Human umbilical blood cord mesenchymal stem cell transplantation combined with injection of ganglioside for cerebral palsy

doi:10.3969/j.issn.2095-4344.2016.19.010

Abstract

Abstract:

BACKGROUND: In recent years, some studies have demonstrated that ganglioside can promote survival and differentiation of umbilical blood cord mesenchymal stem cells in vitro.
OBJECTIVE: To observe the effect of injection of human umbilical blood cord mesenchymal stem cells and ganglioside into rat lateral ventricles on neurological functional recovery from cerebral palsy.
METHODS: Totally 60 cerebral palsy neonatal rats were delivered from pregnant rats which were modes were given intraperitoneal injection of lipopolysaccharide for 2 successive days on day 17 of gestation. Then those neonatal rats were randomly divided into five groups, including model group (n=10), sham transplantation group (n=10), stem cell transplantation group (n=18), ganglioside group (n=10) and combination group (n=12). Under stereotaxic instrument, umbilical blood cord mesenchymal stem cells or ganglioside were injected into left lateral ventricles of the rat brain, respectively, and the sham transplantation group was given the same volume of phosphate buffered saline. Two rats from the stem cell transplantation group were put to death for immunofluorescence staining at 7, 14, 21 and 28 days after transplantation, respectively, and two rats in the combination group were killed for immunofluorescence staining at 14 days. Besides, all rats were underwent neurologic evaluation at 28 days after transplantation.
RESULTS AND CONCLUSION: The umbilical blood cord mesenchymal stem cells could survive, migrate and differentiate, which mainly distributed in the lateral ventricle, hippocampus and cortex. At 14 days after transplantation, positive expressions of BrdU and glial fibrillary acidic protein in the combination group were significantly higher than those in the stem cell transplantation group (P < 0.05). In addition, compared with the model group, the holding time significantly prolonged and foot error times significantly decreased in the latter three groups (P < 0.05), as well as in the combination group compared with the stem cell transplantation and ganglioside groups (P < 0.05). These results indicate that umbilical blood cord mesenchymal stem cells and ganglioside can both improve neurological function of rats with cerebral palsy. Given that ganglioside can promote survival and differentiation of umbilical blood cord mesenchymal stem cells in vivo, the combined transplantation is preferred.

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: Cerebral Palsy, Gangliosides, Cord Blood Stem Cell Transplantation, Tissue Engineering

CLC Number:

R394.2

Yang Zi-jin, Guo Jia-li, Lu Si-guang, Gao Chang-long, Li Hong-mei, Feng Yue. Human umbilical blood cord mesenchymal stem cell transplantation combined with injection of ganglioside for cerebral palsy[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(19): 2803-2809.

Figures/Tables 1

2.1 脐血间充质干细胞的培养及鉴定复苏后细胞生长旺盛，呈形态及大小均一的小长梭形细胞(图1A)，折光性强，倍增时间为5-9 d，约1周融合。细胞传3代后，应用流式细胞仪进行脐血间充质干细胞表面标志性抗原检测，结果显示脐血间充质干细胞高表达CD29(68.2%)、CD44(52.2%)和CD105(65.7%)表面标志, 低表达CD34(3.6%)和CD45(1.0%)表面标志，符合间充质干细胞特征。 2.2 培养细胞免疫荧光结果 BrdU标记脐血间充质干细胞72 h，细胞爬片后，应用直接免疫荧光法检测BrdU阳性细胞率，大部分细胞核被染成鲜绿色(图1B)，标记率为93%，说明培养的脐血间充质干细胞标记成功。 2.3 脑瘫模型制作结果对生后4周的幼鼠进行行为学检测，模型成功率约85%，随机选出符合脑瘫标准的大鼠60只。 2.4 神经行为学功能评定 2.4.1 握持牵引实验时间比较与模型组比较，正常组握持时间明显延长，二者相比差异有显著性意义(P < 0.05)，表明模型组大鼠较正常组握持能力明显下降，提示存在脑损伤。假移植组与模型组相比差异无显著性意义(P > 0.05)，表明PBS及注射本身对脑瘫鼠无明显影响；脐血间充质干细胞移植组、神经节苷脂组、联合移植组握持时间均高于模型组，差异有显著性意义(P < 0.05)，同时联合移植组握持时间高于脐血间充质干细胞移植组，二者相比差异有显著性意义(P < 0.05)，表明脐血间充质干细胞移植及神经节苷脂均可改善脑瘫大鼠的握持能力，联合移植改善程度优于脐血间充质干细胞移植组，脐血间充质干细胞与神经节苷脂有一定协调作用，见表1。 2.4.2 足错误实验模型组较正常组错误次数明显增多，且差异有显著性意义(P < 0.05)，说明脑瘫模型制作成功，模型组大鼠造成了脑部损伤以致肢体运动协调能力发生障碍；假移植组及模型组二者无明显差异，说明移植手术本身对模型无明显影响。脐血间充质干细胞移植组、联合移植组、神经节苷脂组分别与模型组相比，差异有显著性意义(P < 0.05)，表明脐血间充质干细胞移植可提高脑瘫大鼠的肢体运动协调能力，且联合移植组优于脐血间充质干细胞组，差异有显著性意义(P < 0.05)，表明脐血间充质干细胞及神经节苷脂二者有部分协调作用，能改善脑瘫大鼠部分功能，见表1。 2.5 脑实质BrdU阳性细胞迁移、分布、分化情况应用免疫荧光单标染色，在荧光显微镜下，可见FITC标记的BrdU阳性细胞。人脐血间充质干细胞移植后大鼠脑实质BrdU阳性细胞主要分布在大脑皮质区、海马、脑室周围，有局部聚集现象，白质区、基底节、丘脑区可见少量BrdU阳性细胞。脐血间充质干细胞组移植后第7天大鼠脑组织内可见较多BrdU阳性细胞(28.3± 6.2)，主要聚集在大脑皮质、侧脑室及嗅球，左侧(移植侧)多于右侧；第14天BrdU阳性细胞(20.6±3.6)、第21天BrdU阳性细胞(13.7±3.8)逐渐减少，主要分布在海马、侧脑室旁、基底节(图2)，第28天仅见少量BrdU阳性细胞(6.2±2.1)存活。因GFAP抗体及NSE抗体一抗均为抗人抗体，GFAP、NSE分别存在于星形胶质细胞、神经元细胞中，故GFAP、NSE阳性细胞可视为移植细胞分化的星形胶质细胞、神经元细胞。移植14，28 d均可见到GFAP、NSE阳性细胞，但28 d时明显减少。 2.6 脐血间充质干细胞移植组及联合移植组脑实质阳性细胞对比人脐血间充质干细胞移植第14天，联合移植组BrdU阳性细胞为(30.10±2.23)个/视野，高于脐血间充质干细胞移植组(20.00±2.79)个/视野，差异有显著性意义(t=10.28，P < 0.05)；移植第14天，联合移植组GFAP阳性细胞为(17.90±1.52)个/视野，高于脐血间充质干细胞移植组(10.20±1.55)个/视野，差异有显著性意义(t=9.75，P < 0.05)；联合移植组NSE阳性细胞为(7.00±1.82)个/视野，高于脐血间充质干细胞移植组(5.30±2.11)个/视野，差异无显著性意义(t=2.08，P > 0.05)，见图3。移植28 d，两组BrdU、GFAP、NSE阳性细胞均明显减少，差异无显著性意义。"

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