骨髓间质干细胞源性神经干细胞对脊髓损伤修复作用的研究
发布时间：2018-08-16 18:33
【摘要】： 神经干细胞(Neural stem cells,NSCs)因其来源于神经组织、具有分化为神经元和神经胶质潜能、能达到损伤或疾病部位等特点,成为脊髓等中枢神经系统损伤的细胞替代的理想移植材料。成年动物的骨髓间质干细胞(bone marrow mesenchymal stemcells,BMSCs)是一种成体干细胞,具有与胚胎干细胞相当的分化潜能,在一定条件下可诱导分化NSCs,继而分化为神经元,而且可用于自体移植。因此BMSCs可能成为NSCs的广泛的来源,将能克服从脑组织获取NSCs的危险性和局限性,同时又可避免胎儿脑组织移植存在的伦理和免疫排斥问题,可解决脊髓损伤等疾病的临床个体化治疗问题。 目的建立适合本实验室体外分离培养骨髓间质干细胞的方法,诱导骨髓间质干细胞向神经干细胞分化,将获得的神经干细胞用于脊髓损伤的细胞移植,探讨骨髓间质干细胞源性的神经干细胞(BMSCs derived NSCs,BMSCs-D-NSCs)对脊髓损伤(spine cord injury,SCI)的修复作用。 方法取大鼠骨髓,原代贴壁培养,通过多向分化能力鉴定细胞。取第3代BMSCs加入含bFGF及EGF的无血清培液中分化,采用免疫细胞化学法以nestin鉴定神经干细胞,加入血清培养后,以MAP2、GFAP及MBP鉴定分化的神经元样、星形胶质细胞样及少突胶质细胞样细胞。80只成年SD大鼠,随机分为4组:A组为正常组,不行SCI手术,与其他3组同期饲养检测指标;其他三组均行T9脊髓左半侧离断手术,B组为损伤对照组,脊髓损伤区内植入吸附生理盐水的明胶海棉;C组为BMSCs-D-NSCs组,损伤处植入细胞悬液;D组,脑源性NSCs组。移植术后7、14、28d各组以BBB(the Basso,Beattie,Bresnahan locomotor rating scale)评定法进行神经功能评定,并进行BrdU免疫组织化学标记等,观察BrdU在各组脊髓组织中的表达。 结果在体外扩增培养的BMSCs,增殖速度快,扩增能力强,在体外能分化为成骨细胞、脂肪细胞及神经元样细胞。BMSCs在含bFGF及EGF的无血清培液中能分化为神经干细胞,加入血清后能进一步分化为神经元样细胞、星形胶质细胞样细胞及少突胶质细胞样细胞,相应表达神经元、星形胶质细胞与少突胶质细胞的标记物MAP2、GFAP及MBP。SCI细胞移植术后1—4 w,B、C、D组动物后肢神经功能均有不同程度的恢复,C组与B组间有显著性差异(P＜0.05),C与D组之间无显著性差异(P＞0.05),A组BBB功能评分为21分。C组、D组BrdU标记的细胞移植后7d在损伤脊髓可见,14d阳性细胞有所减少,28d时BrdU标记的阳性细胞几乎检测不到。 结论体外扩增纯化的骨髓间质干细胞具有分化为神经干细胞的生物学功能,而且获得的BMSCs-D-NSCs具有进一步分化能力;BMSCs-D-NSCs移植可以促进脊髓损伤后的功能恢复,可成为治疗SCI的细胞来源。
[Abstract]:Neural stem cells (NSCs) have the potential to differentiate into neurons and glia because they are derived from nerve tissue and can reach the injury or disease location. NSCs can be used as an ideal substitute for central nervous system injury (CNS) such as spinal cord. Adult animal bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells) is a kind of adult stem cells, which has the same differentiation potential as embryonic stem cells. Under certain conditions, it can induce the differentiation of NSCs and then differentiate into neurons, and can be used for autologous transplantation. Therefore, BMSCs may become a broad source of NSCs, which will overcome the dangers and limitations of obtaining NSCs from brain tissue, and at the same time avoid the ethical and immune rejection of fetal brain tissue transplantation. It can solve the problem of individualized treatment of spinal cord injury and other diseases. Objective to establish a method suitable for isolation and culture of bone marrow mesenchymal stem cells (BMSCs) in our laboratory in vitro, to induce the differentiation of BMSCs into neural stem cells (NSCs), and to apply the obtained neural stem cells (NSCs) to transplantation of spinal cord injury cells. To investigate the repair effect of bone marrow mesenchymal stem cell derived neural stem cell (BMSCs derived) on spinal cord injury (spine cord injury-induced sci). Methods Rat bone marrow was harvested and primary adherent culture was used to identify the cells by multi-directional differentiation. The third generation of BMSCs was added to the serum-free culture medium containing bFGF and EGF. The neural stem cells were identified by nestin by immunocytochemistry, and the differentiated neurons were identified by MAP2GFAP and MBP after the addition of serum. Astrocyte-like and oligodendrocyte-like cells. 80 adult SD rats were randomly divided into 4 groups: group A as normal group, no SCI operation, and the other three groups were fed and tested at the same time. The other three groups were treated with T9 spinal cord left hemisection. Group B was the control group, group C was BMSCs-D-NSCs group, group C was implanted with normal saline, group D was implanted with cell suspension and group D was implanted with brain-derived NSCs. The nerve function was assessed by BBB (the Basso Beattiean locomotor rating scale) method and BrdU immunohistochemical staining was used to observe the expression of BrdU in spinal cord tissues in each group on the 7th day and 28th day after transplantation. Results BMSCs could differentiate into osteoblasts in vitro, adipocytes and neuron-like cells. BMSCs could be differentiated into neural stem cells in serum-free culture containing bFGF and EGF. After addition of serum, neurons were further differentiated into neuron-like cells, astrocyte-like cells and oligodendrocyte like cells. MAP2GFAP, a marker of astrocytes and oligodendrocytes, and MBP.SCI cell transplantation showed different degrees of recovery of nerve function in hind limbs of rats in group C and group B (P < 0. 05). There was no significant difference between group C and group D (P < 0. 05). The difference was significant (P > 0. 05) the BBB function score of group A was 21. The BrdU labeled cells in group C were almost undetectable 7 days after transplantation. The positive cells of BrdU could not be detected on the 14th day after the spinal cord injury and the number of BrdU positive cells decreased on the 28th day after transplantation. Conclusion the purified bone marrow mesenchymal stem cells have the biological function of differentiation into neural stem cells, and the BMSCs-D-NSCs obtained has further differentiation ability. BMSCs-D-NSCs transplantation can promote the functional recovery after spinal cord injury. Can be used as a cell source for the treatment of SCI.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329
[Abstract]:Neural stem cells (NSCs) have the potential to differentiate into neurons and glia because they are derived from nerve tissue and can reach the injury or disease location. NSCs can be used as an ideal substitute for central nervous system injury (CNS) such as spinal cord. Adult animal bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells) is a kind of adult stem cells, which has the same differentiation potential as embryonic stem cells. Under certain conditions, it can induce the differentiation of NSCs and then differentiate into neurons, and can be used for autologous transplantation. Therefore, BMSCs may become a broad source of NSCs, which will overcome the dangers and limitations of obtaining NSCs from brain tissue, and at the same time avoid the ethical and immune rejection of fetal brain tissue transplantation. It can solve the problem of individualized treatment of spinal cord injury and other diseases. Objective to establish a method suitable for isolation and culture of bone marrow mesenchymal stem cells (BMSCs) in our laboratory in vitro, to induce the differentiation of BMSCs into neural stem cells (NSCs), and to apply the obtained neural stem cells (NSCs) to transplantation of spinal cord injury cells. To investigate the repair effect of bone marrow mesenchymal stem cell derived neural stem cell (BMSCs derived) on spinal cord injury (spine cord injury-induced sci). Methods Rat bone marrow was harvested and primary adherent culture was used to identify the cells by multi-directional differentiation. The third generation of BMSCs was added to the serum-free culture medium containing bFGF and EGF. The neural stem cells were identified by nestin by immunocytochemistry, and the differentiated neurons were identified by MAP2GFAP and MBP after the addition of serum. Astrocyte-like and oligodendrocyte-like cells. 80 adult SD rats were randomly divided into 4 groups: group A as normal group, no SCI operation, and the other three groups were fed and tested at the same time. The other three groups were treated with T9 spinal cord left hemisection. Group B was the control group, group C was BMSCs-D-NSCs group, group C was implanted with normal saline, group D was implanted with cell suspension and group D was implanted with brain-derived NSCs. The nerve function was assessed by BBB (the Basso Beattiean locomotor rating scale) method and BrdU immunohistochemical staining was used to observe the expression of BrdU in spinal cord tissues in each group on the 7th day and 28th day after transplantation. Results BMSCs could differentiate into osteoblasts in vitro, adipocytes and neuron-like cells. BMSCs could be differentiated into neural stem cells in serum-free culture containing bFGF and EGF. After addition of serum, neurons were further differentiated into neuron-like cells, astrocyte-like cells and oligodendrocyte like cells. MAP2GFAP, a marker of astrocytes and oligodendrocytes, and MBP.SCI cell transplantation showed different degrees of recovery of nerve function in hind limbs of rats in group C and group B (P < 0. 05). There was no significant difference between group C and group D (P < 0. 05). The difference was significant (P > 0. 05) the BBB function score of group A was 21. The BrdU labeled cells in group C were almost undetectable 7 days after transplantation. The positive cells of BrdU could not be detected on the 14th day after the spinal cord injury and the number of BrdU positive cells decreased on the 28th day after transplantation. Conclusion the purified bone marrow mesenchymal stem cells have the biological function of differentiation into neural stem cells, and the BMSCs-D-NSCs obtained has further differentiation ability. BMSCs-D-NSCs transplantation can promote the functional recovery after spinal cord injury. Can be used as a cell source for the treatment of SCI.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329
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相关期刊论文 前4条
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