67阳性细胞数量接近。细胞植入后60d时原代细胞治疗组CD34和ki-67的表达少于诱导分化细胞治疗组(P < 0.05)，而神经胶质纤维酸性蛋白阳性细胞数量高于诱导分化细胞治疗组(P < 0.05；图7)。 2.8 脑组织炎症因子的表达变化 14 d时，各组大鼠脑组织白细胞介素1β、白细胞介素6、肿瘤坏死因子β表达水平接近(P > 0.05)。60 d时诱导分化细胞治疗组大鼠脑组织白细胞介素1β和肿瘤坏死因子β表达水平明显低于脑梗死组和原代细胞治疗组(P < 0.05)，而白细胞介素6表达水平与脑梗死组和原代细胞治疗组接近(P > 0.05；图8)。"

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