原代培养许旺细胞的神经营养性作用受神经细胞的调节

许旺细胞(Schwann cells,SCs)是外周神经的重要组成部分,在周围神经系统发育和再生中起着重要作用.为了研究许旺细胞的神经营养性作用及其产生的条件,我们将新生大鼠许旺细胞与14 d胚龄的大鼠腹侧中脑细胞或皮质细胞联合培养,收集许旺细胞条件液并观察它们对培养的腹侧中脑多巴胺能神经元的支持作用.结果显示:与腹侧中脑细胞接触或不接触联合培养的许旺细胞条件液以及与皮质细胞不接触联合培养的许旺细胞条件液均促进多巴胺能神经元的存活和生长,原代单独培养的许旺细胞条件液对神经元的存活和突起的生长却无明显作用.结果表明,原代培养的许旺细胞神经营养性效应受神经细胞的正向调节,这一调节作用可能由可溶性物质介导.     

大鼠胚胎腹侧中脑神经干细胞的分离培养与鉴定

目的探讨大鼠胚胎腹侧中脑神经干细胞分离、培养及鉴定的方法。方法解剖分离E14d大鼠胚胎腹侧中脑组织，经机械吹打制成单细胞悬液，接种于无血清培养基中培养，观察其增殖、分化并进行Nestin免疫细胞化学鉴定和诱导分化后β-Ⅲ-tubulin、GFAP、CNPase免疫细胞化学鉴定。结果原代培养7d后，可形成大量悬浮生长Nestin免疫阳性的神经球，经诱导分化后细胞呈GFAP、CNPase或β-Ⅲ-tubulin免疫阳性。结论建立大鼠胚胎腹侧中脑神经干细胞分离培养与鉴定的方法，为进一步开展帕金森病的细胞移植治疗研究奠定基础。     

培养纯化的人胎儿许旺细胞在脱髓鞘大鼠脊髓内的成活和迁移

许旺细胞是周围神经系统的髓鞘形成细胞，它能修复遗传性或物理，化学因素导致的中枢脱髓鞘损伤，本实验用荧光素Ｈｏｅｃｈｓｔ３３３４２标记体培养的人胎儿许旺细胞并将其移植于１％溶血卵磷酯造成的脱髓鞘大鼠脊髓内，直接观察许旺细胞的成活和迁移，实验结果显示人胎儿许旺细胞特异性地朝脱髓鞘损伤方向迁移，迁移的速度约２ｍｍ／ｄ，迁移途径主要软脊膜，室管膜以及血管周间隙，亦有部分细胞在白质内迁移，移植１个月后，细胞     

去细胞同种异体神经种植许旺细胞的体外实验：组织工程化人工神经应用的可行性

目的：许旺细胞作为种子细胞在组织工程化人工神经制备中的作用已被学术界所接受。胎兔的神经系统已发育完善，而免疫系统尚未成熟，探讨将胎兔许旺细胞植入去细胞同种异体神经桥接体制备组织工程化人工神经修复周围神经缺损的可行性。 方法：实验于2005-03/2006-03在河北医科大学第三医院完成。①实验材料：SPF级怀孕28 d的新西兰白兔2只、成年新西兰白兔1只。②实验过程：包括胎兔许旺细胞培养、去细胞神经桥接体的制备以及许旺细胞与去细胞神经桥接体的体外种植3个步骤。使用双酶消化法培养许旺细胞并将其种植于经3% TritonX-100作用96 h后的同种异体神经中。③实验评估：分别于培养的1，3，5 d通过石蜡切片苏木精-伊红染色观察许旺细胞在桥接体中的生长情况。 结果：①采用双差速贴壁法去除绝大部分成纤维细胞后在细胞培养的初期使用阿糖胞苷抑制成纤维细胞生长，后期培养液中加入神经生长因子促进许旺细胞生长能获取大量许旺细胞且纯度可达90%以上。②用3%Triton X-100作用96 h可去除周围神经中的细胞和髓鞘而保留完整的神经基底膜管和纤维支架结构，并且对神经基底膜主要成分－层粘连蛋白无明显影响。③采用胎兔坐骨神经培养的许旺细胞能在用成年兔坐骨神经制备的去细胞桥神经接体中生长良好，并且有迁移成行的特性。 结论：种植许旺细胞重新细胞化的去细胞同种异体神经将可能成为一种理想的组织工程化人工神经。     

选择性5-HT1A受体拮抗剂WAY-100635抑制帕金森病模型大鼠腹内侧前额叶皮质的神经活动

目的 腹内侧前额叶皮质在随意运动的起始和控制、情感以及认知中具有重要作用.然而,黑质-纹状体通路变性后腹内侧前额叶皮质的神经活动和5-HT1A受体的作用仍不清楚.本研究观察了6-羟基多巴胺(6-hydroxydopamine,6-OHDA)损毁黑质致密部(substantia nigra pars compacta,SNc)后大鼠腹内侧前额叶皮质神经活动的变化和体循环给予选择性5-HT1A受体拮抗剂WAY-100635后神经元活动的改变.方法 采用在体玻璃微电极细胞外记录方法,记录正常大鼠和SNc单侧损毁大鼠的腹内侧前额叶皮质神经元的活动.结果 6-OHDA损毁SNc大鼠的腹内侧前额叶皮质神经元放电频率显著增加,放电形式没有明显改变.体循环给予WAY-100635(0.1 mg/kg,i.v.)不改变正常大鼠腹内侧前额叶皮质神经元的平均放电频率和放电形式,而显著降低了SNc损毁大鼠前额叶皮质神经元的平均放电频率.结论 黑质-纹状体通路的变性可导致腹内侧前额叶皮质神经活动增强,5-HT1A受体拮抗剂WAY-100635可以抑制这种活动增强,提示可能存在腹内侧前额叶皮质5-HT1A受体功能失调.     

短时低频电刺激体外培养许旺细胞髓鞘的形成

周围神经系统损伤后,短时低频电刺激已被证明可显著促进轴突再生和选择性功能修复,但目前对电刺激是否影响周围神经髓鞘的形成还知之甚少,而电刺激发挥作用究竟是通过神经元还是许旺细胞还有待证实。 目的：建立体外背根神经元与许旺细胞联合培养模型,观察短时低频电刺激对许旺细胞髓鞘形成的影响。 方法：体外培养背根神经元,纯化后预先施予电刺激(20 Hz,100 μs,3 V),持续作用1 h,24 h后再加入许旺细胞悬液制成背根神经元/许旺细胞联合培养模型。在此基础上,用L-ascorbic acid诱导髓鞘形成,分别于诱导后第7,14天观察培养体系中髓鞘的形成。 结果与结论：电刺激增强背根神经元分泌脑源性神经营养因子(P < 0.05),经电刺激作用的背根神经元再与许旺细胞联合培养,最终表现为髓鞘形成增多以及髓鞘蛋白表达上调(P < 0.05)。提示短时低频电刺激对体外许旺细胞髓鞘的形成具有促进作用,初步认为该作用至少通过刺激神经元分泌脑源性神经营养因子增多导致。     

骨髓基质细胞条件液联合神经干细胞移植对帕金森病大鼠行为认知功能的影响：效果优于单纯神经干细胞移植吗？

背景：将骨髓基质细胞和神经干细胞分别单独移植于帕金森病模型鼠脑内已取得良好效果,但尚未见联合移植的报道。 目的：观察骨髓基质细胞条件液与神经干细胞联合移植对帕金森病模型大鼠行为认知功能的影响,并与单纯神经干细胞移植效果进行比较。 方法：用Neurobasal+B27培养3~6代的骨髓基质细胞24 h后,收集并离心上清液,即为骨髓基质细胞条件液。体外分离培养大鼠神经干细胞后,行BrdU标记。55只大鼠随机取8只作为正常组,剩余47只采用立体定向仪单侧黑质致密部和腹侧被盖区注射6-羟基多巴胺建立帕金森病动物模型。32只造模成功,随机分为3组,选取右侧纹状体两个坐标点为移植点,单纯神经干细胞组每点注入神经干细胞悬液5 μL,联合组每点注入神经干细胞悬液+骨髓基质细胞条件液5 μL,模型组不注入任何液体。观察帕金森病大鼠旋转行为的改变,通过水迷宫试验对大鼠认知功能进行评价,免疫组织化学染色检测 黑质区酪氨酸羟化酶蛋白的表达及移植区BrdU的表达。 结果与结论：与模型组比较,移植后1~8周单纯神经干细胞组、联合组大鼠旋转次数均显著减少(P < 0.01),移植后第4,8周单纯神经干细胞组、联合组大鼠逃避潜伏期均明显缩短(P < 0.05),在平台象限的游泳时间百分比、游泳距离百分比、穿越平台次数均明显增加(P < 0.05);后2组间各指标比较均无明显差异(P > 0.05)。移植后第8周,各组大鼠6-羟基多巴胺注射侧黑质区酪氨酸羟化酶阳性神经元和神经纤维表达基本缺失;单纯神经干细胞组、联合组移植区可见一定数量的BrdU阳性细胞表达,多数位于针道附近,部分细胞沿胼胝体迁移。提示骨髓基质细胞条件液与神经干细胞联合移植能改善帕金森病模型大鼠的旋转行为和认知能力,与单纯神经干细胞纹状体移植的效果基本相似。     

选择性5-HT1A受体拮抗剂WAY-100635抑制帕金森病模型大鼠腹内侧前额叶皮质的神经活动

目的腹内侧前额叶皮质在随意运动的起始和控制、情感以及认知中具有重要作用。然而,黑质-纹状体通路变性后腹内侧前额叶皮质的神经活动和5-HT_(1A)受体的作用仍不清楚。本研究观察了6-羟基多巴胺(6- hydroxydopamine,6-OHDA)损毁黑质致密部(substantia nigra pars compacta,SNc)后大鼠腹内侧前额叶皮质神经活动的变化和体循环给予选择性5-HT_(1A)受体拮抗剂WAY-100635后神经元活动的改变。方法采用在体玻璃微电极细胞外记录方法,记录正常大鼠和SNc单侧损毁大鼠的腹内侧前额叶皮质神经元的活动。结果6-OHDA损毁SNc大鼠的腹内侧前额叶皮质神经元放电频率显著增加,放电形式没有明显改变。体循环给予WAY-100635 (0.1 mg/kg,i.v.)不改变正常大鼠腹内侧前额叶皮质神经元的平均放电频率和放电形式,而显著降低了SNc损毁大鼠前额叶皮质神经元的平均放电频率。结论黑质-纹状体通路的变性可导致腹内侧前额叶皮质神经活动增强,5-HT_(1A)受体拮抗剂WAY-100635可以抑制这种活动增强,提示可能存在腹内侧前额叶皮质5-HT_(1A)受体功能失调。     

中脑源性神经干细胞培养过程中pitx3以及凋亡基因的表达

目的 探讨胚胎大鼠腹侧中脑源性神经干细胞培养过程中pitx3、TH以及凋亡基因的表达.方法 分离E14.5 d胚胎大鼠腹侧中脑组织,用机械吹打方法形成细胞悬液培养,传代7 d、14 d、28 d后,用real-time PCR检测pitx3、Bax、Bcl-2的表达情况.结果 在培养的神经干细胞克隆中,pitx3、Bax基因7～21 d表达逐渐增加,而Bcl-2 基因表达逐渐下降.结论 胚胎大鼠腹侧中脑源性神经干细胞具有分化为中脑多巴胺能神经的潜能,但是随着培养时间的延长,调亡的细胞也在不断增加.     

中脑腹侧区细胞移植对帕金森病鼠纹状体多巴胺受体敏感 …

大鼠纹状体中多巴胺受体介导的ｃ－ｆｏｓ基因的表达与多巴胺Ｄ１受体的超敏现象有关。本实验将鼠胚胎中脑腹侧区细胞植入帕金森病大鼠模型纹状体后第１２周，用免疫组化法检测阿朴吗啡诱发的ｃ－ｆｏｓ蛋白，同时取相邻切片进行酪氨酸羟化酶检测。     

The effect of microglia on embryonic dopaminergic neuronal survival in vitro: diffusible signals from neurons and glia change microglia from neurotoxic to neuroprotective

When embryonic dopaminergic neurons are transplanted into the adult brain, approximately 95% die within a few days. To assess whether microglia activated during transplantation might be responsible for this rapid death, we examined the effect of microglia on rat embryonic dopaminergic neurons in vitro. Conditioned medium from 7-day-old microglia was found to decrease the number of dopamine neurons surviving in primary culture, but activation of the microglia with N-formyl-methionyl-leucyl-phenylalanine (FMLP) or Zymosan A did not increase the toxicity of the conditioned medium. We next tested the effect of coculturing microglia and dopaminergic neurons by placing microglia in semipermeable well inserts over the neuronal cultures. The presence of microglia now increased dopaminergic neuronal survival, microglial activation again having no effect. To increase yet further the possible interactions between microglia and neurons, the mesencephalic cells and microglia were mixed together and placed as a tissue in three-dimensional culture, and here again the presence of microglia increased dopaminergic neuronal survival with no effect of activation. Contact of microglia with the mesencephalic cells therefore converted them from being toxic to dopaminergic neurons to promoting their survival. The change in microglial effect from toxic to protective was caused by soluble molecules secreted by cells in the neuronal cultures, as conditioned medium derived from microglia-neuronal cocultures also had a dopaminergic neuron survival effect, indicating that microglia in cocultures behave differently from microglia removed from neuronal and glial influence. Microglia cocultured with either neurons or astrocytes downregulated inducible nitric oxide synthase (iNOS), indicating a decrease in the production of nitric oxide and possibly other toxic molecules. These findings indicate that in their natural environment, microglia are likely to be beneficial for the survival of embryonic dopaminergic grafts.     

In vitro and in vivo differentiation of boundary cap neural crest stem cells into mature Schwann cells

Boundary cap cells can generate neurons as well as peripheral glia during embryonic development (Maro, G.S., Vermeren, M., Voiculescu, O., Melton, L., Cohen, J., Charnay, P., Topilko, P., 2004. Neural crest boundary cap cells constitute a source of neuronal and glial cells of the PNS. Nat Neurosci. 7 (9), 930-938), and, recently, the boundary cap was shown to contain multipotent stem cells (Hjerling-Leffler, J., Marmigère, F., Heglind, M., Cederberg, A., Koltzenburg, M., Enerb?ck, S., Ernfors, P., 2005. The boundary cap, a source of neural crest stem cells generating multiple sensory neuron subtypes. Development. 132 (11), 2623-2632). The ability of stem cells to generate mature functional glial phenotypes has not been addressed. In this study, we have explored the competence of boundary neural crest stem cells (bNCSCs) to differentiate into mature functional Schwann cells (SCs) in vitro and in vivo. bNCSCs failed to differentiate into SCs in vitro when cultured in a defined media and in vivo when grafted into adult rat sciatic nerves. However, in the presence of neuregulins, during long-term cultures, the majority of bNCSCs differentiated into SCs. After analysis of the in vivo expression of Sox2, Sox10, S100, GFAP, fibronectin and Krox20 in the glial lineages, we used these markers to characterize differentiation of the bNCSCs. Gliogenesis of bNCSCs proceeded similar to that in vivo by sequentially adopting a SC precursor and immature Schwann cell before maturing into myelinating and non-myelinating SCs. In co-culture with explanted dorsal root ganglia (DRG) as well as in vivo in transplants to the axotomized sciatic nerve, these bNCSC-derived SCs myelinated axons as shown by ensheathing of neuronal processes and expression of myelin basic proteins (MBP). These results show that, under appropriate conditions, bNCSCs can generate mature SCs that are functional and can myelinate axons in regenerating nerves.     

Astrocytoma and Schwann cells in coculture

Glial fibrillary acidic protein (GFAP) is the principal intermediate filament protein found in mature astrocytes. Although the exact function of GFAP is poorly understood, it is presumed to stabilize the astrocyte’s cytoskeleton and help in maintaining cell shape. Previous studies from our laboratory have shown that when astrocytes were cocultured with primary Schwann cells (pSCs), astrocytes became hypertrophied and fibrous with intensely positive GFAP staining and segregated Schwann cells (SCs) into pockets. In order to understand the functional role of GFAP in this already established astrocyte-SC coculture model, we generated GFAP-negative cell lines from a GFAP-positive astrocytoma cell line and cocultured both the cell lines with pSCs. Our studies demonstrate that the GFAP-positive cell line put out processes toward the SCs, whereas the GFAP-negative cells did not form processes and the majority of the cells remained round. The most significant and interesting finding of this study, however, is the formation of elaborate processes by SCs when grown in coculture with the astrocytoma cells, unlike SCs cultured alone, which showed their typical bipolar spindle-shaped morphology. The extent of processes did not seem to be dependent on GFAP, since SCs cultured with both the cell lines formed similar processes. This coculture model may be useful in elucidating the factor(s) responsible for the formation of processes by SCs and can be further help in our understanding of the mechanism of morphological transformation of SCs.     

Environmental cues from CNS, PNS, and ENS cells regulate CNS progenitor differentiation

Cellular origin and environmental cues regulate stem cell fate determination. Neuroepithelial stem cells form the central nervous system (CNS), whereas neural crest stem cells generate the peripheral (PNS) and enteric nervous system (ENS). CNS neural stem/progenitor cell (NSPC) fate determination was investigated in combination with dissociated cultures or conditioned media from CNS, PNS, or ENS. Cells or media from ENS or PNS cultures efficiently promoted NSPC differentiation into neurons, glia, and smooth muscle cells with a similar morphology as the feeder culture. Together with CNS cells or its conditioned medium, NSPC differentiation was partly inhibited and cells remained immature. Here, we demonstrate that secreted factors from the environment can influence CNS progenitor cells to choose a PNS-like cell fate.     

常氧及低氧条件下胶质源性神经营养因子体外诱导中脑神经干细胞向多巴胺能神经元的分化

背景：神经干细胞的定向诱导分化和扩增受细胞自身基因和外来信号的调控。 目的：观察中脑源性神经干细胞在常氧、低氧和胶质源性神经营养因子诱导下向多巴胺能神经元的分化情况。 方法：无菌条件下分离E12小鼠胚胎腹侧中脑组织,胰酶消化和机械吹打制成单细胞悬液,在无血清培养基中培养扩增;Nestin免疫细胞化学染色方法鉴定神经干细胞。在有血清培养基中对纯化神经干细胞自然分化;神经元特异性烯醇化酶和胶质纤维酸性蛋白免疫细胞化学染色方法分别鉴定神经元和星形胶质细胞。建立常氧和低氧环境,设置常氧组、常氧+胶质源性神经营养因子组、低氧组、低氧+胶质源性神经营养因子组,按实验分组在有血清条件下诱导分化。 结果与结论：在低氧条件下,中脑神经干细胞向多巴胺能神经元分化均高于常氧组;尤其是低氧环境和胶质源性神经营养因子诱导下向多巴胺能神经元分化比例更高,表型更成熟。说明低氧环境下胶质源性神经营养因子可明显促进中脑神经干细胞分化为数量足够、形态及功能成熟的多巴胺能神经元。     

Dedifferentiated Schwann cells secrete progranulin that enhances the survival and axon growth of motor neurons

Schwann cells (SCs), the primary glia in the peripheral nervous system (PNS), display remarkable plasticity in that fully mature SCs undergo dedifferentiation and convert to repair SCs upon nerve injury. Dedifferentiated SCs provide essential support for PNS regeneration by producing signals that enhance the survival and axon regrowth of damaged neurons, but the identities of neurotrophic factors remain incompletely understood. Here we show that SCs express and secrete progranulin (PGRN), depending on the differentiation status of SCs. PGRN expression and secretion markedly increased as primary SCs underwent dedifferentiation, while PGRN secretion was prevented by administration of cAMP, which induced SC differentiation. We also found that sciatic nerve injury, a physiological trigger of SC dedifferentiation, induced PGRN expression in SCs in vivo. These results suggest that dedifferentiated SCs express and secrete PGRN that functions as a paracrine factor to support the survival and axon growth of neighboring neurons after injury.     

Bone marrow-derived Schwann cells achieve fate commitment – a prerequisite for remyelination therapy

Schwann cell transplantation improves post-traumatic nerve regeneration in both PNS and CNS but sufficient numbers of immunocompatible cells are required for clinical application. Currently, Schwann cell-like cells derived from the bone marrow lack fate commitment and revert to a fibroblast-like phenotype upon withdrawal of differentiation-inducing factors. In recapitulation of embryonic events leading to Schwann cell maturation, we hypothesize that the Schwann cell-like cells acquire the switch to fate commitment through contact-dependant cues from incipient neurons of the developing dorsal root ganglia. To address this, Schwann cell-like cells derived from adult rat bone marrow were cocultured with neurons purified from embryonic dorsal root ganglia. A cell-intrinsic switch to the Schwann cell fate was achieved consistently and the cell progeny maintained expression of the markers S100β, p75^NTR , GFAP, P0 and Sox 10 even without exogenous differentiation-inducing factors or neurons. In vitro formation of MBP-positive segments under myelinating conditions by the cell progeny was comparable to that by sciatic nerve-derived Schwann cells. Controls in which Schwann cell-like cells were barred from direct contact with neurons in coculture reverted to SMA/CD90-expressing myofibroblasts. We demonstrate therefore for the first time fate commitment among bone marrow-derived Schwann cells. The therapeutic potential of these cells may be tested in future transplantation studies. (206 words)     

Striatal target-induced axonal branching of dopaminergic mesencephalic neurons in culture via diffusible factors

The effects of striatal target cells on the morphological development of dopaminergic neurons were studied in dissociated cultures of embryonic rat mesencephalon. Mesencephalic neurons were cultured for four days in presence of target striatal cells or non target cerebellar ones. The outgrowth of dopaminergic neurons, visualized after tyrosine hydroxylase immunohistochemistry, was examined by quantitative morphometry. In cocultures, the increased complexity of dopaminergic neurites (branching) was the most striking pattern. It was dependent on the presence of target striatal cells as compared to non target ones. Cultures raised in presence or absence of serum lead to suggest the implicaton of striatal neurons rather than glia. Using MAP2 and phosphorylated neurofilaments immunohistochemistry in combination with tyrosine hydroxylase immunolabelling, it could be shown that the target-induced branching effect concerned only axonal and not dendritic processes. To further define whether diffusible factors from the striatal target would participate in the axonal branching effect, mesencephalic cells were cultured in conditioned medium from striatal neurons. Striatal conditioned medium enhanced dopamine uptake and dopamine neuron branching to the same extent as that observed in striatal cocultures. These findings demonstrate that soluble factors secreted by striatal neurons themselves selectively influence the branching of dopaminergic axons in vitro. J. Neurosci. Res. 48:358–371, 1997. © 1997 Wiley-Liss, Inc.