人脊髓内NF样阳性神经元的形态及分布

目的：观察不同胎龄胎儿脊髓神经丝蛋白(NF)阳性神经元的形态、分布和发育变化，为脊髓-脊髓移植选择适宜的胎龄提供形态学依据。方法：胎儿脊髓19例，SP免疫组化染色，图像分析。结果：脊髓侧角内NF阳性神经元由中央管向外迁移；前角神经元由外向内迁移。NF阳性神经元密度在胚胎早期逐渐升高，晚期呈下降趋势。NF阳性神经元在胎龄16周时，胞体呈圆形、卵圆形，突起少沙，胞核大，有偏极现象，至32周时胞体呈锥形、梭形、多角形；胞体逐渐增大，胞浆逐渐增多，胞核多位居中央；突起增多。结论：未发育成熟的神经元内也有NF的存在。脊髓内NF阳性神经元密度随胎龄增加而逐渐增加，形态逐渐成熟，提示人脊髓-脊髓移植时以16周胎龄作供体较为适宜。     

大鼠脊髓发育过程中神经干细胞的分化

目的:研究正常大鼠脊髓发育过程中神经干细胞的分化规律。方法:应用免疫荧光染色技术,检测Nestin、NeuN、MAP2、GFAP、CNPase阳性细胞在大鼠胚胎期及生后脊髓内的分布及变化情况。结果:胚胎发育早期,脊髓中央管、灰质、白质均可检测到Nestin阳性细胞,生后Nestin阳性细胞数量逐渐减少。大鼠脊髓神经元的发生呈现明显的背腹模式,孕14d(E14)脊髓内可检测到NeuN阳性细胞,E16 NeuN阳性细胞逐渐增多,腹侧NeuN阳性细胞核体积较大,分布较稀疏,背侧神经元细胞核体积较小,分布较密集。MAP2染色结果与NeuN一致。胶质细胞的分化、成熟在生后初期进行,P4可检测到GFAP及CNPase阳性细胞,主要分布于脊髓白质内,P30在脊髓灰质内可检测到GFAP阳性细胞,细胞分支较多且短。结论:正常大鼠脊髓发育中神经干细胞的分化呈现一定规律性,向神经元方向化较早且呈明显的背腹模式,胶质细胞的分化较晚。     

人胎蓝斑神经元免疫组织化学研究

为了探讨人蓝斑神经元的胚胎发育特征 ,为蓝斑 -脊髓移植选择适宜胎龄提供形态学根据 ,本研究用免疫组织化学技术系统地观察了人胎蓝斑酪氨酸羟化酶样免疫反应阳性神经元的发育。结果证明 :( 1)蓝斑酪氨酸羟化酶样神经元在胎龄 4个月时已经出现在蓝斑的腹侧部 ;( 2 )蓝斑酪氨酸羟化酶样神经元随胎龄增长逐渐增多 ,以 5个月时增加显著 ;( 3)酪氨酸羟化酶样神经元的密度在胚胎早期升高 ,晚期呈下降趋势 ;( 4)酪氨酸羟化酶样神经元主要分布在蓝斑的背侧部 ,少量散在于腹侧部 ;( 5)酪氨酸羟化酶样神经元开始出现时呈圆形或卵圆形 ,5～ 6个月时呈锥形和梭形 ,7～ 8个月时则以梭形、多角形为主。其胞体逐渐增大 ,胞浆逐渐增多 ,核浆之比由大变小 ,胞突从粗短变为细长平滑。本研究结果提示 ,人胎蓝斑移植以 4个月胎龄者作移植供体较为适宜     

人胎儿脊髓星形胶质细胞形态及分布

目的　观察星形胶质细胞在胎儿脊髓不同发育阶段形态和分布的变化。方法　在引产 19例胎儿脊髓 ,多克隆抗体GFAP ,应用SP免疫组织化学染色和图像分析。结果　星形胶质细胞在中央管、毛细血管周围密度大 ,染色深 ,环绕血管呈辐射状排列。 16W时已有少突起GFAP阳性细胞出现 ,胞体小 ,分布于脊髓白质的周边部 ,细长突起指向中央管方向 ;突起较短的细胞分布于脊髓前后正中沟两侧和中央管周围 ;灰质内阳性细胞主分布于背侧部的两侧 ,突起多而短 ,细胞核大。 2 4W时 ,多突起细胞增多 ,GFAP阳性细胞染色强度、细胞密度接近出生时水平。结论　人胎儿脊髓星形胶质细胞 16W时最多 ,2 4W时逐渐减少至出生时水平     

微囊化兔嗅球细胞悬液移植对脊髓损伤大鼠GFAP及NF200表达的影响

目的:探讨微囊化兔嗅球组织细胞移植对脊髓损伤大鼠胶质原纤维酸性蛋n(GFAP)及神经丝蛋白200(NF200)表达的影响.方法:SD大鼠分为正常组、损伤对照组、细胞悬液组、微囊化移植组.用免疫组织化学染色方法观察损伤脊髓内GFAP及NF200表达,变化.结果:脊髓损伤后NF200及GFAP表达均呈进行性增高,二者有显著的相关性;微囊组术后7、14、21 d胶质原纤维酸性蛋白的表达显著低于损伤对照组;但微囊组脊髓内NF200表达显著升高.结论:微囊化兔嗅球细胞悬液移植可促进损伤脊髓内的NF200表达并抑制GFAP表达,有可能有助于脊髓功能的恢复.     

大鼠腰骶髓和延髓星形胶质细胞及神经元对慢性结肠炎的反应

目的探讨大鼠腰骶髓和延髓星形胶质细胞及神经元对慢性结肠炎的反应,及反应性星形胶质细胞和反应性神经元之间的关系.方法成年雄性SD大鼠,实验组（n=17）给予三硝基苯磺酸（TNBS）灌肠诱导结肠炎;对照组（n=16）给予生理盐水灌肠.免疫组织化学法显示大鼠腰骶髓和延髓内胶质原纤维酸性蛋白（GFAP）阳性星形胶质细胞和Fos阳性神经元.结果TNBS灌肠后,GFAP阳性星形胶质细胞主要分布在脊髓背角浅层（Ⅰ～Ⅱ层）、中间外侧核（Ⅴ层）、后连合核（Ⅹ层）和腹角外侧核（Ⅸ层）.Fos阳性神经元集中分布在背角深层（Ⅲ～Ⅳ,Ⅴ～Ⅵ层）.在延髓,两者均主要分布在由孤束核、中间网状带和腹外侧区组成的延髓内脏带（MVZ）.TNBS灌肠后3、7、14 d,脊髓中GFAP阳性细胞密度明显高于对照组（P＜0.05）.TNBS灌肠后3 d,延髓中GFAP阳性细胞密度明显高于对照组（P＜0.05）.TNBS灌肠后28 d,脊髓和延髓中GFAP阳性细胞密度下降,与对照组无显著性差异（P＞0.05）.结论结肠炎性刺激引起脊髓和延髓中星形胶质细胞激活.随着结肠炎的恢复,星形胶质细胞的反应性下降.在延髓内脏带,反应性星形胶质细胞与反应性神经元关系密切.     

纤维蛋白支架促进神经干细胞向神经元分化并抑制星形胶质细胞增生

目的探讨纤维蛋白支架对神经干细胞和星形胶质细胞分化及增殖的影响。方法分别培养胚胎大鼠脊髓来源的神经干细胞和新生鼠脊髓神经胶质细胞,接种于纤维蛋白支架上,同时用多聚赖氨酸修饰的玻片作为对照。于体外培养不同时间后,用神经丝蛋白(NF200)对神经细胞进行免疫荧光染色,测量各复孔(n=4)内NF阳性细胞的突起长度,计算其平均值;用胶质纤维酸性蛋白(GFAP)对胶质细胞进行染色,各复孔(n=4)内统计5个不同视野的胶质细胞总数和GFAP阳性细胞数,计算GFAP阳性细胞相对数量的平均值。比较在纤维蛋白支架和玻片上神经干细胞分化、神经纤维延伸及神经胶质细胞增殖的差异。同时用免疫印迹技术对荧光染色结果进行验证。上述实验各重复3次。结果纤维蛋白支架组的NF阳性纤维明显长于对照组,GFAP阳性星形胶质细胞相对数量明显少于对照组,GFAP的表达水平明显低于对照组。结论纤维蛋白支架可促进神经干细胞向神经细胞分化,并有利于神经纤维的延伸而抑制星形胶质细胞的增殖和成熟。     

大鼠生后中枢神经系统S100B和胶质纤维酸性蛋白表达的变化

目的 探讨SD大鼠生后中枢神经系统发育过程中S100B和胶质纤维酸性蛋白(GFAP)的表达变化.方法 24只雄性SD大鼠分为生后7d、14d、21d和成年4组,用免疫组织化学方法对脑、脊髓切片进行S100B、GFAP抗体染色,观察不同时间点不同部位中两种阳性细胞平均数.结果 生后7d到成年,前额皮质、海马、纹状体、黑质和脊髓S100B阳性标记的密度和数量逐渐减少,生后2～3周时渐趋于稳定;脑内GFAP阳性星形胶质细胞(AST)随年龄增大逐渐增多,突起增粗增长,生后21d GFAP阳性细胞数量已接近成年;相反,脊髓GFAP阳性标记数则随年龄增长呈现由多到少的趋势;海马CAl区生后各年龄段GFAP和S100B免疫荧光双标显示,生后1周至成年S100B阳性细胞的数量明显减少,尤以分子层明显;随年龄增长,双标阳性细胞的比例逐渐增高,多集中分布于锥体细胞层和多形层.结论 大鼠中枢神经系统中S100B和GFAP两种星形胶质细胞蛋白存在不同的表达模式;同时S100B和GFAP蛋白的表达在发育过程中可能受不同机制的调节,并可能代表星形胶质细胞的不同亚型.     

神经元核心抗原和神经元特异性烯醇化酶在人胚胎脊髓发育阶段的表达

目的 探讨神经元核心抗原（NeuN）和神经元特异性烯醇化酶(NSE)在人胚胎脊髓发育阶段的分布规律及其表达意义。 方法 应用免疫组织化学法检测第2、3、4个月龄段,共16例人胚胎,脊髓前角、后角中NeuN和NSE的表达、分布状况。 结果 在脊髓后角处：第2个月胚龄段,NeuN呈阴性表达, 部分细胞呈NSE阳性表达;第3个月胎龄段,部分细胞开始呈NeuN阳性表达,NSE则呈广泛阳性表达;第4个月胎龄段,NeuN阳性细胞表达数量和强度均较前增高,NSE阳性表达与第3个月龄相似。在人胚胎脊髓前角处： 第2～4个月龄段,NeuN和NSE呈广泛阳性表达。 结论 人胚胎脊髓发育第2～4个月龄段,NeuN和NSE阳性表达在脊髓的前角处早于后角,NeuN和NSE参与脊髓神经元的发育成熟过程。     

小鼠脊髓灰质发育过程中细胞迁移与血管之间的关系

目的:探讨小鼠神经系统发育过程中脊髓成熟神经元迁移与血管发育之间的关系。方法:不同年龄小鼠共计75只,应用免疫荧光及墨汁灌注的技术,标记小鼠胚胎E17到P30脊髓神经元和血管。结果:大约在胚胎E17左右,小鼠脊髓灰质内开始出现NeuN阳性的神经元,白质中神经元较少,且此时灰质和白质内血管分布均匀,管径一致,分支较少。随着年龄的增长,脊髓周围的神经元不断向内迁移,灰质内NeuN阳性的神经元数先增多后减少且血管体密度先增加后减小,而白质内的神经元持续减少,血管逐渐稀疏。P14以后,脊髓灰质内的血管密度明显高于白质内的血管密度。同时,研究中还发现部分NeuN阳性细胞紧贴着血管分布。结论:在小鼠脊髓的发育过程中,NeuN阳性细胞由脊髓周围逐渐向中心迁移,和脊髓灰质H形态的形成密切相关。同时,血管扮演着重要的角色,它可能通过与神经元的相互作用,引导神经元的迁移,并且为神经元的迁移提供路径和支架。     

Myelin formation in myelin-deficient rat spinal cord following transplantation of normal fetal spinal cord

Previous studies of the myelin-deficient rat spinal cord have suggested that astrocytes may play a role in preventing the formation of myelin in this mutant, or causing its breakdown. Comparison of mutant and normal littermate spinal cords shows a marked hypertrophy of astrocytes in the mutant in both gray matter and fiber tract regions. Nevertheless, when normal fetal spinal cord fragments are transplanted into mutant host spinal cord, clusters of normal-looking myelin sheaths develop with no sign of attack by host astrocytes.     

Increased phospho-adducin immunoreactivity in a murine model of amyotrophic lateral sclerosis

Adducins alpha, beta and gamma are proteins that link spectrin and actin in the regulation of cytoskeletal architecture and are substrates for protein kinase C and other signaling molecules. Previous studies have shown that expressions of phosphorylated adducin (phospho-adducin) and protein kinase C are increased in spinal cord tissue from patients who died with amyotrophic lateral sclerosis, a neurodegenerative disorder of motoneurons and other cells. However, the distribution of phospho-adducin immunoreactivity has not been described in the mammalian spinal cord. We have evaluated the distribution of immunoreactivity to serine/threonine-dependent phospho-adducin at a region corresponding to the myristoylated alanine-rich C kinase substrate-related domain of adducin in spinal cords of mice over-expressing mutant human superoxide dismutase, an animal model of amyotrophic lateral sclerosis, and in control littermates. We find phospho-adducin immunoreactivity in control spinal cord in ependymal cells surrounding the central canal, neurons and astrocytes. Phospho-adducin immunoreactivity is localized to the cell bodies, dendrites and axons of some motoneurons, as well as to astrocytes in the gray and white matter. Spinal cords of mutant human superoxide dismutase mice having motoneuron loss exhibit significantly increased phospho-adducin immunoreactivity in ventral and dorsal horn spinal cord regions, but not in ependyma surrounding the central canal, compared with control animals. Increased phospho-adducin immunoreactivity localizes predominantly to astrocytes and likely increases as a consequence of the astrogliosis that occurs in the mutant human superoxide dismutase mouse with disease progression. These findings demonstrate increased immunoreactivity against phosphorylated adducin at the myristoylated alanine-rich C kinase substrate domain in a murine model of amyotrophic lateral sclerosis. As adducin is a substrate for protein kinase C at the myristoylated alanine-rich C kinase substrate domain, the increased phospho-adducin immunoreactivity is likely a consequence of protein kinase C activation in neurons and astrocytes of the spinal cord and evidence for aberrant phosphorylation events in mutant human superoxide dismutase mice that may affect neuron survival.     

Ultrastructural changes in the CNS in amyotrophic leukospongiosis

Various types of cells in the human spinal cord and brain, status spongiosus of the white matter of the spinal cord were studied by electron microscopy; the comparison was made to the status spongiosus in monkeys with experimental amyotrophic leukospongiosis. The alterations in the motor neurons of the spinal cord, pyramidal neurons of the brain hemisphere cortex and cerebellum ganglious neurocytes were revealed. Changes in some cells (astrocytes, olygodendrocytes) were characterized. The possibility of a primary axon damage in this disease is discussed.     

大鼠胎脑细胞脊髓内移植的实验研究

本文是用孕１３天（Ｅ１３）大鼠胎脑细胞悬液，以慢速注入法移植于同种成年大鼠脊髓白质的侧索及后索、中央管及灰质中。移植后分１天、２周、４周、１２周等４组，用光镜检查在脊髓内不同部位移植的胎脑细胞，存活、生长、分化、成熟以及与宿主脊髓嵌合等情况。结果表明：①移植于成鼠脊髓侧索、后索及中央管等不同部位，９０％以上移植成功，胎脑细胞的存活、生长、分化极为良好，术后４周形成神经毡样结构，且多与宿主灰质嵌合；②灰质内移植组的宿主脊髓灰质损伤严重，神经元多变性、坏死消失；③移植于脊髓不同部位的方法将有助于脊髓前、后以及中央等各种类型损伤的修复，探讨是否有可能建立脊髓内不同移植部位的模型。     

低渗刺激后大鼠视上核内星形胶质细胞和神经元的可塑性改变及其相互关系

为观察低渗刺激后大鼠视上核(SON)内星形胶质细胞和神经元的可塑性反应及其两者在反应时间和空间上的相互关系,我们采用大鼠尾静脉注射 5. 5ml/kg低渗盐水(0. 83% 葡萄糖+ 0. 3% NaCl)制作模型,应用抗Fos、抗胶质原纤维酸性蛋白(GFAP)单标记,以及抗Fos/抗GFAP双标记、抗Fos/抗血管加压素 (VP) /抗GFAP三标记的免疫组化方法。结果发现:在SON中GFAP阳性星形胶质细胞数量在刺激后 15min增加,其胞体肥大,突起伸长变粗, 45min达高峰。Fos阳性星形胶质细胞胞核呈蓝黑色小点,刺激后 15~45min达高峰, 90min明显减少;Fos阳性神经元胞核较大,刺激后 15min未见表达, 45min少量表达, 90min表达增加。该结果表明低渗刺激后,SON内星形胶质细胞的Fos表达早于神经元,三重标记显示星形胶质细胞与神经元关系密切,提示SON内星形胶质细胞可能在低渗刺激的调节中发挥一定作用。     

Astrocyte transplantation for repairing the injured spinal cord

Spinal cord injury (SCI) leads to permanent deficits in neural function without effective therapies, which places a substantial burden on families and society. Astrocytes, the major glia supporting the normal function of neurons in the spinal cord, become active and form glial scars after SCI, which has long been regarded as a barrier for axon regeneration. However, recent progress has indicated the beneficial role of astrocytes in spinal repair. During the past three decades, astrocyte transplantation for SCI treatment has gained increasing attention. In this review, we first summarize the progress of using rodent astrocytes as the primary step for spinal repair. Rodent astrocytes can survive well, migrate extensively, and mature in spinal injury; they can also inhibit host reactive glial scar formation, stimulate host axon regeneration, and promote motor, sensory, respiratory, and autonomic functional recovery. Then, we review the progress in spinal repair by using human astrocytes of various origins, including the fetal brain, fetal spinal cord, and pluripotent stem cells. Finally, we introduce some key questions that merit further research in the future, including rapid generation of large amounts of human astrocytes with high purity, identification of the right origins of astrocytes to maximize neural function improvement while minimizing side effects, testing human astrocyte transplantation in chronic SCI, and verification of the long-term efficacy and safety in large animal models.     

Selective stimulation of dendrite outgrowth from identified corticospinal neurons by homotopic astrocytes.

Corticospinal neurons were identified in primary cultures of cortical neurons established from rats that had been injected with a fluorescent tracer to retrogradely label the corticospinal tract. We measured neurite outgrowth from corticospinal neurons after they had been co-cultured with astrocytes derived from either the cerebral cortex (homotopic region) or spinal cord (target region) of postnatal rats. The axon length of corticospinal neurons was increased when they were cultured on astroglial monolayers compared to a control monolayer (fibroblasts). However, no difference in axon length was noted on cortical versus spinal cord-derived astrocytes. On the other hand, total dendritic length was increased on cortical compared to spinal cord astrocytes. This increase in total dendrite length was not the result of differences in the length of primary dendrites, but primarily of a higher number of dendrites and increased branching on the cortical astroglia. If the corticospinal neurons were co-cultured without physical contact with the astrocytes, axonal and dendritic outgrowth were not stimulated when compared to the fibroblast control. The data indicate that dendritic growth from corticospinal neurons is preferentially promoted by astrocytes from the cerebral cortex, whereas axonal growth is not influenced by the anatomical origin of the astrocytes. The impact of these findings on our understanding of the role of astrocytes in the development and regeneration of the corticospinal tract is discussed.     

胚胎大鼠脊髓神经干细胞的培养及鉴定

为了探讨胚胎大鼠脊髓神经管神经干细胞体外培养、鉴定的方法,本实验在解剖显微镜下机械性分离11.5 d的胚胎大鼠脊髓神经管,并制备细胞悬液,无血清培养基培养。应用免疫荧光染色方法对原代和传代细胞进行巢蛋白(nestin)鉴定;加入胎牛血清诱导其分化后分别行神经元特异烯醇化酶(NSE)、胶质原纤维酸性蛋白(GFAP)和髓磷脂碱性蛋白(MBP)鉴定。结果显示:11.5 d胚胎大鼠脊髓神经管来源的神经干细胞经连续传代培养可形成较多克隆球,呈nestin免疫阳性;加入胎牛血清可诱导其分别向神经元、星形胶质细胞和少突胶质细胞分化。本实验结果提示,利用E11.5的胚胎大鼠脊髓神经管可成功培养出大量稳定增殖并有多向分化潜能的神经干细胞,可用于进一步的研究。