乳鼠雪旺氏细胞的纯化培养研究

目的：获得高纯度的乳鼠雪旺氏细胞。方法：采用出生后３～５天乳鼠的坐骨、臂丛神经，植块法培养乳鼠雪旺氏细胞；并通过精细剥除神经外膜、组织块反复再植、低浓度胰酶快速消化、差速贴壁法的有机结合对雪旺氏细胞进行纯化；继用抗Ｓ－１００蛋白单抗通过间接免疫荧光法及ＡＢＣ法进行细胞鉴定。结果：首次植块获得的细胞经纯化后雪旺氏细胞可高达８５％以上，反复植块者可达９５％，甚至高达９８％。结论：本方法简单、稳定，所获得的雪旺氏细胞纯度高、数量大、受非实验因素影响小     

人胎儿雪旺氏细胞的体外培养及其纯化研究

本实验用酶消化法从孕１４～１９周人胎儿周围神经中分离培养雪旺氏细胞，结合差速贴壁和阿糖胞苷处理进行纯化，根据Ｓ－１００蛋白免疫细胞化学染色和形态学特征，分析不同时期雪旺氏细胞的纯化程度。实验结果显示：培养４ｄ，倒置相差显微镜下观察，雪旺氏细胞呈典型的双极或三极形，端对端或旋涡状排列，Ｓ－１００蛋白免疫细胞化学染色呈阳性反应，其比例约占９８％；在２～３周内，雪旺氏细胞的纯度维持在８５％～９０％；培养３５ｄ，雪旺氏细胞约占８０％；单纯采用阿糖胞苷或差速贴壁处理，培养３５ｄ，雪旺氏细胞约占７０％；原代培养９２ｄ，传代１１代细胞生长良好。实验结果表明，多种方法联合使用较单一方法所得的纯度高。本方法快速、简便，能为雪旺氏细胞作为移植材料提供足够的来源。     

乳鼠肋间神经雪旺氏细胞培养

目的 通过12只2至4天大鼠(乳鼠)的肋间神经的雪旺氏细胞(SC)培养,获得原代雪旺氏细胞;方法 组织块反复种植法;结果 所有的肋间神经培养均能成活;结论 肋间神经是培养雪旺氏细胞的又一良好材料,在不增加成本的前提下,一只乳鼠上可获得更多的雪旺氏细胞.     

兔背根神经节植块法的雪旺氏细胞培养

目的:建立一种新的培养雪旺氏细胞(Schwann cell,SC)的方法,为神经移植研究获取高纯度的细胞奠定基础。方法:采用植块培养的方法,分离新生1～2天兔子的背根神经节(Dorsal root ganglion,DRG),弃膜,剪碎进行植块的雪旺氏细胞的培养,然后采用阿糖胞苷(cytosine arabinoside,Ara-C)抑制,差速贴壁法、Forskolin的牛垂体提取液(bovine pituitary extract,BPE)刺激进行细胞纯化,最后S-100免疫组织化学染色进行纯度鉴定。结果:本实验采用背根神经节植块及纯化的方法获得雪旺氏细胞的纯度可达95％以上。结论:这种选用DRG组织块培养及纯化SC的方法效率很高而且简单方便,所获SC的纯度高数量大。     

LPS对大鼠雪旺氏细胞TNF-α合成和释放的影响

目的：探讨内毒素脂多糖（lipopolysaccharide,LPS）的诱导作用对大鼠雪旺氏细胞肿瘤坏死因子α（tumor nec-rosis factor-α,TNF-α）的表达。方法：不同浓度,不同时间用LPS刺激雪旺氏细胞,用酶联免疫吸附实验（ELISA）分别检测细胞胞液和上清中分泌的TNF-α的表达量,同时用间接免疫荧光细胞化学染色检测TNF-α的细胞定位。结果：用LPS10μg/ml刺激后2小时能显著促进雪旺氏细胞浆内TNF-α的表达,同时也检测到培养液上清中有TNF-α的释放。结论：细菌的致病因子LPS的诱导确能促进雪旺氏细胞高效表达和分泌TNF-α,从而为雪旺氏细胞作为免疫活性细胞在周围神经系统中发挥免疫调节作用提供初步依据。     

流式细胞术检测细胞凋亡比较研究

从三方面探讨检测细胞凋亡流式细胞术的方法.选用大鼠皮质神经元细胞,同时使用碘化丙锭(PI)An-nexin V/PI.JC-1法检测细胞凋亡率.结果显示三种方法的凋亡率分别是:2.60%,6.52%和18.56%,两两之间有显著性差异(P＜0.01).JC-1法最敏感,AV/PI次之,PI单染法不适合检测早期细胞凋亡.     

雪旺氏细胞植入Dacron人造管修复周围神经缺损的实验研究

用自体或异体雪旺氏细胞植入Ｄａｃｒｏｎ管，移植修复大鼠坐骨神经缺损，术后７、１５、３０天取材进行光镜观察，见植入自体雪旺氏细胞组，再生轴突数与再生轴恢复率均优于不植入雪旺氏细胞组和异体雪旺氏细胞组，与自体神经全转回值组结果相近。实验表明：自体雪旺氏细胞植入到渗透作用好、无排斥反应的人造Ｄａｃｒｏｎ管中，同时加入自体血浆作营养支持物，对周围神经再生有较好的促进作用，可望有临床应用前景。     

雪旺氏细胞从冷冻人胎周围神经的分离技术及其特性

本研究取１５～２０周人胎周围神经，放在冷冻的含ＤＭＥＭ（４０％）、ＤＭＳＯ（１０％）及小牛血清（５０％）中。细胞活性为９１％。雪旺氏细胞经过分离、培养、保持了活性。经３～６月液氮保存后，细胞活性为９４％，雪旺氏细胞在４℃下经２４小时，活性仍有９２％。从新鲜周围神经取得的和经冷冻保存的以及单个培养的雪旺氏细胞，在培养时生长缓慢，但在培养液中加牛脑垂体提取液后，细胞增殖加快。取自新鲜或冷冻的周围神经的雪旺氏细胞用Ｓ－１００蛋白抗体染色的特性相似。本文还报告了分离出的雪旺氏细胞在扫描及透射电镜下的形态特征。     

聚吡咯和背根神经节及游离细胞联合培养形态学观察

目的：探讨聚吡呼和周围神经组织的生物相容性。方法：取新生大鼠的背根神经节及细胞与聚吡咯膜联合培养,免疫细胞化学双重染色,光镜和扫描电镜观察。结果：背根神经节中,雪旺氏细胞生长迁移活跃;神经细胞突起借助雪旺氏细胞向外延伸,同时雪旺氏细胞和神经元又可借助这些神经突起向外迁移。游离神经细胞能在聚吡咯膜上良好生长,伸出多极突起及分枝。结论：在体外,聚吡咯与大鼠周围神经组织有较好的生物相容性。     

LPS对大鼠雪旺氏细胞iNOS表达的影响

目的：探讨内毒素脂多糖（Lipopolysaccharide,LPS）对大鼠雪旺氏细胞（Schwann cells,Scs）诱导型一氧化氮合酶（Inducible nitric-oxide synthase,iNOS）基因表达及一氧化氮（Nitric oxide,NO）生成的影响。方法：用不同浓度（1、10、100μg/ml）和同一浓度不同时间（1、2、4、6小时）的LPS刺激雪旺氏细胞,分别用RT-PCR和亚硝酸盐含量测定观察细胞iNOS mRNA的表达量和细胞培养液中亚硝酸盐的水平,同时用免疫荧光细胞化学染色检测iNOS的细胞定位。结果：用LPS10μg/ml刺激2小时后,iNOS mRNA的表达增加,4小时表达活性最高。细胞上清中的亚硝酸盐含量高峰在6小时。免疫细胞化学证明LPS诱导雪旺氏细胞iNOS的表达定位在胞浆。结论：LPS可在转录水平上诱导雪旺氏细胞iNOS mRNA表达,促进NO的合成,提示雪旺氏细胞在周围神经系统炎症过程中可能发挥免疫调节作用。     

Delayed nerve repair increases number of caspase 3 stained Schwann cells

Caspase 3 staining in Schwann cells was investigated with immunohistochemistry, as a measure of Schwann cell apoptosis, after transection and immediate (day 0) or delayed rat sciatic nerve repair (30, 90 and 180 days post injury). Cleaved caspase 3 stained Schwann cells significantly increased at the site of lesion (SNL; median [IQR], 15.2 [7.0] %) and in the distal nerve segment (SND; 9.5 [3.6] %) 10 days after immediate repair. The number of cleaved caspase stained Schwann cells also increased significantly after delayed repair, irrespective of length of delay, at both locations (SNL: 22.0–27.1%; SND: 18.5–22.1%; p < 0.05). Some cleaved caspase 3 stained satellite cells were seen in dorsal root ganglia on the injured side, but no stained motor or sensory neurons were observed at any time-point. Delayed nerve repair is associated with more pronounced Schwann cell apoptosis which may explain impaired nerve regeneration after nerve injury and delayed repair.     

Schwann细胞与硫酸肝素-胶原蛋白支架材料的复合

本研究目的是观测硫酸肝素-胶原蛋白支架材料对Schwann细胞的相容性,采用: (1)冷冻干燥法制备硫酸肝素-胶原蛋白支架材料,培养、纯化并鉴定Schwann细胞后,将Hoechst荧光标记的Schwann细胞复合于硫酸肝素-胶原支架材料中,荧光显微镜、扫描电镜及光镜下观察其在材料内部的空间排列形式; (2)以台盼蓝染色、MTT法检测Schwann细胞与硫酸肝素-胶原蛋白共培养时的生物活性。结果显示:作者制备的支架材料具有纵行的、平行排列的微管结构,Schwann细胞在支架材料微管内成线性平行排列,类似于神经基底膜与Schwann细胞形成的Büngner带;台盼蓝染色Schwann细胞死亡率仅6. 47%,MTT法证明Schwann细胞与硫酸肝素-胶原蛋白材料共培养时保有高度的生物活性。本研究结果表明:以硫酸肝素复合胶原蛋白结合特定条件下冷冻干燥技术可以制备在组分和内部空间结构上高度仿生神经的新型神经组织工程支架材料,与Schwann细胞有良好的细胞相容性。     

坐骨神经瓦勒变性大鼠许旺细胞生物学特性及分泌功能变化

背景：研究表明外周神经损伤后,许旺细胞在基底膜管内形成Bunger带,引导再生轴突延伸,但具体作用机制目前尚不清楚。 目的：观察大鼠坐骨神经损伤后瓦勒变性对许旺细胞生物学特性及分泌功能的影响。 方法：建立大鼠坐骨神经横切模型,分为坐骨神经瓦勒变性组(坐骨神经横断组)和手术对照组。采用神经段单酶消化法分离培养许旺细胞,光镜下观察细胞形态变化,S-100免疫荧光鉴定。取第1代许旺细胞,利用计数法绘制14 d内许旺细胞的生长曲线,MTT法检测14 d内许旺细胞增殖活性,酸性磷酸酶法检测许旺细胞黏附能力,ELISA法检测神经生长因子浓度。 结果与结论：坐骨神经段培养第14天,坐骨神经横断组神经段边缘可见大量许旺细胞,呈线形排列;手术对照组许旺细胞数量少,呈散在分布,两组许旺细胞S-100均呈阳性表达。许旺细胞传代培养第3天,两组许旺细胞均进入对数增长期,随时间延长,细胞数及细胞增殖吸光度值均呈上升趋势,坐骨神经横断组细胞数及增殖吸光度值明显高于手术对照组(P < 0.05);坐骨神经横断组许旺细胞黏附能力明显高于手术对照组(P < 0.05);ELISA法检测示,坐骨神经横断组神经生长因子浓度在培养第4,6,8,10,12,14天时均高于手术对照组(P < 0.05)。结果表明大鼠坐骨神经损伤后两三周,瓦勒变性对许旺细胞生物学功能具有显著影响,可诱导许旺细胞幼稚化,促使许旺细胞在短期内迅速分裂增殖,并分泌大量神经营养因子及细胞外黏附成分,为再生轴突的延伸提供适宜的神经微环境;并增加细胞黏附能力,为外周神经损伤修复提供适宜的神经微环境。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

Visualization of regenerating sciatic nerve fibres by neurofilament immunohistochemistry

Regeneration of injured sciatic nerve in rats was studied with an immunohistochemical technique visualizing neurofilaments in nerve fibres as well as S-100 protein in Schwann cells. Outgrowing axons with delicate sprouts could be demonstrated along pathways formed by Schwann cells. In contrast, axons which had stopped growing or were degenerating showed bulb-like swellings in their terminal parts. The use of immunohistochemical techniques offers advantages over conventional neurohistochemical staining methods, enabling more detailed observations of nerve regeneration mechanisms in animals.     

Expression of Toll-like receptor 2 on human Schwann cells: a mechanism of nerve damage in leprosy

Nerve damage is a clinical hallmark of leprosy and a major source of patient morbidity. We investigated the possibility that human Schwann cells are susceptible to cell death through the activation of Toll-like receptor 2 (TLR2), a pattern recognition receptor of the innate immune system. TLR2 was detected on the surface of human Schwann cell line ST88-14 and on cultured primary human Schwann cells. Activation of the human Schwann cell line and primary human Schwann cell cultures with a TLR2 agonist, a synthetic lipopeptide comprising the N-terminal portion of the putative Mycobacterium leprae 19-kDa lipoprotein, triggered an increase in the number of apoptotic cells. The lipopeptide-induced apoptosis of Schwann cells could be blocked by an anti-TLR2 monoclonal antibody. Schwann cells in skin lesions from leprosy patients were found to express TLR2. It was possible to identify in the lesions Schwann cells that had undergone apoptosis in vivo. The ability of M. leprae ligands to induce the apoptosis of Schwann cells through TLR2 provides a mechanism by which activation of the innate immune response contributes to nerve injury in leprosy.     

一种新的组织块培养新生大鼠雪旺细胞方法(英文)

目的:为了建立一种组织块培养雪旺细胞的有效方法,为周围神经组织工程修复的研究提供种子细胞。方法:本研究从新生大鼠的周围神经分离雪旺细胞,经组织块法培养并纯化,观察并记录其形态特征;经HE染色以及S-100免疫组化染色鉴定并计算其纯度。结果:结果显示,4 d后,获得的雪旺细胞突起呈双极或三极,S-100阳性细胞纯度达到95%以上。结论:上述结果表明该方法快速有效,所获得的雪旺细胞纯度高、活性好。     

感觉神经和运动神经Schwann细胞的培养研究

为了建立应用于神经组织工程的感觉神经和运动神经的 Schwann细胞的体外培养方法 ,采用联合酶消化法对 SD乳鼠后根神经和股神经运动支的 Schwann细胞进行了培养 ,并经抗 S10 0荧光染色鉴定和双抗体夹心间接 Elisa法测量两种 Schwann细胞培养基中 NGF的表达。结果表明 ,两种培养细胞经荧光染色证明均为 Schwann细胞 ,细胞纯度均超过 95 % ,光镜观察未见形态学差异 ,但 NGF的表达量和表达模式均有显著性差别 (P<0 .0 5 )。提示 ,本实验方法可以获得高纯度的感觉神经和运动神经的 Schwann细胞 ,两者的生物学功能有一定差异。     

GAP-43 and p75NGFR immunoreactivity in presynaptic cells following neuromuscular blockade by botulinum toxin in rat

Summary Peripheral nerve lesion results in changes in protein expression by neurons and denervated Schwann cells. In the present study we have addressed the question whether similar changes take place following functional denervation. Using immunohistochemistry and immunoelectron microscopy we examined changes in growth-associated protein (GAP-43) and low-affinity nerve growth factor receptor (p75^NGFR) in rat gastrocnemius muscle following botulinum toxin-induced paralysis. GAP-43 and p75^NGFR were selected because they are not expressed by mature intact motor neurons or Schwann cells, but are expressed following nerve lesion in both motor neurons and denervated Schwann cells. In control muscle, GAP-43 and p75^NGFR immunoreactivity was seen only in nerve fibres near blood vessels. Two weeks after toxin injection, GAP-43 immunoreactivity could be seen at the motor endplates and in axons. Intensity of staining increased with longer survival and reached a peak between 4 and 8 weeks post-injection. Ultrastructurally, GAP-43 immunoreactivity was confined to nerve terminals and axons, whereas Schwann cells remained negative. Immunostaining for p75^NGFR also increased following toxin injection and was detected in some terminal Schwann cells and in perineurial cells of small nerve fascicles near the paralyzed target cells, but not in axons. These results show that changes in expression of GAP-43 in motor neurons following functional denervation closely resemble the changes following anatomical interruption of nerve-muscle contact. GAP-43 was not expressed in Schwann cells, indicating that its upregulation in these cells is induced by loss of axonal contact or nerve degeneration products. There is no support for a role of p75^NGFR in incorporation of neurotrophins in axons. The restriction of p75^NGFR expression to terminal Schwann cells and perineurial cells in close proximity to the paralyzed target suggests a role for a target-derived signal or, alternatively, macrophages in eliciting this expression.     

miR-21在周围神经损伤时调控雪旺细胞凋亡

目的:探讨周围神经损伤时,微小RNA-21(microRNA-21,miR-21)与雪旺细胞(SC)凋亡的关系及相关分子机制。方法:采用实时荧光定量PCR(real-time PCR)检测动物模型中miR-21以及人第10号染色体缺失的磷酸酶及张力蛋白同源基因(phosphatase and tensin homologue deleted on chromosome 10,PTEN)的表达情况;通过将miR-21类似物(miR-21-mimic)、miR-21抑制物(miR-21-inhibitor)和阴性对照miRNA(negative control miRNA,NC-miRNA)转染入RSC96细胞中,构建出过表达miR-21的雪旺细胞株(MI-SC)、抑制miR-21表达的雪旺细胞株(IN-SC)及表达对照miRNA的雪旺细胞株(NC-SC);采用流式细胞仪检测3组细胞的凋亡情况;real-time PCR检测3组细胞中miR-21以及PTEN的表达情况;Western blot检测3组细胞中PTEN及凋亡相关蛋白激活型caspase-3(cleaved caspase-3)的表达情况。结果:神经损伤组miR-21的表达量与对照组相比明显升高,神经损伤组PTEN mRNA的表达水平与对照组相比明显降低;与NC-SC相比,上调miR-21组细胞的凋亡比例减少,PTEN mRNA及蛋白的表达水平降低,cleaved caspase-3的表达水平降低,下调miR-21组细胞的凋亡比例增加,PTEN mRNA及蛋白的表达水平升高,cleaved caspase-3的表达水平升高(P0.05)。结论:miR-21可能通过下调PTEN的表达抑制雪旺细胞的凋亡,从而可能在周围神经的损伤修复中发挥作用。