Transplantation of neural stem cells from different sources in the treatment of cerebral ischemia injury in rats

doi:10.3969/j.issn.2095-4344.0881

Abstract

Abstract:

BACKGROUND: The underlying mechanism of neurological dysfunction caused by cerebral ischemia is neuronal damage, and facilitating neuronal regeneration is the key to neural function recovery. However, endogenous neural stem cells have limited use in nerve repair. Transplantation of exogenous neural stem cells brings hope for tissue remodeling and functional recovery after cerebral ischemia, but systematic comparative studies on the selection of seed cells are still lacking.
OBJECTIVE: To compare the therapeutic effect of neural stem cells from three sources in the treatment of cerebral ischemia injury in rats.
METHODS: Adipose-derived mesenchymal stem cells (ADMSCs), bone marrow mesenchymal stem cells (BMSCs), and skin-derived induced pluripotent stem cells (iPSCs) from Sprague-Dawley rats were isolated and cultured, and induced in vitro to differentiate into neural stem cells. Conversion efficiency was detected at 7 days of neuronal induction. One hundred rat models of cerebral ischemia were randomized into model, BMSCs, iPSCs, ADMSCs, and PBS groups, with 20 rats in each group. Then, corresponding cell suspension or PBS solution was transplanted in each group. Another 40 Sprague-Dawley rats were taken as sham operation group (n=20) and control group (n=20) with no intervention. Index measurements were performed at 1 and 4 weeks after cell transplantation.
RESULTS AND CONCLUSION: After 7 days of in vitro induction, the conversion efficiency of iPSCs was significantly higher than that of ADMSCs and BMSCs (P < 0.01). At 1 and 4 weeks after transplantation, neuronal apoptosis and infarction size were significantly reduced in the iPSCs, ADMSCs and BMSCs groups as compared with the PBS and model groups (P < 0.05). Moreover, neurological severity score, infarction size and neuronal apoptosis were significantly reduced in the iPSCs group as compared with the ADMSCs and BMSCs groups (P < 0.05). At 4 weeks after cell transplantation, neurological severity score, infarction size and neuronal apoptosis in each group showed significant reduction as compared with the values at 1 week after cell transplantation (P < 0.01). Numbers of Nissl bodies in neurons of the striatum in the three cell transplantation groups were significantly higher than those in the control and sham operation groups. Our findings from this study reveal that neural stem cells differentiated from three sources can significantly improve the symptoms of neurological deficits, and reduce infarction size, neuronal degeneration and neuronal apoptosis. iPSCs especially have better differentiation ability and therapeutic effects than BMSCs and ADMSCs.

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: Mesenchymal Stem Cells, Bone Marrow, Adipose Tissue, Induced Pluripotent Stem Cells, Neural Stem Cells, Cell Differentiation, Brain Infarction, Tissue Engineering

CLC Number:

R394.2

Zhong Bo, Liu Xia, Wu Wei . Transplantation of neural stem cells from different sources in the treatment of cerebral ischemia injury in rats[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(21): 3349-3356.

Figures/Tables 2

2.1 诱导性多能干细胞、脂肪间充质干细胞、骨髓间充质干细胞、神经干细胞的鉴定倒置相差显微镜下，传代脂肪间充质干细胞和骨髓间充质干细胞呈典型的成纤维细胞样形态，多边形，轮廓清晰，骨髓间充质干细胞表面标志物CD29、CD34、CD90和CD45表达率依次为99.56%，62.73%，97.92%，73.77%，脂肪间充质干细胞表面标志物CD29、CD44、CD90和CD105表达率依次为98.62%，96.75%，100%和97.24%；诱导性多能干细胞克隆光镜下可见边界清晰，细胞聚集成团，免疫荧光染色细胞中SSEA-4呈绿色荧光为阳性表达；传代神经干细胞呈大小不等的细胞团悬浮于培养基中，免疫荧光染色细胞中巢蛋白呈绿色荧光为阳性表达，见图1。 2.2 不同来源神经干细胞的转化率比较诱导分化7 d后，诱导性多能干细胞来源的神经干细胞的转化率为(89.46±2.63)%，明显高于脂肪间充质干细胞来源(65.32± 3.77)%和骨髓间充质干细胞来源(67.28±4.51)%，差异有显著性意义(P < 0.01)，脂肪间充质干细胞来源的神经干细胞的转化率与骨髓间充质干细胞来源差异无显著性意义(P > 0.05)。 2.3 实验动物数量分析用于研究的SD大鼠共150只，随机选取110只进行脑缺血模型建立，其中10只SD大鼠造模不理想予以剔除，将100只造模成功的SD大鼠随机分为5组进行后续实验，每组20只；另将剩余40只SD大鼠随机分为对照组和假手术组，每组20只；最终140只SD大鼠的研究数据进入结果分析，实验过程顺利，无中途脱落。 2.4 各组大鼠神经功能缺损评分比较移植1周和4周后，脂肪间充质干细胞来源组、骨髓间充质干细胞来源组、诱导性多能干细胞来源组、模型组和磷酸盐缓冲液组大鼠的神经功能缺损评分均明显高于对照组和假手术组(P < 0.01)；模型组和磷酸盐缓冲液组大鼠的神经功能缺损评分均明显高于脂肪间充质干细胞来源组、骨髓间充质干细胞来源组和诱导性多能干细胞来源组(P < 0.01)；移植4周时脂肪间充质干细胞来源组、骨髓间充质干细胞来源组、诱导性多能干细胞来源组和磷酸盐缓冲液组大鼠的神经功能缺损评分均明显低于移植1周(P < 0.01)；移植4周后，诱导性多能干细胞来源组神经功能缺损评分明显低于脂肪间充质干细胞来源组和骨髓间充质干细胞来源组(P < 0.05)，见表1。 2.5 各组大鼠脑梗死体积比较移植1周和4周后，脂肪间充质干细胞来源组、骨髓间充质干细胞来源组和诱导性多能干细胞来源组的大鼠脑梗死体积明显小于磷酸盐缓冲液组和模型组(P < 0.01)；移植4周时，脂肪间充质干细胞来源组、骨髓间充质干细胞来源组、诱导性多能干细胞来源组、模型组和磷酸盐缓冲液组大鼠脑梗死体积明显低于移植1周(P < 0.01)；移植4周时，诱导性多能干细胞来源组的大鼠脑梗死体积明显低于脂肪间充质干细胞来源组、骨髓间充质干细胞来源组(P < 0.01)，见表2。 2.6 各组大鼠神经元凋亡数量比较移植1周和4周后，脂肪间充质干细胞来源组、骨髓间充质干细胞来源组和诱导性多能干细胞来源组的大鼠神经元凋亡数量明显小于磷酸盐缓冲液组和模型组(P < 0.01)；移植4周时，脂肪间充质干细胞来源组、骨髓间充质干细胞来源组、诱导性多能干细胞来源组、模型组和磷酸盐缓冲液组大鼠神经元凋亡数量明显低于移植1周(P < 0.01)，见表3。 2.7 各组大鼠神经元丢失情况比较尼氏染色显示，对照组纹状体的神经元形态正常，尼氏体呈蓝紫色斑块或颗粒状，分布均匀；假手术组纹状体的神经元形态正常，数目无明显改变，尼氏体分布正常；模型组、磷酸盐缓冲液组纹状体构松散、神经元排列紊乱、神经元数量明显减少；脂肪间充质干细胞来源组和骨髓间充质干细胞来源组神经元数量增多，聚集成团；诱导性多能干细胞来源组神经元数量明显增多，排列较整齐，见图2。 2.8 各组大鼠移植的神经干细胞分化率比较移植1周和4周后，诱导性多能干细胞来源组高于脂肪间充质干细胞来源组、骨髓间充质干细胞来源组(P < 0.01)；移植4周时，脂肪间充质干细胞来源组、骨髓间充质干细胞来源组、诱导性多能干细胞来源组大鼠神经干细胞分化率均明显低于移植1周(P < 0.05)，见表4。"

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