中国组织工程研究

中国组织工程研究 ›› 2013, Vol. 17 ›› Issue (23): 4256-4263.doi: 10.3969/j.issn.2095-4344.2013.23.011

• 干细胞移植 stem cell transplantation • 上一篇    下一篇

骨髓间充质干细胞诱导成神经元样细胞移植治疗脊髓损伤

高  平1,2，孙占胜1，王伯珉1，李连欣1，王  甫1，牟乐明1   

  1. 1山东大学附属省立医院创伤骨科，山东省济南市  250021
    2山东大学医学院，山东省济南市  250012
  • 出版日期:2013-06-04 发布日期:2013-06-04
  • 作者简介:高平★，男，1987年生，山东省高密市人，汉族，山东大学医学院骨外科在读硕士，主要从事骨髓间充质干细胞治疗脊髓损伤的研究。 Gaopinggp125@163.com
  • 基金资助:

    山东省科技攻关计划项目(2006GG3202046)。

Transplantation of neuron-like cells from bone marrow mesenchymal stem cells for treatment of spinal cord injury

Gao Ping1,2, Sun Zhan-sheng1, Wang Bo-min1, Li Lian-xin1, Wang Fu1, Mu Le-ming1
   

  1. 1 Department of Traumatic Orthopedics, Provincial Hospital Affiliated to Shandong University, Jinan  250021, Shandong Province, China
    2 Medical College of Shandong University, Jinan  250012, Shandong Province, China
  • Online:2013-06-04 Published:2013-06-04
  • About author:Gao Ping★, Studying for master’s degree, Department of Traumatic Orthopedics, Provincial Hospital Affiliated to Shandong University, Jinan 250021, Shandong Province, China; Medical College of Shandong University, Jinan 250012, Shandong Province, China Gaopinggp125@163.com
  • Supported by:

    Key Scientific and Technological Project of Shandong Province, No. 2006GG3202046*

PDF

798

被引次数

28

摘要：

背景：骨髓间充质干细胞诱导成为神经元样细胞移植治疗脊髓损伤已被证实有效，但不同诱导方法之间的差别尚无报道。
目的：通过对脊髓损伤模型大鼠的行为对比及生化指标的检测，观察采用不同诱导方法将骨髓间充质干细胞诱导分化为神经元样细胞后移植对脊髓损伤疗效的差别。
方法：取4周龄雄性Wistar大鼠骨髓分离培养骨髓间充质干细胞，对第3代骨髓间充质干细胞分别进行化学诱导和生物因子诱导后，收集备用。8周龄雄性Wistar大鼠48只，采用脊髓半横切法建立大鼠的脊髓损伤模型，随机分为4组：1周后骨髓间充质干细胞组损伤部位局部注射第3代骨髓间充质干细胞，化学诱导组局部注射化学诱导成的神经元样细胞，生物诱导组局部注射生物诱导成的神经元样细胞，DMEM培养液组局部注射细胞培养液。对48只大鼠脊髓损伤模型分别于伤后1，2，3，4，6，8，10，12周进行BBB评分，并于第12周末对损伤部位进行取材做组织切片，观察脊髓损伤的修复情况。
结果与结论：模型建立后12周，骨髓间充质干细胞组、化学诱导组和生物诱导组大鼠后肢功能恢复明显优于DMEM培养液组(P < 0.05)，骨髓间充质干细胞组和化学诱导组功能恢复无明显差别(P=0.436 3)，生物诱导组恢复效果好于前2组(P < 0.05)。生物诱导组大鼠运动功能持续恢复显著好于其他3组。脊髓组织切片苏木精-伊红染色显示骨髓间充质干细胞组和化学诱导组近似，脊髓胶质细胞增生，神经元样细胞崩解、空洞形成少于DMEM培养液组，生物诱导组神经损伤修复效果最佳。提示化学诱导后的骨髓间充质干细胞与未经过诱导的骨髓间充质干细胞在修复神经损伤的效果上没有明显差别；而经过生物诱导的骨髓间充质干细胞分化为神经元样细胞后移植治疗脊髓损伤的疗效明显优于未经诱导和化学诱导的骨髓间充质干细胞移植的疗效。

关键词: 干细胞, 干细胞移植, 脊髓损伤, 细胞移植, 诱导分化, 生物诱导, 化学诱导, 省级基金, 干细胞图片文章

Abstract:

BACKGROUND: The therapeutic effects of transplantation of neuron-like cells from bone marrow mesenchymal stem cells in the treatment of spinal cord injury have been confirmed. Howver, the efficiency differences among different methods of inducing neuron-like cell differentiation of bone marrow mesenchymal stem cells remain poorly understood.
OBJECTIVE: After behavioral observation and biochemical index measurements of spinal cord injury rat models, the curative effects of transplantation of neuron-like cells induced by different methods from bone marrow mesenchymal stem cells in the treatment of spinal cord injury were investigated.
METHODS: Bone marrow mesenchymal stem cells were isolated from 4-week-old male Wistar rats. Passage 3 bone marrow mesenchymal stem cells were induced by chemical methods and biological factors for later use. Spinal cord injury was induced in 48 male Wistar rats aged 8 weeks by spinal cord hemisection method. Then the rats were randomly divided into four groups: bone marrow mesenchymal stem cells, chemically-induced, biological factor-induced and Dulbecco's modified Eagle’s medium (DMEM) groups, in which, passage 3 bone marrow mesenchymal stem cells, chemically-induced or biological factor-induced, or DMEM-treated bone marrow mesenchymal stem cells were injected into the injury region 1 week later, respectively. All 48 spinal cord injury rats were scored by the Basso, Beattie and Bresnahan locomotor rating scale at various time points (1, 2, 3, 4, 6, 8, 10, 12 weeks after injury). At the end of 12 weeks after injury, tissue sections from the injury region were prepared for observation of repair of spinal cord injury.
RESULTS AND CONCLUSION: At 12 weeks after spinal cord injury induction, recovery of hindlimb motor functions was superior in the bone marrow mesenchymal stem cells, chemically-induced, biological factor-induced groups to that in the DMEM group (P < 0.05). There was no obvious difference in recovery of hindlimb motor function between bone marrow mesenchymal stem cells group and chemically-induced group (P = 0.436 3), and the hindlimb motor function recovery was better in the biological factor-induced group than that in the bone marrow mesenchymal stem cells group and chemically-induced group (P < 0.05). The motor function recovery was superior in the biological factor-induced group to that in the other three groups. Hematoxylin-eosin staining results demonstrated that bone marrow mesenchymal stem cells group and chemically-induced group showed similar outcomes: glial cells proliferation, neuron-like cell disruption and cavitation. The biological factor-induced group yielded best recovery of hindlimb motor function. These findings indicate that in the treatment of spinal cord injury by cell transplantation, chemically-induced and non-induced bone marrow mesenchymal stem cells have no obvious difference, while biological factor-induced bone marrow mesenchymal stem cells exhibit better therapeutic effects than non-induced and chemically-induced bone marrow mesenchymal stem cells.

Key words: stem cells, stem cell transplantation, spinal cord injury, cell transplantation, induced differentiation, biological induction, chemical inducation, provincial grants-supported paper, stem cell photographs-containing paper

中图分类号: 





细胞免疫化学染色在显微镜下可以见到CD44呈阳性反应，整个细胞呈棕黄色；而CD34呈阴性反应，细胞没有着色。符合骨髓间充质干细胞表面特异性标志的特点[10] 。见图3。



2.3  骨髓间充质干细胞来源的神经元样细胞的鉴定
细胞形态学观察：在诱导培养基条件下，持续镜下观察细胞形态的变化。化学诱导组：预诱导24 h后，细胞形态改变不明显；正式诱导12 h后，细胞形态发生明显改变，胞体变圆、变小，折光性增强，多个突起产生，24 h后，有长的类似轴突的结构出现。生物诱导组：开始诱导后3 d，细胞由原来的长梭形逐渐变为三角形、多边形；5 d后，大部分细胞胞体收缩，折光性增强，有多个长的突起形成；9 d时，细胞胞体变圆，有数个突起，具有长的轴突样结构，交织成网状，神经元样细胞的形态显现。见图4。



免疫细胞化学法鉴定：化学诱导组和生物诱导组细胞，免疫细胞化学染色结果相似，显示神经元特异性烯醇化酶和神经丝蛋白表达阳性，而胶质纤维酸性蛋白表达为阴性。说明骨髓间充质干细胞经过两种诱导方法所获得的细胞都表达了神经元样细胞的特异性蛋白。见图5。
PCR检测：化学诱导组和生物诱导组细胞，PCR结果相似，神经元特异性烯醇化酶、神经丝蛋白在电泳图像中表现为阳性，胶质纤维酸性蛋白为阴性。以神经胶质细胞作为对照，其神经元特异性烯醇化酶、胶质纤维酸性蛋白、神经丝蛋白在电泳图像中均为阳性表达。说明骨髓间充质干细胞经过两种诱导方法定向诱导所获得的细胞在基因水平上表现了神经元样细胞的特异性。见图6。




2.4  动物模型行为学评分  造模前，各大鼠活动正常，BBB评分21分[9，11] 。造模后表现右后肢及尾无活动，针刺皮肤无反应，排尿障碍，排便未观察到明显异常。伤后1-12周BBB评分见表1。



伤后1-4周：伤后1周，针刺右后肢皮肤有回缩反应，伤后2周偶见髋关节活动，伤后3周髋膝关节皆有活动。伤后4周，骨髓间充质干细胞组和化学诱导组可见到后肢三关节活动，而DMEM培养液组踝关节未见活动。
 
伤后6周：骨髓间充质干细胞组和化学诱导组见掌面非承重着地，生物诱导组掌面承重着地，但后肢活动不协调，DMEM培养液组较前恢复不明显。
 
伤后8周：骨髓间充质干细胞组和化学诱导组见掌面承重着地，但活动不协调，生物诱导组有持续性掌面承重移动和前后肢协调动作，常见掌面移动，持续型前后肢协调动作，偶有爪背侧移动，DMEM培养液组无继续恢复。
 
伤后10周：骨髓间充质干细胞组、化学诱导组、生物诱导组3组较前略有恢复但不明显，DMEM培养液组无继续恢复。伤后12周，4组均无继续恢复，骨髓间充质干细胞组、化学诱导组与DMEM培养液组比较差异有显著性意义(t=22.81/23.70，P < 0.05)；生物诱导组与骨髓间充质干细胞组、化学诱导组比较差异有显著性意义(t=26.08/25.38，P < 0.05)；化学诱导组与骨髓间充质干细胞组比较差异无显著意义(t=0.792 8，P=0.436 3)。
在伤后的前4-6周，随着损伤部位水肿和炎症的消退及周围轻度受损神经元的恢复，部分神经传导重新建立，模型动物的运动功能也部分恢复。6周后，DMEM培养液组功能再无继续恢复；骨髓间充质干细胞组和化学诱导组继续恢复但不明显，伤后12周与伤后6周比较差异无显著性意义(t=1.586/3.023，P=0.127 1/0.063 0)；生物诱导组继续恢复至10周，且恢复明显，伤后12周与伤后6周比较差异有显著意义(t=13.70，P < 0.05)。见表1。
 
脊髓切片苏木精-伊红染色：见图7。
损伤12周后损伤部位脊髓切片苏木精-伊红染色显示DMEM培养液组脊髓灰质与白质分界不清，结构混乱，且有较多大的坏死空洞存在，神经元样细胞崩解广泛，胶质细胞增生不明显。骨髓间充质干细胞组与化学诱导组相似，灰质与白质分界清楚，坏死空洞较DMEM培养液组小且少，神经元样细胞崩解较DMEM培养液组轻，胶质细胞增生。说明骨髓间充质干细胞组和化学诱导组功能恢复较DMEM培养液组好。生物诱导组脊髓结构清晰，坏死空洞较其他3组都少，胶质细胞增生明显，神经元样细胞崩解少。说明生物诱导组脊髓损伤后，较快的进入恢复期，且神经恢复程度高于骨髓间充质干细胞组、化学诱导组和DMEM培养液组。