急性运动轴索型吉兰—巴雷综合征患者血清对体外培养脊髓运动神经元的影响
|
摘要
吉兰－巴雷综合征（Guillain-Barre syndrome,GBS）是周围神经系统中引起弛缓性瘫痪的常见疾病之一，临床表现为对称性进行性瘫痪，症状持续一段时间后可逐渐恢复。GBS是指急性炎性脱髓鞘性多发性神经病（acute inflammatory demyelinating polyneuropathies, AIDP），其病理特征是主要累及周围神经脊髓前根、后根及其远端神经纤维的节段性脱髓鞘，严重者伴继发性轴索变性，病灶中可以有大量炎性单核细胞浸润。电生理学特征为周围神经传导速度明显减慢，F波异常。近年来发现有一类临床表现与AIDP相似的GBS患者，病理改变仅累及脊髓前根和周围运动神经，以轴索变性为主，未见明显的脱髓鞘，且病灶中少量或无炎性单核细胞浸润；电生理检查发现感觉、运动神经传导速度正常，但运动诱发波幅明显降低，有学者将之称为急性运动性轴索型吉兰－巴雷综合征，即AMAN(acute motor axonal neuropathy)。现在认为AMAN是GBS的一个新亚型。与AIDP相比，临床症状相对较重，可累及呼吸肌，部分遗留有后遗症，严重者引起死亡。目前还不明确AMAN的病因和发病机制。有证据表明AMAN与空肠弯曲菌（campylobacter jejuni, Cj）的前驱感染有密切关系，是周围神经系统的自身免疫性疾病。国内李春岩等对AMAN进行了一系列研究，在AMAN病人粪便中分离出致病Cj菌株，经美国疾病控制与预防中心鉴定为PennerO:19型，随后应用PennerO:19型Cj菌株及其脂多糖诱发出动物模型，并证实AMAN病人血清中存在抗PennerO:19型Cj及其脂多糖抗体。另外，一些（不是所有的）病人经血浆交换或／和静脉点滴免疫球蛋白治疗后有效，这也说明病人的循环血清中存在某些致病成分，它（们）对AMAN的发病及治疗均有重要的意义。虽然AMAN的发病机制尚不清楚，但免疫机制（包括细胞免疫和体液免疫）在该病发生、发展过程中起着至关重要的作用。有研究表明，在AMAN的病理中，发现神经纤维的郎飞结有补体存在，轴索周围间隙有补体活
化标记物的表达，电镜下可发现有巨噬细胞包绕等。为了进一步研究AMAN的病因和发病机制，实验中建立并优化了体外胚胎大鼠脊髓前角运动神经元的原代培养体系。这种体外脊髓神经元培养中，缺乏在体实验研究中难以避免的淋巴细胞、单核巨噬细胞以及补体成分等，这对研究免疫系统中免疫机制参与运动神经元轴索的损害提供了便利条件；除此之外，实验中培养的胚胎大鼠脊髓前角运动神经元发出的轴突样突起的外面没有髓鞘包绕，可直接观察到致病因素对轴索损害的动态变化，为阐明AMAN的致病机制提供一定的理论基础。
神经元作为一种高度分化的细胞，形态上有胞体胞突之分。在单细胞培养中，相对于机体的其他细胞，在体外难以存活和生长，尤其是脊髓神经元，作为下运动神经元，对营养要求极高，对环境变化也极其敏感。因此，在实验的前两部分中，首先进行体外脊髓运动神经元的分离与鉴定，建立体外原代培养体系。为了优化培养方法，我们观察了不同包被基质和神经营养因子等对培养脊髓运动神经元生长状况的影响，寻找较适合神经元存活并利于以后实验研究的条件。第三部分是观察AMAN病人血清对培养脊髓运动神经元的作用。下面简述这三部分。
一、不同生长底物对体外培养脊髓运动神经元生长的影响
目的：首先建立脊髓运动神经元的体外培养，并对分离的细胞进行鉴定，以确定为脊髓运动神经元；在此基础上，观察了不同溶剂溶解稀释的多聚赖氨酸（poly-l-lysine, PLL）对脊髓神经元体外生长的影响，确定一种较好的方法以便进行以后的实验；随后，选取多种包被底物如PLL、Ⅰ型胶原、层粘连蛋白(laminin, LN)以及PLL和LN联合等进行包被，观察不同底物对胚胎大鼠脊髓运动神经元在体外存活生长的影响。
方法：在SD大鼠怀孕15天时，无菌条件下取出胚胎，解剖显微镜下取脊髓，取其腹侧组织切成小块，消化后制成单细胞悬液，差速贴壁以去除成纤维细胞，置于CO2培养箱中培养。培养细胞经神经元特异性烯醇化酶（NSE）抗体和抗神经丝单克隆抗体－SMI-32的免疫组织化学染色，对培养细胞进行鉴定。多聚赖氨酸分别用蒸馏水、磷酸盐缓冲液（PBS）、两种浓度的硼酸溶液溶解后对培养板进行包被，
接种制备的细胞悬液进行培养观察。不同的包被底物选取PLL、Ⅰ型胶原、LN、PLL联合LN等几种进行包被，对照组以蒸馏水代替。观察几种方法中培养神经元的存活数目、神经元突起的平均长度以及神经元的生长分布等状况。
结果：本实验建立的细胞培养体系为脊髓运动神经元的原代分散细胞培养，NSE抗体染色显示神经元的纯度达90％以上，SMI-32抗体染色能很好的显示脊髓运动神经元的形态：胞体大，为多极性，具有单个长的轴索样突起；以大的SMI-32阳性细胞进行计数，表明脊髓运动神经元占培养细胞总数的70％以上；神经元体外培养必须要求包被底物，否则将难以存活，对照组中细胞在24～36小时后死亡；当培养板包被底物后，神经元贴壁良好，部分细胞在短时间内长出突起，初步具有神经元形态，一周内达到生长旺盛期，各时间段包被组在细胞存活数、突起的平均长度等方面与对照组相比差异显著；PLL各组之间的细胞生长情况相比，在24小时观察，双蒸水配制?
Guillain-Barre syndrome (GBS) is the most common cause of acute flaccid paralysis and an autoimmune disorder of the peripheral nervous system characterized by the progressive weakness, usually symmetrical, evolving over a period of several days or more. The classical is also called acute inflammatory demyelinating polyneuropathy (AIDP) characterized by an immune-mediated demyelination and varying degrees of lymphocytic infiltration in pathologic techniques and decreased conductive velocity of motor and sensory nerve by electrophysiological examination. Until recently, a new type of GBS with the same clinical manifestation has been found with different electrophysiological and pathological characters, especially in North China. The axon of ventral roots and distal motor fibers appears wallerian-like degeneration with myelin sheath relatively preserved and less infiltration of inflammatory mononuclear cells. This pattern was termed as acute motor axonal neuropathy (AMAN) with a relatively severe clinical course and a poor recovery. The etiology and pathogenesis of the condition has not been established. In recent years, the association between AMAN and preceding campylobacter jejuni (Cj) infection has been noticed. Professor Li in our department isolated campylobacter jejuni with O serotyping O:19 from an AMAN patient and developed an animal model of AMAN using this Cj strains with O serotyping O:19 and one of Cj structure-lipopolysaccharide (LPS). Anti-Cj or LPS antibodies were also found in the serum from AMAN patients. Plasmapheresis and intravenous human immunoglobulin administration are effective to lower the patient fatality, indicating some pathogenetic factor(s) exist in the circulation of AMAN patients and play an important role in the etiology and pathogenesis, even treatment in the AMAN. AMAN is an autoimmune disorder of the
peripheral nervous system and the target of immune attack is axon of motor nerve. How the immune factors exert the damage is unclear, but the host immune system also play an even more important role in the developing AMAN. In some studies, complement C3d was found existing on the node of Ranvier using immunostain and complement activation presented in the periaxonal space, electron micrograph showing a macrophage surrounding the motor axon in patient with AMAN form of GBS. We established primary dissociated spinal motor neuron culture from embryonic rat to investigate the pathogenesis of AMAN. This culture system may take advantage in observing the axon damage from AMAN serum due to no limphocytes, mononuclear cells and complements exited which could not be eliminated in vivo and the axon-like neuritis extended from motor neurons without myelin out.
Neuron is difficult to survive in vitro in dissociated cell culture because it is a kind of cell with high degree of differentiation and has soma and neuritis, especially for spinal motor neurons (SMNs) that are sensitive to environment and need nutrition as the lowest motor neurons. So in the first two parts, we established the SMNs dissociated culture in vitro and compared the influence of different substrates coated and a kind of neurotrophin-glial cell line-derived neurotrophic factor (GDNF) on the growth condition of SMNs, the third part is to investigate the effect of AMAN serum on the SMNs cultured.
Part Ⅰ Influence of different substrate coated on spinal motor neurons cultured from embryonic rats
Objective: To isolate and incubate spinal motor neurons from embryonic rat and identify motor neurons in the culture firstly to develop the primary SMNs dissociated cell culture, then observe the influence of poly-L-lysine (PLL) dissolved in different solution on the growth of SMNs, and last to investigate the effect of different substrates coated including PLL, collagenⅠ, laminin and PLL combined with laminin on the cell survival and neurite outgrowth of SMNs.
Methods: The spinal cords were carefully isolated aseptically from embryo of pregnant 15 days Sprague-Dawley rat with all the roots and meninges dis
化标记物的表达，电镜下可发现有巨噬细胞包绕等。为了进一步研究AMAN的病因和发病机制，实验中建立并优化了体外胚胎大鼠脊髓前角运动神经元的原代培养体系。这种体外脊髓神经元培养中，缺乏在体实验研究中难以避免的淋巴细胞、单核巨噬细胞以及补体成分等，这对研究免疫系统中免疫机制参与运动神经元轴索的损害提供了便利条件；除此之外，实验中培养的胚胎大鼠脊髓前角运动神经元发出的轴突样突起的外面没有髓鞘包绕，可直接观察到致病因素对轴索损害的动态变化，为阐明AMAN的致病机制提供一定的理论基础。
神经元作为一种高度分化的细胞，形态上有胞体胞突之分。在单细胞培养中，相对于机体的其他细胞，在体外难以存活和生长，尤其是脊髓神经元，作为下运动神经元，对营养要求极高，对环境变化也极其敏感。因此，在实验的前两部分中，首先进行体外脊髓运动神经元的分离与鉴定，建立体外原代培养体系。为了优化培养方法，我们观察了不同包被基质和神经营养因子等对培养脊髓运动神经元生长状况的影响，寻找较适合神经元存活并利于以后实验研究的条件。第三部分是观察AMAN病人血清对培养脊髓运动神经元的作用。下面简述这三部分。
一、不同生长底物对体外培养脊髓运动神经元生长的影响
目的：首先建立脊髓运动神经元的体外培养，并对分离的细胞进行鉴定，以确定为脊髓运动神经元；在此基础上，观察了不同溶剂溶解稀释的多聚赖氨酸（poly-l-lysine, PLL）对脊髓神经元体外生长的影响，确定一种较好的方法以便进行以后的实验；随后，选取多种包被底物如PLL、Ⅰ型胶原、层粘连蛋白(laminin, LN)以及PLL和LN联合等进行包被，观察不同底物对胚胎大鼠脊髓运动神经元在体外存活生长的影响。
方法：在SD大鼠怀孕15天时，无菌条件下取出胚胎，解剖显微镜下取脊髓，取其腹侧组织切成小块，消化后制成单细胞悬液，差速贴壁以去除成纤维细胞，置于CO2培养箱中培养。培养细胞经神经元特异性烯醇化酶（NSE）抗体和抗神经丝单克隆抗体－SMI-32的免疫组织化学染色，对培养细胞进行鉴定。多聚赖氨酸分别用蒸馏水、磷酸盐缓冲液（PBS）、两种浓度的硼酸溶液溶解后对培养板进行包被，
接种制备的细胞悬液进行培养观察。不同的包被底物选取PLL、Ⅰ型胶原、LN、PLL联合LN等几种进行包被，对照组以蒸馏水代替。观察几种方法中培养神经元的存活数目、神经元突起的平均长度以及神经元的生长分布等状况。
结果：本实验建立的细胞培养体系为脊髓运动神经元的原代分散细胞培养，NSE抗体染色显示神经元的纯度达90％以上，SMI-32抗体染色能很好的显示脊髓运动神经元的形态：胞体大，为多极性，具有单个长的轴索样突起；以大的SMI-32阳性细胞进行计数，表明脊髓运动神经元占培养细胞总数的70％以上；神经元体外培养必须要求包被底物，否则将难以存活，对照组中细胞在24～36小时后死亡；当培养板包被底物后，神经元贴壁良好，部分细胞在短时间内长出突起，初步具有神经元形态，一周内达到生长旺盛期，各时间段包被组在细胞存活数、突起的平均长度等方面与对照组相比差异显著；PLL各组之间的细胞生长情况相比，在24小时观察，双蒸水配制?
Guillain-Barre syndrome (GBS) is the most common cause of acute flaccid paralysis and an autoimmune disorder of the peripheral nervous system characterized by the progressive weakness, usually symmetrical, evolving over a period of several days or more. The classical is also called acute inflammatory demyelinating polyneuropathy (AIDP) characterized by an immune-mediated demyelination and varying degrees of lymphocytic infiltration in pathologic techniques and decreased conductive velocity of motor and sensory nerve by electrophysiological examination. Until recently, a new type of GBS with the same clinical manifestation has been found with different electrophysiological and pathological characters, especially in North China. The axon of ventral roots and distal motor fibers appears wallerian-like degeneration with myelin sheath relatively preserved and less infiltration of inflammatory mononuclear cells. This pattern was termed as acute motor axonal neuropathy (AMAN) with a relatively severe clinical course and a poor recovery. The etiology and pathogenesis of the condition has not been established. In recent years, the association between AMAN and preceding campylobacter jejuni (Cj) infection has been noticed. Professor Li in our department isolated campylobacter jejuni with O serotyping O:19 from an AMAN patient and developed an animal model of AMAN using this Cj strains with O serotyping O:19 and one of Cj structure-lipopolysaccharide (LPS). Anti-Cj or LPS antibodies were also found in the serum from AMAN patients. Plasmapheresis and intravenous human immunoglobulin administration are effective to lower the patient fatality, indicating some pathogenetic factor(s) exist in the circulation of AMAN patients and play an important role in the etiology and pathogenesis, even treatment in the AMAN. AMAN is an autoimmune disorder of the
peripheral nervous system and the target of immune attack is axon of motor nerve. How the immune factors exert the damage is unclear, but the host immune system also play an even more important role in the developing AMAN. In some studies, complement C3d was found existing on the node of Ranvier using immunostain and complement activation presented in the periaxonal space, electron micrograph showing a macrophage surrounding the motor axon in patient with AMAN form of GBS. We established primary dissociated spinal motor neuron culture from embryonic rat to investigate the pathogenesis of AMAN. This culture system may take advantage in observing the axon damage from AMAN serum due to no limphocytes, mononuclear cells and complements exited which could not be eliminated in vivo and the axon-like neuritis extended from motor neurons without myelin out.
Neuron is difficult to survive in vitro in dissociated cell culture because it is a kind of cell with high degree of differentiation and has soma and neuritis, especially for spinal motor neurons (SMNs) that are sensitive to environment and need nutrition as the lowest motor neurons. So in the first two parts, we established the SMNs dissociated culture in vitro and compared the influence of different substrates coated and a kind of neurotrophin-glial cell line-derived neurotrophic factor (GDNF) on the growth condition of SMNs, the third part is to investigate the effect of AMAN serum on the SMNs cultured.
Part Ⅰ Influence of different substrate coated on spinal motor neurons cultured from embryonic rats
Objective: To isolate and incubate spinal motor neurons from embryonic rat and identify motor neurons in the culture firstly to develop the primary SMNs dissociated cell culture, then observe the influence of poly-L-lysine (PLL) dissolved in different solution on the growth of SMNs, and last to investigate the effect of different substrates coated including PLL, collagenⅠ, laminin and PLL combined with laminin on the cell survival and neurite outgrowth of SMNs.
Methods: The spinal cords were carefully isolated aseptically from embryo of pregnant 15 days Sprague-Dawley rat with all the roots and meninges dis
引文
1 Williams PL, Bannister LH, Berry MM, et al Gray'sAnatomy[M]. 杨琳，高英茂主译。格氏解剖学。沈阳：辽宁教育出版社，1999年
2 薛庆善主编。体外培养的原理与技术。北京：科学出版社，2001年
3 Deloulme JC, Baudier J, Sensenbrenner M. Establishment of pure neuronal cultures from fetal rat spinal cord and proliferation of the neuronal precursor cells in the presence of fibroblast growth factor. J Neurosci Res, 1991, 29:499~509
4 Sadahiko M, Hiroshi K, Yutaka S. Isolation and culture of motoneurons from embryonic chicken spinal cords. Proc Natl Acad Sci USA, 1979, 76:3537~3541
5 Erkman L, Touzeau G, Bertrand D, et al Characterization of dissociated monolayer cultures of human spinal cord. Brain research Bulletin, 1989, 22: 57~65
6 Michikawa M, Lim KT, McLarnon JG, et al Oxygen Radical-induced Neurotoxicity in spinal cord neuron cultures. J Neurosci Res, 1994, 37: 62~70
李芳，何风慈，李东江，等 胎鼠脊髓神经细胞的分离培养与生长分
7 化。第三军医大学学报，1993，15：544～545
8 许汉鹏，梁哲，肖华胜，等 原核表达的Janusin片段对体外培养培养脊髓神经细胞形态的影响。第四军医大学学报，1998，19：336～339
9 郭畹华，林启祥。胶质细胞成熟因子支持原代培养的小脑皮质神经元的生存。解剖学报，1989，20:50～55
10 Bottenstein JE. Growth and differentiation of neural cells in defined media. In: Bottenstein JE, Sato G eds. Cell culture in the neurosciences. New York: Plenum Press, 1985, 1: 1~43
11 Sean GC, Hong ZY, John HW. Motor neurons are selectively vulnerable to AMPA/Kainate receptor-mediated injury in vitro. J Neurosci, 1996, 16: 4069~4079
12 Sean GC, Stefano LS, Hong XY, et al AMPA exposure induce mitochondrial ca2+ overload and ROS generation in spinal motor neurons in vitro. J Neurosci, 2000, 20: 240~250
13 Josee R, Sandra M, Lichun D, et al Glutamate potentiates the toxicity of mutant Cu/Zn-superoxide dismutase in motor neurons by postsynaptic calcium-dependent mechanisms. J Neurosci, 1998, 18: 9673~9684
14 Schaffner AE, St John PA, Barker JL. Fluorescence-activated cell sorting of embryonic mouse and rat motoneurons and their long-term survival in vitro. J Neurosci, 1987, 7: 3088~3104
15 Gotow T, Tanaka J. Phosphorylation of neurofilament H subunit as related to arrangement of neurofilament. J Neurosci Res, 1994, 37: 691~713
16 Carrido SG, Yin HZ, Lamberta R, et al In vitro kainite injury to large SMI-32 (+) spinal neurons is ca2+ dependent. NeuroReport, 1995, 6:945~948
17 Geffard M, McRae-Degueurce A, Souan ML. Immunocytochemical detection of acetylcholine in the rat central nervous system. Science, 1985, 229: 77~79
Hietanen M, Pelto-Huiko M, Rechardt L. Immunocytochemical study of
18 the relations of acetylcholinesterase, enkephalin-, substance P-, choline acetyltransferase- and calcitonin gene-related peptide-immunoreactive structures in the ventral horn of rat spinal cord. Histochemistry, 1990, 93: 473~477
19 Escurat M, Djabali K, Gumpel M, et al Differential expression of two neuronal intermediate-filament proteins, peripherin and the low- molecular -mass neurofilament protein (NF-L), during the development of the rat. J Neurosci, 1990, 10:764~784
20 Carriedo SG, Yin HZ, Weiss JH. Motor neurons are selectively vulnerable to AMPA/Kainate receptor-mediated injury in vitro. J Neurosci, 1996, 16:4069~4079
21 Rogers SL, Letourneau PC, Palm SL, et al Neurite extension by peripheral and central nervous system neurons in response to substratum-bound fibronectin and laminin. Dev Biol, 1983, 98: 212~20
22 Lein PJ, Banker GA, Higgins D. Laminin selectively enhances axonal growth and accelerates the development of polarity y hippocampal neurons in culture. Brain Res Dev Brain Res, 1992, 69: 191~197
23 Carri NG, Rubin K, Gullberg D, et al Neuritogenesis on collagen substrates. Involvement of integrin-like matrix receptors in retinal fibre outgrowth on collagen. Int J Dev Neurosci, 1992, 10: 393~405
24 O'Connor SM, Stenger DA, Shaffer KM, et al Survival and neurite outgrowth of rat cortical neurons in three-dimensional agarose and collagen gel matrices. Neurosci Lett, 2001, 304: 189~193
25 Liesi P. Laminin and fibronectin in normal and malignant neuroecto -dermal cells. Med Biol, 1984, 62: 163~180
26 Tisay KT, Key B. The extracellular matrix modulates olfactory neurite outgrowth on ensheathing cells. J Neurosci, 1999, 19: 9890~9899
27 Lein PJ, Higgins D. Laminin and a basement membrane extract have different effects on axonal and dendritic outgrowth from embryonic rat sympathetic neurons in vitro. Dev Biol, 1989, 136: 330~345
28 Fredrick JS. The extracellular matrix molecule, laminin, induces Purkinje cell dendritic spine proliferation in granule cell depleted cerebellar cultures. Brain Res, 1998, 795: 112~120
2 薛庆善主编。体外培养的原理与技术。北京：科学出版社，2001年
3 Deloulme JC, Baudier J, Sensenbrenner M. Establishment of pure neuronal cultures from fetal rat spinal cord and proliferation of the neuronal precursor cells in the presence of fibroblast growth factor. J Neurosci Res, 1991, 29:499~509
4 Sadahiko M, Hiroshi K, Yutaka S. Isolation and culture of motoneurons from embryonic chicken spinal cords. Proc Natl Acad Sci USA, 1979, 76:3537~3541
5 Erkman L, Touzeau G, Bertrand D, et al Characterization of dissociated monolayer cultures of human spinal cord. Brain research Bulletin, 1989, 22: 57~65
6 Michikawa M, Lim KT, McLarnon JG, et al Oxygen Radical-induced Neurotoxicity in spinal cord neuron cultures. J Neurosci Res, 1994, 37: 62~70
李芳，何风慈，李东江，等 胎鼠脊髓神经细胞的分离培养与生长分
7 化。第三军医大学学报，1993，15：544～545
8 许汉鹏，梁哲，肖华胜，等 原核表达的Janusin片段对体外培养培养脊髓神经细胞形态的影响。第四军医大学学报，1998，19：336～339
9 郭畹华，林启祥。胶质细胞成熟因子支持原代培养的小脑皮质神经元的生存。解剖学报，1989，20:50～55
10 Bottenstein JE. Growth and differentiation of neural cells in defined media. In: Bottenstein JE, Sato G eds. Cell culture in the neurosciences. New York: Plenum Press, 1985, 1: 1~43
11 Sean GC, Hong ZY, John HW. Motor neurons are selectively vulnerable to AMPA/Kainate receptor-mediated injury in vitro. J Neurosci, 1996, 16: 4069~4079
12 Sean GC, Stefano LS, Hong XY, et al AMPA exposure induce mitochondrial ca2+ overload and ROS generation in spinal motor neurons in vitro. J Neurosci, 2000, 20: 240~250
13 Josee R, Sandra M, Lichun D, et al Glutamate potentiates the toxicity of mutant Cu/Zn-superoxide dismutase in motor neurons by postsynaptic calcium-dependent mechanisms. J Neurosci, 1998, 18: 9673~9684
14 Schaffner AE, St John PA, Barker JL. Fluorescence-activated cell sorting of embryonic mouse and rat motoneurons and their long-term survival in vitro. J Neurosci, 1987, 7: 3088~3104
15 Gotow T, Tanaka J. Phosphorylation of neurofilament H subunit as related to arrangement of neurofilament. J Neurosci Res, 1994, 37: 691~713
16 Carrido SG, Yin HZ, Lamberta R, et al In vitro kainite injury to large SMI-32 (+) spinal neurons is ca2+ dependent. NeuroReport, 1995, 6:945~948
17 Geffard M, McRae-Degueurce A, Souan ML. Immunocytochemical detection of acetylcholine in the rat central nervous system. Science, 1985, 229: 77~79
Hietanen M, Pelto-Huiko M, Rechardt L. Immunocytochemical study of
18 the relations of acetylcholinesterase, enkephalin-, substance P-, choline acetyltransferase- and calcitonin gene-related peptide-immunoreactive structures in the ventral horn of rat spinal cord. Histochemistry, 1990, 93: 473~477
19 Escurat M, Djabali K, Gumpel M, et al Differential expression of two neuronal intermediate-filament proteins, peripherin and the low- molecular -mass neurofilament protein (NF-L), during the development of the rat. J Neurosci, 1990, 10:764~784
20 Carriedo SG, Yin HZ, Weiss JH. Motor neurons are selectively vulnerable to AMPA/Kainate receptor-mediated injury in vitro. J Neurosci, 1996, 16:4069~4079
21 Rogers SL, Letourneau PC, Palm SL, et al Neurite extension by peripheral and central nervous system neurons in response to substratum-bound fibronectin and laminin. Dev Biol, 1983, 98: 212~20
22 Lein PJ, Banker GA, Higgins D. Laminin selectively enhances axonal growth and accelerates the development of polarity y hippocampal neurons in culture. Brain Res Dev Brain Res, 1992, 69: 191~197
23 Carri NG, Rubin K, Gullberg D, et al Neuritogenesis on collagen substrates. Involvement of integrin-like matrix receptors in retinal fibre outgrowth on collagen. Int J Dev Neurosci, 1992, 10: 393~405
24 O'Connor SM, Stenger DA, Shaffer KM, et al Survival and neurite outgrowth of rat cortical neurons in three-dimensional agarose and collagen gel matrices. Neurosci Lett, 2001, 304: 189~193
25 Liesi P. Laminin and fibronectin in normal and malignant neuroecto -dermal cells. Med Biol, 1984, 62: 163~180
26 Tisay KT, Key B. The extracellular matrix modulates olfactory neurite outgrowth on ensheathing cells. J Neurosci, 1999, 19: 9890~9899
27 Lein PJ, Higgins D. Laminin and a basement membrane extract have different effects on axonal and dendritic outgrowth from embryonic rat sympathetic neurons in vitro. Dev Biol, 1989, 136: 330~345
28 Fredrick JS. The extracellular matrix molecule, laminin, induces Purkinje cell dendritic spine proliferation in granule cell depleted cerebellar cultures. Brain Res, 1998, 795: 112~120