三色染色法和免疫细胞化学法显示培养的神经轴索及雪旺细胞

本研究目的在于用三色染色法和免疫细胞化学反应显示培养的周围神经中的轴索及雪旺细胞。将大鼠背根神经节体外培养于聚吡咯膜上 2周 ;用苏木精、固绿 FCF、变色素 2 R及磷钨酸等配制的染色液染色 ;或用抗 S-10 0蛋白和抗神经微丝蛋白抗体进行免疫细胞化学法反应。结果证明 ,在三色染色法中神经节神经元发出的长突起呈蓝绿色 ,细胞核呈红色或紫红色 ,胞质呈浅灰色。免疫细胞化学反应证明神经节发出的长突起为轴索 ,紫红色核和浅灰色胞质的细胞为雪旺细胞。本文结果提示 ,三色染色法能区别显示培养的周围神经组织中的轴索及雪旺细胞。     

雪旺氏细胞对中枢神经轴突生长的支持和促进作用

胚胎大鼠脊髓植块接种于纯度〉９５％的单层人胎儿雪旺氏细胞表面进行培养，观察雪旺氏细胞对神经突起生长的支持作用，脊髓神经突起在雪旺氏细胞表面活跃生长，突起生长速度〉４５０μｍ／天，与对照组相比有显著性差异。将长于鼠尾胶上的雪旺氏细胞移植于成年大鼠脊髓损伤腔内，移植后２１天后，透射电镜观察，有髓或无髓纤维长人移植物中，移植后３０天，长入移植物中的轴突更丰富，而无细胞的胶原移植物中未见轴突长入。结果表明     

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。     

细胞外基质对培养神经元细胞存活和轴突生长的影响

目的：探讨不同的细胞外基质对体外培养胚胎大鼠脊髓运动神经元和背根神经节感觉神经元生长的影响。方法：取大鼠胚胎脊髓腹侧组织和背根神经节体外分离培养，选取不同生长底物包括多聚赖氮酸(PLL)、Ⅰ型胶原(CoⅠ)、层粘连蛋白(LN)、PLL联合LN进行包被培养板，观察运动神经元和感觉神经元的体外生长状况。结果：PLL和LN联合包被时，神经元存活率高，细胞分散良好。CoⅠ包被时细胞聚集成团．突起粗长。结论：不同的细胞外基质影响神经元的生长方式，PLL联合LN包被是体外研究单一神经元胞体和突起的较好方法。     

GDNF促进大鼠背根神经元的存活和突起生长

探讨胶质细胞源性神经营养因子(GDNF)对正常胚胎大鼠背根神经节(DRGn)的存活及突起生长的作用。本实验采用神经组织原代分离培养的方法建立体外胚胎大鼠背根神经节单细胞培养体系，从细胞形态学及应用MTT。法观察1μg／L、10μg／L、50μg／L和100μg／L GDNF对体外培养的正常感觉神经元生长的影响。结果表明：GDNF能明显促进体外培养的正常大鼠背根神经节感觉神经元的存活及突起生长。提示GDNF对正常大鼠胚胎发育期感觉神经元具有神经营养作用。     

神经再生素促大鼠背根神经节生长作用的研究

目的研究神经再生素（NRF）对体外培养新生大鼠背根神经节生长及NF—H表达的影响。方法体外大鼠背根神经节（DRG）培养;免疫荧光细胞化学、实时荧光定量PCR和Western blot等方法。结果免疫荧光细胞化学结果提示,NRF能促进背根神经节神经突起的生长,浓度为2．0mg／L时生长状况最佳;实时荧光定量PCR和Western blot结果提示,NRF能增加体外培养的背根神经节NF-H mRNA和蛋白的表达,在浓度为2．0mg／L时表达最高。结论NRF能促进体外培养背根神经节神经突起的生长和NF—H的表达,表明NRF对发育期感觉神经元具有神经营养作用。     

坐骨神经慢性卡压损伤模型大鼠背根神经节纤维化改变

文题释义：组织纤维化：是结缔组织过度增生和细胞外基质沉积的结果,纤维化过程是一个异常的、不受控制的组织修复过程,其中组织损伤和自身免疫疾病是导致组织纤维化的主要因素,过度的组织纤维化最终导致组织结构和功能改变。 慢性神经卡压损伤：指周围神经在特定部位受到慢性卡压引起的相应神经功能障碍。 背景：既往的研究主要集中于周围神经慢性卡压损伤所导致的靶器官——骨骼肌萎缩及其纤维化发生的机制研究,关于周围神经慢性卡压损伤信号向上引起背根神经节功能改变的研究较少。 目的：观察大鼠坐骨神经慢性卡压损伤对背根神经节纤维化的影响。方法：按照Mackinnon设计的方法制备大鼠坐骨神经慢性卡压模型,术后3周,分别取大鼠坐骨神经卡压侧和对侧L_4-6背根神经节,采用RT-PCR、Western blot及免疫荧光检测大鼠卡压侧和对照侧背根神经节内转化生长因子β1、结缔组织生长因子及胶原蛋白Ⅰ的表达变化。结果与结论：①损伤3周后,相比于对照侧,背根神经节内转化生长因子β1、结缔组织生长因子及胶原蛋白Ⅰ的mRNA及蛋白表达均具有相同的升高趋势(P < 0.05);②损伤3周后,大鼠卡压侧和对照侧背根神经节内转化生长因子β1和结缔组织生长因子主要表达在背根神经元内和轴突内,而胶原蛋白Ⅰ主要表达在背根神经元和轴突的周围,形成包绕背根神经元和轴突的网状结构;③上述数据证实,大鼠坐骨神经慢性卡压损伤可以导致背根神经节纤维化的改变,并且背根神经节纤维化的改变可能与背根神经元内转化生长因子β1及结缔组织生长因子表达升高有关。ORCID: 0000-0003-1632-0837(黎琴文) 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     

导电聚合物聚吡咯/聚苯乙烯硫酸酯刺激体外神经轴突生长的生物活性及组织相容性

背景:导电聚合物的合成较具灵活性,通过改变合成条件,可以使导电聚合物表现出不同的、更适宜的表面特性。目的:观察导电聚合物聚吡咯/聚苯乙烯硫酸酯的生物相容性以及对体外神经细胞轴突生长的刺激效应。方法:应用电化学聚合聚吡咯/聚苯乙烯硫酸酯膜片,检测导电膜片的电化学指标;然后通过体外电刺激,分4组:电刺激组、无点刺激组、空白对照组和溶液对照组观察聚吡咯/聚苯乙烯硫酸酯对嗜铬细胞瘤细胞轴突生长的生物效应;制备聚吡咯/聚苯乙烯硫酸酯-聚乳酸-聚羟基乙酸共聚物膜片,并移植到大鼠体内,观察膜片的短期和长期炎性反应,评价其组织相容性。结果与结论:体外细胞培养图像显示,在未接受电刺激条件下,对照组和聚吡咯/聚苯乙烯硫酸酯膜上嗜铬细胞瘤细胞贴附、分化情况相似。通过聚吡咯/聚苯乙烯硫酸酯膜基质进行电刺激实验定性观察,聚吡咯/聚苯乙烯硫酸酯膜上的细胞如处在电流环境中则可使神经轴突延伸的更长,而未接受电刺激的神经轴突长度明显短于前者。此外,接受电刺激组的细胞的铺展形态明显要好于对照组,但均未见任何细胞毒性反应。体内实验现实该膜片没有诱导出明显的炎性反应。提示,聚吡咯/聚苯乙烯硫酸酯是一种组织相容性较好的材料。     

雪旺氏细胞的功能研究进展

雪旺氏细胞是周围神经系统的胶质细胞，具有多种生理功能，当周围神经受损时，反应性地分裂增殖，形成Ｂｕｎｇｎｅｒ带，为轴突再生提供通道；同时雪旺氏细胞分泌数种神经营养因子，防止受损神经经元死亡，促进轴突再生；雪旺氏细胞还分泌细胞外基质成分和细胞粘附分子等，为轴突体供良好的再生环境，支持和引导再生的轴突重新支配靶组织。将体外培养的雪旺氏细胞进行移植，不仅能促进周围神经和中枢神经再生，而且能修复脱髓鞘损伤。     

胶质细胞源性神经营养因子对培养的背根神经节的影响

目的 探讨胶质细胞源性神经营养因子(GDNF)在体外促进正常胚胎大鼠背根神经节(DRGn)的存活及突起生长情况。方法 用原代分离培养法建立体外胚胎大鼠背根神经节单细胞培养体系，通过活体观察、MTT微量比色法、NSE免疫组织化学染色观察不同浓度GDNF对体外培养的正常感觉神经元的影响。结果 GDNF组培养的DRG神经元存活数量增加，神经元突起的长度比对照组明显增长。结论 GDNF能明显促进体外培养的正常大鼠胚胎背根神经节感觉神经元的存活及突起生长，表明GDNF对正常大鼠胚胎发育期感觉神经元具有神经营养作用。     

Stimulation of neurite outgrowth using positively charged hydrogels

Autologous nerve grafts are currently the best option for the treatment of segmental peripheral nerve defects. However, autografts have several drawbacks including size mismatch and loss of sensation in the donor nerve's sensory distribution. In this work, we have investigated the development of a synthetic hydrogel that contains positive charge for use as a substrate for nerve cell attachment and neurite outgrowth in culture. We have demonstrated that modification of oligo-(polyethylene glycol) fumarate (OPF) with a positively charged monomer improves primary sensory rat neuron attachment and differentiation in a dose-dependent manner. Positively charged hydrogels also supported attachment of dorsal root ganglion (DRG) explants that contain sensory neurons, Schwann cells and neuronal support cells. Furthermore, charged hydrogels were analyzed for the appearance of myelinated structures in a co-culture containing DRG neurons and Schwann cells. DRGs and Schwann cells remained viable on charged hydrogels for a time period of three weeks and neurites extended from the DRGs. Sudan black staining revealed that neurites emerging from DRGs were accompanied by migrating Schwann cells. These findings suggest that charged OPF hydrogels are capable of sustaining both primary nerve cells and the neural support cells that are critical for regeneration.     

鸡后根神经节多巴胺细胞在体及体外培养中的免疫细胞化学观察

应用免疫细胞化学PAP法,研究了鸡胚和雏鸡后根神经节细胞内多巴胺递质的表现与胚胎发育过程中的变化,并在细胞培养条件下研究了周围靶组织——皮肤对后根节细胞胚胎发育的影响。鸡后根节细胞内多巴胺的免疫反应最先出现于E_(10),只占后根节细胞的0.8%,E_(18)时达5.6%,出壳后雏鸡为8.6%。在鸡后根节内,多巴胺免疫反应阳性细胞主要为较大的A类细胞,和极少较小的B类细胞。免疫反应阳性的周围神经末梢位于皮肤和交感干内。当取自E_9的鸡胚后根节细胞经7天培养后,部分神经细胞出现多巴胺免疫反应阳性;而取自E_6的鸡胚后根节细胞虽经10天培养后仍为免疫反应阴性;然而,将取自E_6后根节细胞与皮肤组织联合培养10天后,少量神经细胞及轴突出现多巴胺免疫阳性反应。作者认为:鸡后根节多巴胺细胞在胚胎时期的发育有赖于周围靶组织的作用。     

Biocompatibility evaluation of silk fibroin with peripheral nerve tissues and cells in vitro

Silk-based materials have been used in the field of bone or ligament tissue engineering. In order to explore the feasibility of using purified silk fibroin to construct artificial nerve grafts, it is necessary to evaluate the biocompatibility of silk fibroin material with peripheral nerve tissues and cells. We cultured rat dorsal root ganglia (DRG) on the substrate made up of silk fibroin fibers and observed the cell outgrowth from DRG during culture by using light and electron microscopy coupled with immunocytochemistry. On the other hand, we cultured Schwann cells from rat sciatic nerves in the silk fibroin extract fluid and examined the changes of Schwann cells after different times of culture. The results of light microscopy, MTT test and cell cycle analysis showed that Schwann cells cultured in the silk fibroin extract fluid showed no significant difference in their morphology, cell viability and proliferation as compared to that in plain L15 medium. Furthermore, no significant difference was found in expression of the factors secreted by Schwann cells, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and S-100, between Schwann cells cultured in the silk fibroin extraction fluid and in plain L15 medium by the aid of immunocytochemistry, RT-PCR and Western analysis. Collectively, these data indicate that silk fibroin has good biocompatibility with DRG and is also beneficial to the survival of Schwann cells without exerting any significant cytotoxic effects on their phenotype or functions, thus providing an experimental foundation for the development of silk fibroin as a candidate material for nerve tissue engineering applications.     

在体和离体的鸡背根节神经细胞5-羟色胺的免疫组织化学研究

用PAP免疫组织化学方法研究了鸡胚和雏鸡背根节内5-HT神经细胞的形态与胚胎发育,并在离体细胞培养条件下研究了靶周围组织(皮肤)对其胚胎发育的影响。在鸡腰骶部背根节,5-HT免疫反应阳性细胞最先出现于鸡胚13d(E_(13)),占0.4%;孵出后2d(AH_2)占6.2%。阳性细胞主要为大的A类细胞和极少的小B类细胞。免疫反应阳性的周围神经末梢位于皮肤和跟腱中。取自E_9鸡胚的背根节细胞培养7d后有部分出现5-HT阳性免疫反应,取自E_6鸡胚的背根节细胞培养10d仍为阴性反应;然而,当取自E_6鸡胚的背根节细胞与皮肤组织联合培养10d后,则出现5-HT阳性免疫反应细胞。本文还对脊髓5-HT细胞的出现进行了讨论。     

Neurons and satellite glial cells in adult rat lumbar dorsal root ganglia express connexin 36

Previous studies have shown that following peripheral nerve injury there was a downregulation of the gap junction protein connexin 36 (Cx36) in the spinal cord; however, it is not known whether Cx36 protein is expressed in the dorsal root ganglia (DRGs), nor if its levels are altered following peripheral nerve injuries. Here we address these aspects in the adult rat lumbar DRG. Cx36 mRNA was detected using qRT-PCR, and Cx36 protein was identified in DRG sections using immunohistochemistry (IHC) and immunofluorescence (IF). Double staining revealed that Cx36 co-localizes with both anti-β-III tubulin, a neuronal marker, and anti-glutamine synthetase, a satellite glial cell (SGC) marker. In neurons, Cx36 staining was mostly uniform in somata and fibers of all sizes and its intensity increased at the cell membranes. This labeling pattern was in contrast with Cx36 IF dots mainly found at junctional membranes in islet beta cells used as a control tissue. Co-staining with anti-Cx43 and anti-Cx36 showed that whereas mostly uniform staining of Cx36 was found throughout neurons and SGCs, Cx43 IF puncta were localized to SGCs. Cx36 mRNA was expressed in normal lumbar DRG, and it was significantly down-regulated in L4 DRG of rats that underwent sciatic nerve injury resulting in persistent hypersensitivity. Collectively, these findings demonstrated that neurons and SGCs express Cx36 protein in normal DRG, and suggested that perturbation of Cx36 levels may contribute to chronic neuropathic pain resulting from a peripheral nerve injury.     

Survival and regeneration of cutaneous and muscular afferent neurons after peripheral nerve injury in adult rats

Peripheral nerve injury induces the retrograde degeneration of dorsal root ganglion (DRG) cells, which affects predominantly the small-diameter cutaneous afferent neurons. This study compares the time-course of retrograde cell death in cutaneous and muscular DRG cells after peripheral nerve transection as well as neuronal survival and axonal regeneration after primary repair or nerve grafting. For comparison, spinal motoneurons were also included in the study. Sural and medial gastrocnemius DRG neurons were retrogradely labeled with the fluorescent tracers Fast Blue (FB) or Fluoro-Gold (FG) from the homonymous transected nerves. Survival of labeled sural and gastrocnemius DRG cells was assessed at 3 days and 1–24 weeks after axotomy. To evaluate axonal regeneration, the sciatic nerve was transected proximally at 1 week after FB-labeling of the sural and medial gastrocnemius nerves and immediately reconstructed using primary repair or autologous nerve grafting. Twelve weeks later, the fluorescent tracer Fluoro-Ruby (FR) was applied 10 mm distal to the sciatic lesion in order to double-label sural and gastrocnemius neurons that had regenerated across the repair site. Counts of labeled gastrocnemius DRG neurons did not reveal any significant retrograde cell death after nerve transection. In contrast, sural axotomy induced a delayed loss of sural DRG cells, which amounted to 22% at 4 weeks and 43–48% at 8–24 weeks postoperatively. Proximal transection of the sciatic nerve at 1 week after injury to the sural or gastrocnemius nerves neither further increased retrograde DRG degeneration, nor did it affect survival of sural or gastrocnemius motoneurons. Primary repair or peripheral nerve grafting supported regeneration of 53–60% of the spinal motoneurons and 47–49% of the muscular DRG neurons at 13 weeks postoperatively. In the cutaneous DRG neurons, primary repair or peripheral nerve grafting increased survival by 19–30% and promoted regeneration of 46–66% of the cells. The present results suggest that cutaneous DRG neurons are more sensitive to peripheral nerve injury than muscular DRG cells, but that their regenerative capacity does not differ from that of the latter cells. However, the retrograde loss of cutaneous DRG cells taking place despite immediate nerve repair would still limit the recovery of cutaneous sensory functions.     

大鼠脊神经节神经元对其中枢突与外周突刺激的反应

本文应用细胞内记录技术在大鼠腰骶段脊神经节（DRG）记录到68个单位，其中对刺激坐骨神经和背根，盆神经和背根，坐骨神经、盆神经和背报以及时侧背报发生反应的单位分别为33、11、16和8个；同侧坐骨神经、盆神经和背根刺激诱发反应的传导速度分别平均为36．80±17．86m／s（x±s，下同）；36．76±17．66m／s和32．11±16．39m／s．刺激一侧背根，在对侧脊神经节记录到短、长和短长潜伏期兼有的单位分别为3、2和3个．结果提示：大鼠脊神经节神经元具有分叉的外周突和中枢突；两侧脊种经节神经元之间存在交互支配．     

大鼠颈脊神经节中降钙素基因相关肽免疫阳性神经元分支至颈椎关节突关节囊和前肢

目的探讨颈椎关节突关节疼痛综合征并发上臂痛的神经解剖学机制。方法以抗降钙素基因相关肽（CGRP）抗体对SD大鼠C4-T1关节突关节囊作免疫组织化学染色。将荧光标记物快蓝（FB）和核黄（NY）分别注射到SD大鼠右侧桡神经和C1～T1颈椎关节突关节囊。显微镜下观察颈脊神经背根节（DRG）内被逆行荧光标记的神经元，再用抗CGRP抗体行免疫组织化学染色，观察DRG内的CGRP免疫阳性神经元。最后比较荧光照片和免疫组织化学照片上的神经元。结果C4～T1关节突关节囊上分布着丰富的CGRP免疫阳性神经纤维，它们或独立走行或交织成网。在C5～T1各节段的DRG中均可观测到被FB单标（76％）、NY单标（16％）和FB／NY双标（8％）的神经元。约40％的荧光逆行标记神经元在免疫组织化学染色的切片上呈CGRP免疫反应阳性。结论大鼠颈部DRG内一部分含GRP的感觉神经元的周围突分两支，一支支配颈椎关节突关节囊，另一支随桡神经分布到前（上）肢。提示，颈椎关节突关节疼痛综合征并发上臂牵涉痛的神经形态学基础之一可能发生在DRG水平。     

The occurrence of nitric oxide synthase-containing axonal baskets surrounding large neurons in rat dorsal root ganglia after sciatic nerve ligation

To clarify the possible role of nitric oxide (NO) induced in primary sensory neurons after peripheral axotomy, NO synthase (NOS) immunohistochemistry was carried out on rat L5 dorsal root ganglia after sciatic nerve ligation. The results were compared with the expression of 27-kDa heat shock protein (HSP27), a neuroprotective molecule. In intact animals, NOS-immunoreactive neurons represented about 2% of all dorsal root ganglion (DRG) neurons, whereas HSP27-immunoreactive neurons comprised about 14%. After sciatic nerve ligation, both neurons increased, in number and immunoreactivity, reaching a maximum at 2 weeks, when NOS- and HSP27-immunoreactive neurons represented about 33 and 66%, respectively. NOS-immunoreactive neurons then remained unchanged until 7 weeks although HSP27-immunoreactive neurons showed a slight decline. The increased NOS-immunoreactive neurons were preferentially small (100-500 microm(2)) and coexpressed with HSP27 (about 87%). On the other hand, in the proximal stump of sciatic nerves, numerous NOS-immunoreactive fibers with a regenerative profile appeared transiently (2-4 weeks). At higher magnification, an axonal sprout from the NOS-immunoreactive small DRG neurons was found to form a basket-like structure (or basket) mostly around the cell body of NOS-negative large neurons. Retrograde labeling with a fluorescent tracer showed that both neurons sent peripheral axon collaterals to the sciatic nerve. The appearance of this unique structure was most prominent after depletion of the NOS-immunoreactive regenerating fibers in the sciatic nerve (at 7-9 weeks). The findings suggest that NO might be involved in not only axonal regeneration but also the rewiring of two classes of DRG neurons after peripheral nerve injury.