神经干细胞移植联合电针治疗对大鼠脊髓损伤的修复作用研究
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摘要
目的：
探讨胚胎脊髓源性神经干细胞(Neural stem cells, NSCs)移植联合电针治疗对大鼠脊髓全横断损伤的修复作用。
方法：
实验1、取11.5 d的SD胎鼠分离神经管后段,分散细胞进行原代和传代培养,倒置相差显微镜下观察细胞生长状态。对其进行神经干细胞标志抗原Nestin免疫细胞化学染色鉴定；用含10%胎牛血清的DMEM/F12诱导,并进行神经元特异烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)、髓磷脂碱性蛋白(MBP)的免疫细胞化学染色,激光共聚焦显微镜下观察其分化情况。用BrdU标记第二代培养7-8 d的NSCs悬液,进行BrdU免疫细胞化学染色,观察标记情况,收集已标记的细胞备用。
实验2、将54只成年雌性SD大鼠随机分为三组：脊髓损伤(SCI)组；NSCs移植组；NSCs移植联合电针治疗(E+NSCs)组。制作脊髓全横断损伤模型,在大鼠脊髓横断处填放明胶海绵,将BrdU标记的NSCs各10μl分别吸附到NSCs移植组和E+NSCs组损伤处的明胶海绵上,SCI组用生理盐水代替NSCs。E+NSCs组在细胞移植后每天的固定时间给予电针治疗。各组在7 d、14 d、28 d后分别进行BBB运动功能评分观察大鼠后肢运动功能恢复情况；取损伤脊髓分别进行大体观察、光镜观察和电镜观察,分析脊髓形态学改变,通过免疫荧光染色检测脊髓损伤处的神经元、星形胶质细胞及少突胶质细胞的变化情况。
结果：
1、11.5 d胎鼠的神经管后段培养的细胞,有良好的增殖能力,易获得大量细胞；其表达神经干细胞特异抗原Nestin,且诱导分化后表达NSE、GFAP. MBP阳性；BrdU免疫荧光观察显示,新形成的细胞克隆球中的细胞呈BrdU免疫阳性。
2、脊髓损伤后大鼠后肢BBB运动功能评分显示,随时间推移,NSCs联合电针治疗组与其他各组之间均存在显著差异(P<0.01),动物肢体功能恢复显著。
3、大体观察显示,损伤区的脊髓由于瘢痕化颜色变深,植入脊髓断端的明胶海绵有不同程度的吸收,损伤区近头端脊髓轻度萎缩,近尾端明显萎缩。NSCs移植组与E+NSCs组明胶海绵吸收程度较好,基本与脊髓组织融合,且萎缩程度较轻。而SCI组部分大鼠损伤处脊髓变性坏死,明胶海绵吸收较差。
4、光镜观察显示,术后28 d,SCI组脊髓损伤区形态不完整,神经组织残缺；有大量泡沫细胞和明显空洞形成以及大量炎症细胞浸润。NSCs组部分神经元细胞形态较规则,少数神经元可见细胞核及核仁,但部分神经元内尼氏小体较少,分布不均匀。E+NSCs组神经元细胞数量较多,形态基本正常,核仁清晰可见,尼氏小体较多,且分布均匀,神经纤维排列较规则。
5、透射电镜观察显示,SCI组髓鞘松散、破裂,少部分完全崩解,轴突固缩,部分轴突空泡变,线粒体含量较少且有肿胀；NSCs移植组髓鞘松散程度减轻,散在分布,仅有少量髓鞘破裂,轴突消失,其间有薄髓的再生髓鞘；E+NSCs组再生纤维增多,髓鞘松散程度明显减轻,部分髓鞘结构规整,排列有序,线粒体肿胀减轻,微管空泡样变较轻,可发现大量突触,薄髓的再生髓鞘明显增多。
6、免疫荧光染色检测脊髓损伤处的神经元、星形胶质细胞及少突胶质细胞数量随时间推移逐渐增加,E+NSCs组与其他各组之间均存在差异(P＜0.01)。
结论：
1、从11.5 d胎鼠的神经管后段分离、培养,可获得足量的NSCs,血清诱导能够分化为神经元和胶质细胞；
2、NSCs移植联合电针治疗能促进脊髓损伤大鼠后肢运动功能的恢复；
3、NSCs移植联合电针治疗能促进损伤处脱髓的轴突再髓鞘化和神经纤维再生；
4、NSCs移植联合电针治疗能促进细胞的分化,增加损伤脊髓神经元、胶质细胞的数量,改善损伤处的微环境,促进结构的修复。
Objective:
To explore the effect of neural stem cells transplantation in combination with electro-acupuncture treatment on repair of traumatic spinal cord injury rats.
Methods:
Experiment 1, The posterior segment of neural tube was dissected from SD embryonic 11.5d rats, cultured primarily in vitro and observed the growth state of cells under inverted phase contrast microscope. They were identified with immunocytochemistry for nestin which is the marker antigen of neural stem cell. Meanwhile, the differentiation was induced by 10% fetal bovine serum and cells were examined by immunocytochemical stain including neuron specific enolase (NSE), glial fibrillary acidic protein (GFAP), and myelin basic protein (MBP) under confocal laser scanning microscopy. Neural stem cells (made from the culturing cells of the second generation 7-8d) were marked by BrdU immunocytochemical stain, and marked cells were collect to reserve.
Experiment 2,54 adult female SD rats were randomly divided into three groups:spinal cord injury group (SCI), NSCs transplantation group, electro-acupuncture treatment with NSCs transplantation group (E+NSCs). SCI rat model was constructed by transecting spinal cord thoroughly. NSCs labeled with BrdU were adsorbed to the gelatin sponge and then transplanted into injured part of both NSCs and E+NSCs groups, yet normal saline into SCI group instead correspondingly. After transplantation, rats in E+NSCs group were given electro-acupuncture treatment at a fixed time every day. The motor functions of recovery of rat hind limb in each group were detected in 7d, 14d,28d, respectively, by methods of BBB score. The injury spinal cords were admitted to analyze morphological changes through general observation, light microscope and electron microscope respectively, and changes of neurons, astrocytes and oligodendrocytes keratinocyte were detected through immunofluorescence.
Results:
1. The cells derived from the posterior segment of neural tube of E11.5d rat were successfully isolated and cultured with good proliferation. The cultured cells were identified as NSCs by expressing specific antigen Nestin of NSCs and can be induced to differentiate to neurons, astrocytes and oligodendrocytes. The cells in neurosphere newly formed were proved BrdU-positive cells by immunofluorescence.
2. Difference between E+NSCs group and other groups respective were obviously observed by methods of BBB score of hind limb motor function over time (P<0.01). The recovery of animal limb function was significant.
3. It was observed that the color of the spinal cord in the injured site became darker as a result of scarring, the gelatin sponge migrated into the injured site was absorbed in various degree, the atrophy of the injury spinal cord close to the head atrophies was mildly while the part far from the head atrophies markedly. The gelatin sponge was absorbed in a better degree in both NSCs group and E+NSCs group and integrated nearly with the spinal cord tissue, and the spinal cord atrophied mildly. While the injury spinal cord of some rats in SCI group was metamorphic and necrotic with poor absorption of gelatin sponge.
4. Under light microscope, HE staining showed that the spinal cord injury zone in SCI group was incomplete after 28d and the nervous tissue was fragmentary. Large numbers of foam cells and marked cavities emerged and the injury area was intiltrated with many chronic inflammatory cells. In NSCs group, the morphological changes of some neurons was relatively regular, nucleus and nucleolus in the minority of neurons could be seen, but Nissl bodies within some neurons were fairly less and uneven. In E+NSCs group, the number of neurons increased obviously, the shape of neurons was close to normal, nucleolus were clearly visible, more Nissl bodies with even distribution, and the nerve fibers were regular.
5. Transmission Electron Microscope (TEM) showed:In SCI group, the myelin was loose, distorted and broken, with axonal shrinkage and some axonal vacuolate, mitochondrial swelling and there was less content. In NSCs group, the extent of myelin loose reduced and scattered, only a few myelin breakdown, and the axon disappeared and there were thin myelin regeneration of myelinated. In E+NSCs group, regenerated fiber increased, the extent of myelin loose reduced distinctly, the structure of myelin was clear and neat and the arrangement orderly, the swelling of mitochondria became lightened, vacuolar degeneration of microtubules got less, a lot of synapses were found, thin myelin sheath regeneration increased significantly.
6. The number of neurons, astrocytes and oligodendrocytes keratinocyte in injured site gradually increased over time by immunofluorescence stain. Difference between E+NSCs group and other groups respective was obvious (P<0.01).
Conclusion:
1. NSCs derived from the posterior segment of neural tube of E11.5d rat were successfully isolated, cultured to sufficient quantities and induced to differentiate to neurons and glial cells by serum.
2. NSCs transplantation with electro-acupuncture treatment increased the recovery of hind limb motor function of spinal cord injury in rats
3. The treatment of NSCs transplanted with electro-acupuncture promoted demyelination axons in injured site re-myelination and nerve fibers regeneration.
4. NSCs transplantation with electro-acupuncture treatment promoted cells differentiation, increased the number of neurons and glial cells in injured spinal cord, improved the micro-environment of the damaged site and promoted structural repair.
探讨胚胎脊髓源性神经干细胞(Neural stem cells, NSCs)移植联合电针治疗对大鼠脊髓全横断损伤的修复作用。
方法：
实验1、取11.5 d的SD胎鼠分离神经管后段,分散细胞进行原代和传代培养,倒置相差显微镜下观察细胞生长状态。对其进行神经干细胞标志抗原Nestin免疫细胞化学染色鉴定；用含10%胎牛血清的DMEM/F12诱导,并进行神经元特异烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)、髓磷脂碱性蛋白(MBP)的免疫细胞化学染色,激光共聚焦显微镜下观察其分化情况。用BrdU标记第二代培养7-8 d的NSCs悬液,进行BrdU免疫细胞化学染色,观察标记情况,收集已标记的细胞备用。
实验2、将54只成年雌性SD大鼠随机分为三组：脊髓损伤(SCI)组；NSCs移植组；NSCs移植联合电针治疗(E+NSCs)组。制作脊髓全横断损伤模型,在大鼠脊髓横断处填放明胶海绵,将BrdU标记的NSCs各10μl分别吸附到NSCs移植组和E+NSCs组损伤处的明胶海绵上,SCI组用生理盐水代替NSCs。E+NSCs组在细胞移植后每天的固定时间给予电针治疗。各组在7 d、14 d、28 d后分别进行BBB运动功能评分观察大鼠后肢运动功能恢复情况；取损伤脊髓分别进行大体观察、光镜观察和电镜观察,分析脊髓形态学改变,通过免疫荧光染色检测脊髓损伤处的神经元、星形胶质细胞及少突胶质细胞的变化情况。
结果：
1、11.5 d胎鼠的神经管后段培养的细胞,有良好的增殖能力,易获得大量细胞；其表达神经干细胞特异抗原Nestin,且诱导分化后表达NSE、GFAP. MBP阳性；BrdU免疫荧光观察显示,新形成的细胞克隆球中的细胞呈BrdU免疫阳性。
2、脊髓损伤后大鼠后肢BBB运动功能评分显示,随时间推移,NSCs联合电针治疗组与其他各组之间均存在显著差异(P<0.01),动物肢体功能恢复显著。
3、大体观察显示,损伤区的脊髓由于瘢痕化颜色变深,植入脊髓断端的明胶海绵有不同程度的吸收,损伤区近头端脊髓轻度萎缩,近尾端明显萎缩。NSCs移植组与E+NSCs组明胶海绵吸收程度较好,基本与脊髓组织融合,且萎缩程度较轻。而SCI组部分大鼠损伤处脊髓变性坏死,明胶海绵吸收较差。
4、光镜观察显示,术后28 d,SCI组脊髓损伤区形态不完整,神经组织残缺；有大量泡沫细胞和明显空洞形成以及大量炎症细胞浸润。NSCs组部分神经元细胞形态较规则,少数神经元可见细胞核及核仁,但部分神经元内尼氏小体较少,分布不均匀。E+NSCs组神经元细胞数量较多,形态基本正常,核仁清晰可见,尼氏小体较多,且分布均匀,神经纤维排列较规则。
5、透射电镜观察显示,SCI组髓鞘松散、破裂,少部分完全崩解,轴突固缩,部分轴突空泡变,线粒体含量较少且有肿胀；NSCs移植组髓鞘松散程度减轻,散在分布,仅有少量髓鞘破裂,轴突消失,其间有薄髓的再生髓鞘；E+NSCs组再生纤维增多,髓鞘松散程度明显减轻,部分髓鞘结构规整,排列有序,线粒体肿胀减轻,微管空泡样变较轻,可发现大量突触,薄髓的再生髓鞘明显增多。
6、免疫荧光染色检测脊髓损伤处的神经元、星形胶质细胞及少突胶质细胞数量随时间推移逐渐增加,E+NSCs组与其他各组之间均存在差异(P＜0.01)。
结论：
1、从11.5 d胎鼠的神经管后段分离、培养,可获得足量的NSCs,血清诱导能够分化为神经元和胶质细胞；
2、NSCs移植联合电针治疗能促进脊髓损伤大鼠后肢运动功能的恢复；
3、NSCs移植联合电针治疗能促进损伤处脱髓的轴突再髓鞘化和神经纤维再生；
4、NSCs移植联合电针治疗能促进细胞的分化,增加损伤脊髓神经元、胶质细胞的数量,改善损伤处的微环境,促进结构的修复。
Objective:
To explore the effect of neural stem cells transplantation in combination with electro-acupuncture treatment on repair of traumatic spinal cord injury rats.
Methods:
Experiment 1, The posterior segment of neural tube was dissected from SD embryonic 11.5d rats, cultured primarily in vitro and observed the growth state of cells under inverted phase contrast microscope. They were identified with immunocytochemistry for nestin which is the marker antigen of neural stem cell. Meanwhile, the differentiation was induced by 10% fetal bovine serum and cells were examined by immunocytochemical stain including neuron specific enolase (NSE), glial fibrillary acidic protein (GFAP), and myelin basic protein (MBP) under confocal laser scanning microscopy. Neural stem cells (made from the culturing cells of the second generation 7-8d) were marked by BrdU immunocytochemical stain, and marked cells were collect to reserve.
Experiment 2,54 adult female SD rats were randomly divided into three groups:spinal cord injury group (SCI), NSCs transplantation group, electro-acupuncture treatment with NSCs transplantation group (E+NSCs). SCI rat model was constructed by transecting spinal cord thoroughly. NSCs labeled with BrdU were adsorbed to the gelatin sponge and then transplanted into injured part of both NSCs and E+NSCs groups, yet normal saline into SCI group instead correspondingly. After transplantation, rats in E+NSCs group were given electro-acupuncture treatment at a fixed time every day. The motor functions of recovery of rat hind limb in each group were detected in 7d, 14d,28d, respectively, by methods of BBB score. The injury spinal cords were admitted to analyze morphological changes through general observation, light microscope and electron microscope respectively, and changes of neurons, astrocytes and oligodendrocytes keratinocyte were detected through immunofluorescence.
Results:
1. The cells derived from the posterior segment of neural tube of E11.5d rat were successfully isolated and cultured with good proliferation. The cultured cells were identified as NSCs by expressing specific antigen Nestin of NSCs and can be induced to differentiate to neurons, astrocytes and oligodendrocytes. The cells in neurosphere newly formed were proved BrdU-positive cells by immunofluorescence.
2. Difference between E+NSCs group and other groups respective were obviously observed by methods of BBB score of hind limb motor function over time (P<0.01). The recovery of animal limb function was significant.
3. It was observed that the color of the spinal cord in the injured site became darker as a result of scarring, the gelatin sponge migrated into the injured site was absorbed in various degree, the atrophy of the injury spinal cord close to the head atrophies was mildly while the part far from the head atrophies markedly. The gelatin sponge was absorbed in a better degree in both NSCs group and E+NSCs group and integrated nearly with the spinal cord tissue, and the spinal cord atrophied mildly. While the injury spinal cord of some rats in SCI group was metamorphic and necrotic with poor absorption of gelatin sponge.
4. Under light microscope, HE staining showed that the spinal cord injury zone in SCI group was incomplete after 28d and the nervous tissue was fragmentary. Large numbers of foam cells and marked cavities emerged and the injury area was intiltrated with many chronic inflammatory cells. In NSCs group, the morphological changes of some neurons was relatively regular, nucleus and nucleolus in the minority of neurons could be seen, but Nissl bodies within some neurons were fairly less and uneven. In E+NSCs group, the number of neurons increased obviously, the shape of neurons was close to normal, nucleolus were clearly visible, more Nissl bodies with even distribution, and the nerve fibers were regular.
5. Transmission Electron Microscope (TEM) showed:In SCI group, the myelin was loose, distorted and broken, with axonal shrinkage and some axonal vacuolate, mitochondrial swelling and there was less content. In NSCs group, the extent of myelin loose reduced and scattered, only a few myelin breakdown, and the axon disappeared and there were thin myelin regeneration of myelinated. In E+NSCs group, regenerated fiber increased, the extent of myelin loose reduced distinctly, the structure of myelin was clear and neat and the arrangement orderly, the swelling of mitochondria became lightened, vacuolar degeneration of microtubules got less, a lot of synapses were found, thin myelin sheath regeneration increased significantly.
6. The number of neurons, astrocytes and oligodendrocytes keratinocyte in injured site gradually increased over time by immunofluorescence stain. Difference between E+NSCs group and other groups respective was obvious (P<0.01).
Conclusion:
1. NSCs derived from the posterior segment of neural tube of E11.5d rat were successfully isolated, cultured to sufficient quantities and induced to differentiate to neurons and glial cells by serum.
2. NSCs transplantation with electro-acupuncture treatment increased the recovery of hind limb motor function of spinal cord injury in rats
3. The treatment of NSCs transplanted with electro-acupuncture promoted demyelination axons in injured site re-myelination and nerve fibers regeneration.
4. NSCs transplantation with electro-acupuncture treatment promoted cells differentiation, increased the number of neurons and glial cells in injured spinal cord, improved the micro-environment of the damaged site and promoted structural repair.
引文
[1]Stevens RD, Bhardwaj A, Kirsh JR, et al. Critical care and perioperative mamagement in traumatic spinal cord injury[J]. J Neurosurg Anesthesiol,2003,15(3):215-229.
[2]Dobkin BH, Havton LA. Basic advances and new avenues in therapy of spinal cord injury [J]. Annu Rev Med,2004,55:255-282.
[3]Obermair FJ, Schroter A, Thallmair M. Endogenous neural progenitor cells as therapeutic target after spinal cord injury[J]. Physiology (Bethesda),2008,23:296-304.
[4]Murray M, Fischer I. Transplantation and gene therapy:combined approaches for repair of spinal cord injury[J]. Neuroscientist,2001,7(1):28-41.
[5]Horiguchi S, Takahashi J, Kishi Y. Neural precursor cells derived from human embryonic brain retain regional specificity[J]. J Neurosci Res,2004,75(6):817-824.
[6]Gao J, Coggeshall RE, Tarasenko YI, et al. Human neural stem cell-derived cholinergic neurons innervate muscle in motoneuron deficient adult rats[J]. Neuroscience,2005,131:257-262.
[7]孔尧其,任兴生,沈斌.瘫痪病的针灸治疗[M].北京：中医古籍出版社,1993.80-88.
[8]许健鹏,王明久,刘学茹,等.以督脉电针为主治疗脊髓损伤80例的临床观察[J].针灸临床杂志,1994,10(6)：13-15.
[9]陈育春,齐伟力,孔抗美.电针干预急性脊髓损伤大鼠神经生长因子及其受体的表达变化[J].中国临床康复,2006,10(11)：129-131.
[10]张志英,党瑞山,张传森,等.督脉电针上调脊髓损伤早期大鼠Bcl-2mRNA及蛋白的表达[J].中国临床康复,2006,10(31)：185-187.
[11]李连欣,张进禄,周东生,等.电针对脊髓创伤后脊髓组织中氧自由基含量影响及意义[J].中国临床康复,2004,8(5)：912-913.
[12]杨成,刘同慎,吴洪华,等.电针对脊髓损伤后少突胶质细胞再髓鞘化的影响[J].解剖学杂志,2005,28(4)：39 1-394.
[13]杨成,李滨,刘同慎,等.电针对脊髓损伤后星形胶质细胞增生的影响[J].中国针灸,2005,25(8)：569-572.
[14]杨成,徐宁,仇继胜,等.电针对脊髓损伤星形胶质细胞增生及其NGF表达的影响[J].中国组织化学与细胞化学杂志,2005,14(5)：562-565.
[15]Reynolds BA, weiss S. Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell[J]. Dev Biol,1996,175 (1):1-13.
[16]Reynolds BA, Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system[J]. Science,1992,255(5052):1707-1710.
[17]Doetsch F, Scharf C. Challenges for brain repair:Insights from adult neurogenesis in birds and mammals[J]. Brain Behav Evol,2001,58(5):306-322.
[18]Williams PL, Bannister LH, Berry M M, et al. Gray.sAnatomy[M].38thed. Edinburth: Churchill Living stone,1995:217-20.杨琳,高英茂,主译.格氏解剖学[M].第38版.沈阳：辽宁教育出版社,1999：217-220.
[19]Hayashi T, Uchida K, Mine Y, et al. Feasibility of using early mesencephalic neural plate for intracerebral grafting[J]. Cell Transplant,2002, 11(5):465-470.
[20]Uchida K, Okano H,Hayashi T, et al. Grafted swine neuroepithelial stem cells can form myelinated axons and both efferent and afferent synapses with xenogeneic rat neurons[J]. J Neurosci Res,2003,72(6):661-669.
[21]Ghosh A, Greenberg ME. Distinct roles for bFGF and NT-3 in the regulation of cortical neurogenesis[J]. Neuron,1995,15(1):89-103.
[22]Bogler O, Wren D, Barnett SC, et al. Cooperation between two growth factors promotes extended self-renewal and inhibits differentiation of oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells[J]. Neuron,1990,87(16):6368-6372.
[23]Whittemore SR, Morasutti DJ, Walters WM, et al. Mitogen and substrate differentially affect the lineage restriction of adult rat subventricular zone neural precursor cell populations [J]. Exp Cell Res,1999,252(1):75-95.
[24]李峰,刘玉光,朱树干,等.机械分离与胰酶消化分离对长期培养的神经干细胞神经发生能力的影响[J].山东医药,2008,48(21)：22-25.
[25]Lendahl U, Zimmerman LB, McKay RD. CNS stem cells express a new class of intermediate filament protein[J]. Cell,1990,60(4):585-595.
[26]Chalfie M, Tu Y, Euskirchen G, et al. Green fluorescent protein as a marker for gene expression [J]. Science,1994,263(5148):802-805.
[27]Gerrard L, Zhao D, Clark AJ, et al. Stably transfected human embryonic stem cell clones express OCT4-specific green fluorescent protein and maintain self-renewal and pluripotency[J]. Stem Cells,2005,23(1):124-133.
[28]陈秉耀,游思维,王颖.大鼠脊髓全横断术后护理措施[J].实验动物科学与管理,2000,17(4)：55-56.
[29]Basso DM, Beattie MS, Bresnahan JC. A sensitive and reliable locomotor rating scale for open field testing in rats[J]. J Neurotrauma,1995,12(1):1-21.
[30]Young W. Role of calcium in central nervous system injuries [J]. J Neurotrauma,1992, (Suppl 1):S9-25.
[31]Faden AL, Halt P. Platelet-activating factor reduces spinal cord blood flow and causes behavioral deficits after intrathecal administration in rats through a specific receptor
mechanism[J]. J Pharmacol Exp Ther,1992,261(3):1064-1670.
[32]Moriya T, Hassan AZ, Young W, et al. Dynamics of extracellular calcium activity following contusion of the rat spinal cord[J]. J Neurotrauma,1994,11(3):255-263.
[33]Siesjo BK. Historical overview. Calcium, ischemia, and death of brain cells[J]. Ann N Y Acad Sci,1988,522:638-661.
[34]Woerly S. Restorative surgery of the central nervous system by means of tissue engineering using NeuroGel implants[J]. Neurosurg Rev,2000,23(2):59-79.
[35]Nakamura M, Houghtling RA, MacArthur L, et al. Differences in cytokine gene exp ression p rofile between acute and secondary injury in adult rat sp inal cord. Exp Neurol,2003, 184(1):313-325.
[36]陈通,夏鹤春.继发性脊髓损伤病理生理机制[J].中国煤炭工业医学杂志,2008,11(4)：577-581.
[37]Zurita M, Vaquero J, Oya S. Effects of dexamethasone on apoptosis-related cell death after spinal cord injury[J]. J Neurosurg,2002,96(1 Suppl):83-89.
[38]杨东波,金立,李永利,等.神经干细胞移植治疗大鼠局灶性脑缺血：移植部位与移植时间的比较[J].中国临床康复,2006,10(17)：30-33.
[39]Guo JS, Zeng YS, Li HB, et al. Cotransplant of neural stem cells and NT-3 gene modified Schwann cells promote the recovery of transected spinal cord injury[J]. Spinal Cord,2007, 45(1):15-24.
[40]Neuhuber B, Timothy Himes B, Shumsky JS. Axon growth and recovery of function supported by human bone marrow stromal cells in the injured spinal cord exhibit donor variations [J]. Brain Res,2005,1035(1):73-85.
[41]Ogawa Y, Sawamoto K, Miyata T, et al. Transplantation of in vitro-expanded fetal neural progenitor cells results in neurogenesis and functional recovery after spinal cord contusion injury in adult rats[J]. J Neurosci Res,2002,69(6):925-933.
[42]Okano H, Ogawa Y, Nakamura M, et al. Transp lantation of neural stem cells into the sp inal cord after injury. Semin Cell Dev Biol,2003,14(3):191-198.
[43]Jing WL, Yan FX, Zuo YZ. Comparison of various mesenchymal stem cell transplantation pathways for treatment of spinal cord injury in rats [J]. Journal of CRTER,2008,12(51):10045-10048.
[44]Pranq p, Muller R, Eljaouhari A, et al. The promotion of oriented axonal regrowth in the
injured spinal cord by alginatebased anisotropic capillary hydrogels[J]. Biomaterials, 2006,27(19):3560-3569.
[45]Ahmed TA, Dare EV, Hincke M. Fibrin:a versatile scaffold for tissue engineering applications[J]. Tissue Eng Part B Rev,2008,14(2):199-215.
[46]Li X, Yang Z, Zhang A. Repair of thoracic spinal cord injury by chitosan tube implantation in adult rats[J]. Biomaterials,2009,30(6):1121-1132.
[47]Teng YD, Lavik EB, Qu X, et al. Functional recovery following traumatic spinal cord injury mediated by a unique polymer scaffold seeded with neural stem cells. PNAS,2002, 99:3024-3029.
[48]Pluchino S, Zanotti L, Deleidi M,et al. Neural stem cells and their use as therapeutic tool in neurological disorders. Brain Research Reviews 2005;48(2):211-219.
[49]McDonald JW, Liu XZ, Qu Y, et al. Transplanted embryonic stem cells survive, differentiate and promote recovery in injured rat spinal cord[J]. Nat Med,1999,5(12):1410-1412.
[50]Nakamura M, Okano H, Toyama Y, et al. Transplantation of embryonic spinal cord-derived neurospheres support growth of supraspinal projections and functional recovery after spinal cord injury in the neonatal rat[J]. J Neurosci Res,2005,81(4):457-468.
[51]Silver J,Miller JH.Regeneration beyond the glial scar[J].Nat Rav Neurosci,2004,5(2):146-156.
[52]Derksen PW, Tjin E, Meijer HP, et al. Illegitimate WNT signaling promotes proliferation of multiple myeloma cells[J]. Proc Natl Acad Sci USA,2004,101(16):6122-6127.
[53]Norrbrink Budh C, Lundeberg T. Non-pharmacological pain-relieving therapies in individuals with spinal cord injury:a patient perspective[J]. Complement Ther Med,2004,12(4):189-197.
[54]李祖剑,葛林宝,王美娟,等.电针对中度脊髓损伤大鼠模型运动功能和红核神经元的影响[J].上海针灸杂志,2005,24(3)：37-39.
[55]李晓宁,田旭升,刘芳.电针对大鼠脊髓损伤后细胞凋亡相关基因Caspase-9的研究[J].中医药信息,2009,26(1)：61-63.
[56]孙为增,王新家,林丽艳.电针对急性脊髓损伤大鼠白细胞介素-1β表达变化的影响[J].中国康复理论与实践,2009,15(3)：208-210.
[57]杜旭,王瑞辉.电针对脊髓损伤大鼠神经生长因子和神经功能的影响[J].针灸临床杂志,2009,25(20)：42-44.
[58]王瑞辉,席莉.电针对实验性脊髓损伤大鼠胶质纤维酸性蛋白影响的实验研究[J].陕西中医,2008,29(11)：1556-1557.
[1]Gage FH. Mammalian neural stem cell[J]. Science,2000,287(5457):1433-1438.
[2]Doetsch F, Scharf C. Challenges for brain repair:Insights from adult neurogenesis in birds andmammals[J]. Brain Behav Evol,2001,58(5):306-322.
[3]Ostenfeld T, Caldwell MA, Prowse KR, et al. Human neural precursor cells express low levels of telomerase in vitro and show diminishing cell proliferation with extensive axonal outgrowth following transplantation[J]. Exp Neurol,2000,164(1):215-226.
[4]Lu P, Jones LL, Snyder EY, et al. Neural stem cells constitutively secrete neurotrophic factors and promote extensive host axonal growth after spinal cord injury[J]. Exp Neurol,2003,181(2):115-129.
[5]王岩峰,吕刚,李雷,等.神经干细胞移植对大鼠脊髓损伤后胶质细胞源性神经营养因子与生长相关蛋白43基因表达的影响[J].中国修复重建外科杂志,2005,19(6)：416-419.
[6]Adam L, Johan W, Eva L, et al. Neurotrophic properties of olfactory ensheathing glia[J]. Exp Neurol,2003,180: 167-171.
[7]Yin Guodong, Tang Xun, Lin Yueqiu, et al. Recovery of adult rat spinal cord injury by co-transplantion of human embryonic olfactory ensheathing cells and neuro stem cells[J]. Chinese Journal of Rehabilitation Medicine,2006, 21(8):680-682.
[8]Kojun T, Hirchito FT, Akira T, et al. Basic behavior migratory Schwann cells in peripheral nerve regeneration[J]. Exp Neurol,1997,137(2):301-308.
[9]Ding Ying, Zeng Yuanshan,Wu Lizhi, et al. Effects of Schwann cells on survival and differentiation of neural stem cells transplanted into injured spinal cord[J]. Acta anatomica sinica.2003,34(6):589-593.
[10]Li J, Sun CR, Zhang H, et al. Induction of functional recovery by co-transplantation of neural stem cells and Schwann cells in a rat spinal cord contusion injury model[J]. Biomed Environ Sci.2007,20(3):242-249.
[11]Guo JS, Zeng YS, Li HB, et al. Cotransplant of neural stem cells and NT-3 gene modified Schwann cells promote the recovery of transected spinal cord injury[J]. Spinal Cord.2007,45(1):15-24.
[12]李志刚,付平,刘书坤,等.电针对大鼠急性脊髓损伤保护作用的机理研究[J].中国中医基础医学杂志,2005,11(11)：856-858.
[13]李连欣,张进禄,周东生,等.电针对脊髓创伤后脊髓组织中央自由基含量影响及意义[J].中国临床康复,2004,8(5)：912-913.
[14]朱政.电针对大鼠脊髓损伤后层粘连蛋白表达影响[J].中国中西医结合杂志,2002,22(7)：525-527.
[15]王新家,孔抗美,齐伟力.针刺影响慢性脊髓损伤神经递质的表达[J].中国正骨,2002,14(6)：325-327.
[16]张志英,余安胜.电针对脊髓损伤早期bcl-2mRNA及蛋白表达的影响[J].中国针灸,2003,23(8)：473-476.
[17]曾园山,李晓滨,郭家松,等.督脉电针与神经干细胞移植在脊髓损伤修复中的作用[J].中国康复医学杂志,2005,20(6)：468-469.
[18]崔晓军,李伊为,陈东风,等.督脉电针对脊髓损伤大鼠神经干细胞的作用[J].解剖学研究,2002,24(3)：180-183.
[19]李晓滨,曾园山,陈玉玲,等.督脉电针与神经干细胞移植对脊髓全横断大鼠后肢运动功能恢复的影响[J].解剖学报,2004,35(6)：582-588.
[20]Kamei N, Tanaka N, Oishi Y. BDNF, NT-3, and NGF released from transplanted neural progenitor cells promote corticospinal axon growth in organotypic cocultures[J]. Spine.2007,32(12):1272-1278.
[21]Qin DX, Zou XL, Luo W, et al. Expression of some neurotrophins in the spinal motoneurons after cord hemisection in adult rats[J]. Neurosci Lett.2006,410(3):222-227.
[22]Liu Y, Himes BT, Solowska J, et al. Intraspinal delivery of neurotrophin-3 using neural stem cells genetically modified by recombinant retrovirus[J]. Exp Neurol,1999,158(1):9-26.
[23]Vicario-Abejon C, Collin C, Tsoulfas P, et al. Hippocampus stem cells differentiate into excitatory and inhibitory neurons[J]. European Journal of Neuroscience,2000,12(2):677-688.
[24]Zhang L, Gu S, Zhao C, et al. Combined treatment of neurotrophin-3 gene and neural stem cells is propitious to functional recovery after spinal cord injury[J]. Cell Transplant.2007,16(5):475-481.
[25]Chow SY,Moul J, Tobias CA, et al. Characterization and intraspinal grafting of EGF/bFGF-dependent neurospheres derived from embryonic rat spinal cord[J].Brain Res,2000,874 (2):87-106
[26]Yan Jing-feng,Yue chang-bo. Effect of Implantation of Neural Stem Cell Modified by BDNF on Apoptosis of Neur al Cells after Spinal Cord Injury[J]. Chin J Clin Neurosurg,2006,11(9):547-560.
[2]Dobkin BH, Havton LA. Basic advances and new avenues in therapy of spinal cord injury [J]. Annu Rev Med,2004,55:255-282.
[3]Obermair FJ, Schroter A, Thallmair M. Endogenous neural progenitor cells as therapeutic target after spinal cord injury[J]. Physiology (Bethesda),2008,23:296-304.
[4]Murray M, Fischer I. Transplantation and gene therapy:combined approaches for repair of spinal cord injury[J]. Neuroscientist,2001,7(1):28-41.
[5]Horiguchi S, Takahashi J, Kishi Y. Neural precursor cells derived from human embryonic brain retain regional specificity[J]. J Neurosci Res,2004,75(6):817-824.
[6]Gao J, Coggeshall RE, Tarasenko YI, et al. Human neural stem cell-derived cholinergic neurons innervate muscle in motoneuron deficient adult rats[J]. Neuroscience,2005,131:257-262.
[7]孔尧其,任兴生,沈斌.瘫痪病的针灸治疗[M].北京：中医古籍出版社,1993.80-88.
[8]许健鹏,王明久,刘学茹,等.以督脉电针为主治疗脊髓损伤80例的临床观察[J].针灸临床杂志,1994,10(6)：13-15.
[9]陈育春,齐伟力,孔抗美.电针干预急性脊髓损伤大鼠神经生长因子及其受体的表达变化[J].中国临床康复,2006,10(11)：129-131.
[10]张志英,党瑞山,张传森,等.督脉电针上调脊髓损伤早期大鼠Bcl-2mRNA及蛋白的表达[J].中国临床康复,2006,10(31)：185-187.
[11]李连欣,张进禄,周东生,等.电针对脊髓创伤后脊髓组织中氧自由基含量影响及意义[J].中国临床康复,2004,8(5)：912-913.
[12]杨成,刘同慎,吴洪华,等.电针对脊髓损伤后少突胶质细胞再髓鞘化的影响[J].解剖学杂志,2005,28(4)：39 1-394.
[13]杨成,李滨,刘同慎,等.电针对脊髓损伤后星形胶质细胞增生的影响[J].中国针灸,2005,25(8)：569-572.
[14]杨成,徐宁,仇继胜,等.电针对脊髓损伤星形胶质细胞增生及其NGF表达的影响[J].中国组织化学与细胞化学杂志,2005,14(5)：562-565.
[15]Reynolds BA, weiss S. Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell[J]. Dev Biol,1996,175 (1):1-13.
[16]Reynolds BA, Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system[J]. Science,1992,255(5052):1707-1710.
[17]Doetsch F, Scharf C. Challenges for brain repair:Insights from adult neurogenesis in birds and mammals[J]. Brain Behav Evol,2001,58(5):306-322.
[18]Williams PL, Bannister LH, Berry M M, et al. Gray.sAnatomy[M].38thed. Edinburth: Churchill Living stone,1995:217-20.杨琳,高英茂,主译.格氏解剖学[M].第38版.沈阳：辽宁教育出版社,1999：217-220.
[19]Hayashi T, Uchida K, Mine Y, et al. Feasibility of using early mesencephalic neural plate for intracerebral grafting[J]. Cell Transplant,2002, 11(5):465-470.
[20]Uchida K, Okano H,Hayashi T, et al. Grafted swine neuroepithelial stem cells can form myelinated axons and both efferent and afferent synapses with xenogeneic rat neurons[J]. J Neurosci Res,2003,72(6):661-669.
[21]Ghosh A, Greenberg ME. Distinct roles for bFGF and NT-3 in the regulation of cortical neurogenesis[J]. Neuron,1995,15(1):89-103.
[22]Bogler O, Wren D, Barnett SC, et al. Cooperation between two growth factors promotes extended self-renewal and inhibits differentiation of oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells[J]. Neuron,1990,87(16):6368-6372.
[23]Whittemore SR, Morasutti DJ, Walters WM, et al. Mitogen and substrate differentially affect the lineage restriction of adult rat subventricular zone neural precursor cell populations [J]. Exp Cell Res,1999,252(1):75-95.
[24]李峰,刘玉光,朱树干,等.机械分离与胰酶消化分离对长期培养的神经干细胞神经发生能力的影响[J].山东医药,2008,48(21)：22-25.
[25]Lendahl U, Zimmerman LB, McKay RD. CNS stem cells express a new class of intermediate filament protein[J]. Cell,1990,60(4):585-595.
[26]Chalfie M, Tu Y, Euskirchen G, et al. Green fluorescent protein as a marker for gene expression [J]. Science,1994,263(5148):802-805.
[27]Gerrard L, Zhao D, Clark AJ, et al. Stably transfected human embryonic stem cell clones express OCT4-specific green fluorescent protein and maintain self-renewal and pluripotency[J]. Stem Cells,2005,23(1):124-133.
[28]陈秉耀,游思维,王颖.大鼠脊髓全横断术后护理措施[J].实验动物科学与管理,2000,17(4)：55-56.
[29]Basso DM, Beattie MS, Bresnahan JC. A sensitive and reliable locomotor rating scale for open field testing in rats[J]. J Neurotrauma,1995,12(1):1-21.
[30]Young W. Role of calcium in central nervous system injuries [J]. J Neurotrauma,1992, (Suppl 1):S9-25.
[31]Faden AL, Halt P. Platelet-activating factor reduces spinal cord blood flow and causes behavioral deficits after intrathecal administration in rats through a specific receptor
mechanism[J]. J Pharmacol Exp Ther,1992,261(3):1064-1670.
[32]Moriya T, Hassan AZ, Young W, et al. Dynamics of extracellular calcium activity following contusion of the rat spinal cord[J]. J Neurotrauma,1994,11(3):255-263.
[33]Siesjo BK. Historical overview. Calcium, ischemia, and death of brain cells[J]. Ann N Y Acad Sci,1988,522:638-661.
[34]Woerly S. Restorative surgery of the central nervous system by means of tissue engineering using NeuroGel implants[J]. Neurosurg Rev,2000,23(2):59-79.
[35]Nakamura M, Houghtling RA, MacArthur L, et al. Differences in cytokine gene exp ression p rofile between acute and secondary injury in adult rat sp inal cord. Exp Neurol,2003, 184(1):313-325.
[36]陈通,夏鹤春.继发性脊髓损伤病理生理机制[J].中国煤炭工业医学杂志,2008,11(4)：577-581.
[37]Zurita M, Vaquero J, Oya S. Effects of dexamethasone on apoptosis-related cell death after spinal cord injury[J]. J Neurosurg,2002,96(1 Suppl):83-89.
[38]杨东波,金立,李永利,等.神经干细胞移植治疗大鼠局灶性脑缺血：移植部位与移植时间的比较[J].中国临床康复,2006,10(17)：30-33.
[39]Guo JS, Zeng YS, Li HB, et al. Cotransplant of neural stem cells and NT-3 gene modified Schwann cells promote the recovery of transected spinal cord injury[J]. Spinal Cord,2007, 45(1):15-24.
[40]Neuhuber B, Timothy Himes B, Shumsky JS. Axon growth and recovery of function supported by human bone marrow stromal cells in the injured spinal cord exhibit donor variations [J]. Brain Res,2005,1035(1):73-85.
[41]Ogawa Y, Sawamoto K, Miyata T, et al. Transplantation of in vitro-expanded fetal neural progenitor cells results in neurogenesis and functional recovery after spinal cord contusion injury in adult rats[J]. J Neurosci Res,2002,69(6):925-933.
[42]Okano H, Ogawa Y, Nakamura M, et al. Transp lantation of neural stem cells into the sp inal cord after injury. Semin Cell Dev Biol,2003,14(3):191-198.
[43]Jing WL, Yan FX, Zuo YZ. Comparison of various mesenchymal stem cell transplantation pathways for treatment of spinal cord injury in rats [J]. Journal of CRTER,2008,12(51):10045-10048.
[44]Pranq p, Muller R, Eljaouhari A, et al. The promotion of oriented axonal regrowth in the
injured spinal cord by alginatebased anisotropic capillary hydrogels[J]. Biomaterials, 2006,27(19):3560-3569.
[45]Ahmed TA, Dare EV, Hincke M. Fibrin:a versatile scaffold for tissue engineering applications[J]. Tissue Eng Part B Rev,2008,14(2):199-215.
[46]Li X, Yang Z, Zhang A. Repair of thoracic spinal cord injury by chitosan tube implantation in adult rats[J]. Biomaterials,2009,30(6):1121-1132.
[47]Teng YD, Lavik EB, Qu X, et al. Functional recovery following traumatic spinal cord injury mediated by a unique polymer scaffold seeded with neural stem cells. PNAS,2002, 99:3024-3029.
[48]Pluchino S, Zanotti L, Deleidi M,et al. Neural stem cells and their use as therapeutic tool in neurological disorders. Brain Research Reviews 2005;48(2):211-219.
[49]McDonald JW, Liu XZ, Qu Y, et al. Transplanted embryonic stem cells survive, differentiate and promote recovery in injured rat spinal cord[J]. Nat Med,1999,5(12):1410-1412.
[50]Nakamura M, Okano H, Toyama Y, et al. Transplantation of embryonic spinal cord-derived neurospheres support growth of supraspinal projections and functional recovery after spinal cord injury in the neonatal rat[J]. J Neurosci Res,2005,81(4):457-468.
[51]Silver J,Miller JH.Regeneration beyond the glial scar[J].Nat Rav Neurosci,2004,5(2):146-156.
[52]Derksen PW, Tjin E, Meijer HP, et al. Illegitimate WNT signaling promotes proliferation of multiple myeloma cells[J]. Proc Natl Acad Sci USA,2004,101(16):6122-6127.
[53]Norrbrink Budh C, Lundeberg T. Non-pharmacological pain-relieving therapies in individuals with spinal cord injury:a patient perspective[J]. Complement Ther Med,2004,12(4):189-197.
[54]李祖剑,葛林宝,王美娟,等.电针对中度脊髓损伤大鼠模型运动功能和红核神经元的影响[J].上海针灸杂志,2005,24(3)：37-39.
[55]李晓宁,田旭升,刘芳.电针对大鼠脊髓损伤后细胞凋亡相关基因Caspase-9的研究[J].中医药信息,2009,26(1)：61-63.
[56]孙为增,王新家,林丽艳.电针对急性脊髓损伤大鼠白细胞介素-1β表达变化的影响[J].中国康复理论与实践,2009,15(3)：208-210.
[57]杜旭,王瑞辉.电针对脊髓损伤大鼠神经生长因子和神经功能的影响[J].针灸临床杂志,2009,25(20)：42-44.
[58]王瑞辉,席莉.电针对实验性脊髓损伤大鼠胶质纤维酸性蛋白影响的实验研究[J].陕西中医,2008,29(11)：1556-1557.
[1]Gage FH. Mammalian neural stem cell[J]. Science,2000,287(5457):1433-1438.
[2]Doetsch F, Scharf C. Challenges for brain repair:Insights from adult neurogenesis in birds andmammals[J]. Brain Behav Evol,2001,58(5):306-322.
[3]Ostenfeld T, Caldwell MA, Prowse KR, et al. Human neural precursor cells express low levels of telomerase in vitro and show diminishing cell proliferation with extensive axonal outgrowth following transplantation[J]. Exp Neurol,2000,164(1):215-226.
[4]Lu P, Jones LL, Snyder EY, et al. Neural stem cells constitutively secrete neurotrophic factors and promote extensive host axonal growth after spinal cord injury[J]. Exp Neurol,2003,181(2):115-129.
[5]王岩峰,吕刚,李雷,等.神经干细胞移植对大鼠脊髓损伤后胶质细胞源性神经营养因子与生长相关蛋白43基因表达的影响[J].中国修复重建外科杂志,2005,19(6)：416-419.
[6]Adam L, Johan W, Eva L, et al. Neurotrophic properties of olfactory ensheathing glia[J]. Exp Neurol,2003,180: 167-171.
[7]Yin Guodong, Tang Xun, Lin Yueqiu, et al. Recovery of adult rat spinal cord injury by co-transplantion of human embryonic olfactory ensheathing cells and neuro stem cells[J]. Chinese Journal of Rehabilitation Medicine,2006, 21(8):680-682.
[8]Kojun T, Hirchito FT, Akira T, et al. Basic behavior migratory Schwann cells in peripheral nerve regeneration[J]. Exp Neurol,1997,137(2):301-308.
[9]Ding Ying, Zeng Yuanshan,Wu Lizhi, et al. Effects of Schwann cells on survival and differentiation of neural stem cells transplanted into injured spinal cord[J]. Acta anatomica sinica.2003,34(6):589-593.
[10]Li J, Sun CR, Zhang H, et al. Induction of functional recovery by co-transplantation of neural stem cells and Schwann cells in a rat spinal cord contusion injury model[J]. Biomed Environ Sci.2007,20(3):242-249.
[11]Guo JS, Zeng YS, Li HB, et al. Cotransplant of neural stem cells and NT-3 gene modified Schwann cells promote the recovery of transected spinal cord injury[J]. Spinal Cord.2007,45(1):15-24.
[12]李志刚,付平,刘书坤,等.电针对大鼠急性脊髓损伤保护作用的机理研究[J].中国中医基础医学杂志,2005,11(11)：856-858.
[13]李连欣,张进禄,周东生,等.电针对脊髓创伤后脊髓组织中央自由基含量影响及意义[J].中国临床康复,2004,8(5)：912-913.
[14]朱政.电针对大鼠脊髓损伤后层粘连蛋白表达影响[J].中国中西医结合杂志,2002,22(7)：525-527.
[15]王新家,孔抗美,齐伟力.针刺影响慢性脊髓损伤神经递质的表达[J].中国正骨,2002,14(6)：325-327.
[16]张志英,余安胜.电针对脊髓损伤早期bcl-2mRNA及蛋白表达的影响[J].中国针灸,2003,23(8)：473-476.
[17]曾园山,李晓滨,郭家松,等.督脉电针与神经干细胞移植在脊髓损伤修复中的作用[J].中国康复医学杂志,2005,20(6)：468-469.
[18]崔晓军,李伊为,陈东风,等.督脉电针对脊髓损伤大鼠神经干细胞的作用[J].解剖学研究,2002,24(3)：180-183.
[19]李晓滨,曾园山,陈玉玲,等.督脉电针与神经干细胞移植对脊髓全横断大鼠后肢运动功能恢复的影响[J].解剖学报,2004,35(6)：582-588.
[20]Kamei N, Tanaka N, Oishi Y. BDNF, NT-3, and NGF released from transplanted neural progenitor cells promote corticospinal axon growth in organotypic cocultures[J]. Spine.2007,32(12):1272-1278.
[21]Qin DX, Zou XL, Luo W, et al. Expression of some neurotrophins in the spinal motoneurons after cord hemisection in adult rats[J]. Neurosci Lett.2006,410(3):222-227.
[22]Liu Y, Himes BT, Solowska J, et al. Intraspinal delivery of neurotrophin-3 using neural stem cells genetically modified by recombinant retrovirus[J]. Exp Neurol,1999,158(1):9-26.
[23]Vicario-Abejon C, Collin C, Tsoulfas P, et al. Hippocampus stem cells differentiate into excitatory and inhibitory neurons[J]. European Journal of Neuroscience,2000,12(2):677-688.
[24]Zhang L, Gu S, Zhao C, et al. Combined treatment of neurotrophin-3 gene and neural stem cells is propitious to functional recovery after spinal cord injury[J]. Cell Transplant.2007,16(5):475-481.
[25]Chow SY,Moul J, Tobias CA, et al. Characterization and intraspinal grafting of EGF/bFGF-dependent neurospheres derived from embryonic rat spinal cord[J].Brain Res,2000,874 (2):87-106
[26]Yan Jing-feng,Yue chang-bo. Effect of Implantation of Neural Stem Cell Modified by BDNF on Apoptosis of Neur al Cells after Spinal Cord Injury[J]. Chin J Clin Neurosurg,2006,11(9):547-560.