体外获得高纯度大鼠背根神经节神经元的原代培养

目的：建立一种切实可行的胚胎大鼠背根神经节神经元培养及纯化方法。方法：用显微解剖获取足够数量的胚胎大鼠背根神经节,通过胰蛋白酶+EDTA消化、交替使用NB培养基与加入了5-氟-2-脱氧尿嘧啶核苷酸/尿苷的抗有丝分裂NB培养基等方法,在体外获得纯化的背根神经节神经元,采用神经丝蛋白免疫细胞化学染色法与DAPI染核的方法鉴定并测定神经元的纯度。结果：获得的背根神经节神经元在体外生长良好,纯度可达到96%以上。结论：本实验方法可以获得大量高度纯化的大鼠背根神经节神经元。     

牛磺酸对大鼠背根神经节神经元GABA激活电流的调制作用

牛磺酸可能是中枢神经系统中一种抑制性神经递质或神经调质。本实验目的在于观察牛磺酸是否引起背根神经节（ＤＲＧ）神经元膜产生反应以及是否对ＧＡＢＡ引起的背根神经节神经元奄流有调制作用。应用全细胞膜片钳技术,观察未检测到牛磺酸对ＤＲＧ神经元膜电流的改变,但预先给牛磺酸对ＧＡＢＡ激活的膜内向电流有明显的帽作用,并具明显的浓度依赖性。如１０＾－５ｍｏｌ／Ｌ的牛磺酸可使１０＾－５ｍｏｌ／Ｌ ＧＡＢＡ激活的膜电     

背根神经节细胞分离培养法评价神经生长因子的生物活性

本文介绍应用胎鼠背根神经节(Dorsal root ganglion,DRG)细胞分离培养法评价神经生长因子(Nerve Growth Factor,NGF)生物活性及培养细胞的鉴定方法,并扼要叙述DRG组织培养法鉴定NGF生物活性过程中存在的问题。     

背根神经节电穿孔转染神经元技术的改进

文题释义：背根神经节：具有一组不同的感觉神经元,向大脑传递不同的感觉刺激,如疼痛、温度、触摸和身体姿势。背根神经节电穿孔是基因转染中通过非病毒的方式以物理方法转染神经元的技术。周围神经损伤：各种原因导致周围神经支配区域的感觉、运动及营养障碍。文献报道周围神经损伤常伴随损伤近端的神经元胞体的肿胀、尼氏体消失、细胞核偏移;损伤远端轴突发生华勒氏变性、许旺细胞增殖等现象。背景：背根神经节电穿孔技术是一种研究神经再生的高效基因转染方法,以往背根神经节电穿孔的电压条件使得标记的神经元及轴突数目较少,统计误差较高。目的：提高神经元及其轴突的标记率,为周围神经再生的研究提供理论依据。方法：以增强型绿色荧光蛋白为观察指标优化背根神经节电穿孔技术在神经元轴突再生方面的应用。将ICR小鼠随机分为2组,分别行背根神经节电穿孔手术,检测在35 V和60 V电压干预下神经元及其轴突的标记率。结果与结论：①35 V和60 V电压未造成神经元明显死亡;②与35 V电压相比,60 V电压条件下标记的神经元及其轴突数量显著增加,且通过损伤部位的轴突数量明显增多(P < 0.05);③60 V电压对实验动物未造成行为功能损害;④结果表明,60 V电压能够使神经元及其轴突的标记率增高,其结果能够为周围神经元轴突再生研究提供依据。ORCID: 0000-0001-8681-1346(齐士斌)中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

二脱氧肌苷引发培养的背根神经节神经元的形态学改变

为探讨二脱氧肌苷(ddI)对培养的背根神经节(DRG)神经元的影响,我们对分散培养的胎鼠DRG神经元培养3d后,再分别以不同浓度的ddI(1μg/ml,5μg/ml,10μg/mll和20μg/ml)孵育3d。终止培养后,行微管相关蛋白2(MAP2)标记,用共聚焦激光扫描显微镜观察神经元胞体和突起的改变。结果表明,DRG神经元用ddI孵育3d,神经元突起的数目减少和长度变短,呈剂量依赖性,而神经元的直径则没有变化。本研究的结果表明,ddI可影响培养的DRG神经元突起的再生和生长。     

一氧化氮对脊髓和背根神经节神经元的体外存活及活性作用研究

本文应用免疫细胞化学及ＮＳＥ－ＥＬＩＳＡ方法观察了一氧化氮对体外培养脊髓和背根神经节神经元的存活及对活性的影响。结果表明：一氧化氮合酶抑制剂Ｎ－Ａｒｇ组（１００、２００μｍｏｌ／Ｌ）ＮＳＥ免疫反应阳性神经元数目、面积（ＡＦ值）及活性（ＯＤ值）明显大于空白对照组（Ｐ＜０．０１）。而一氧化氮合酶底物Ｌ－Ａｒｇ组（１ｍｍｏｌ／Ｌ）神经元面积积分和活性则小于对照组（Ｐ＜０．０１）。Ｌ－Ａｒｇ的细胞毒性作用可为Ｎ－Ａｒｇ逆转。     

建立一种简便的大鼠背根神经节神经元原代培养方法的建立

目的:建立一种简便易行地获取大鼠背根神经节(dorsal root ganglion,DRG)神经元原代培养的方法。方法:窒息法处死大鼠,打开胸腔经心脏放血后,从背部取出一段脊柱。将脊柱稍加修剪后,从中轴线处打开脊柱,暴露脊髓,在其两侧找到椎间孔内的为DRG,用器械离断神经纤维,取出DRG。Ⅰ胶原酶联合Trypsin-EDTA先消化DRG,再用吹法DRG以获得单个神经元;用TNB-100 basal Medium的复合培养液在经过0.1 mg/ml PolyD-lysine hydrobromide和0.01 mg/ml lamina包被的培养皿内,于5%CO2,37℃孵箱内培养48 h。免疫组织化学方法检测神经元内标志物BetaⅢTubulin的表达,用Alexa flour 488标记的二抗进行显色,倒置荧光显微镜下观察。结果:获得了单层贴壁的神经元,其胞体形状多样,突起生长良好,细胞之间正在逐步形成网络连接;免疫荧光染色显示,细胞的胞体和突起中均可高表达BetaⅢTubulin,且荧光染色较强。结论:运用该方法可以简便地从大鼠DRG内获得单层贴壁的原代神经元。     

高糖对体外培养背根神经节神经元影响的研究进展

背根神经节神经元(DRGn)在糖尿病周围神经病变发展过程中起重要作用,近年来探讨高糖对体外培养背根神经节神经元(DRGn)的影响成为研究热点.高糖环境可致DRGn细胞凋亡;同时高糖环境下DRGn在氧化应激、膜受体mGluRs及TRPV1激活、DRGn/SCs共培养等方面均与正常培养基不同,具体机制仍需深入研究.     

多巴胺对大鼠背根神经节神经元GABA-激活电流的影响

用全细胞膜片箝记录技术在大鼠分离背根节神经元上观察预加多巴胺对 GABA-激活电流的调控作用。发现大部分受检细胞 ( 4 0 / 47)对外加 GABA敏感。 10 - 6 ～ 10 - 3mol/ L GABA引起一剂量依赖性、有明显去敏感作用的内向电流。在对 GABA敏感的 40个细胞中 ,多巴胺引起一小的、无去敏感的外向电流 ( 2 6/ 40 ) ,其他无明显反应 ( 14 / 40 )。预加多巴胺 10 - 7～ 10 - 4 mol/ L 3 0 s后再加 GABA有 68% ( 2 7/ 40 )的细胞 GABA-激活电流幅值抑制 ,以多巴胺的浓度 10 - 5 mol/ L的抑制最明显 ( 3 3 .3 % )。     

野木瓜调制背根神经节细胞钠离子通道的实验与计算机模拟

采用计算机模拟的方法, 排除膜片钳实验观察中除钠通道以外的其它离子通道对实验结果的干扰,从野木瓜注射液对背根神经节细胞钠离子通道电流和动作电位影响的角度,研究野木瓜注射液的镇痛机制.用Hodgkin Huxley模型拟合全细胞膜片钳实验所得的10%,25%和50%野木瓜注射液作用前后的膜片钳钠离子通道电流数据,进行参数估计,评价拟合优度,并将所获参数用于建立模拟神经元,在只改变模型钠离子通道参数的情况下,计算机仿真野木瓜注射液作用前后的背根神经节细胞动作电位.膜片钳实验结果表明,野木瓜注射液浓度依赖地抑制背根神经节细胞钠通道电流峰值,并影响通道的激活失活过程.计算机模拟结果表明,野木瓜注射液作用后,钠通道半激活电压向去极化方向偏移,神经元产生动作电位的刺激电流阈值提高.说明野木瓜注射液可能通过影响钠离子通道激活过程从而阻滞初级感觉神经元动作电位,干预痛觉信息的传导,从而产生镇痛效果.     

神经节苷脂对损伤胎鼠背根神经节的保护作用

目的观察神经节苷脂(GM1)对受损伤胎鼠背根神经节(DRG)神经元形态改变的影响,探讨其可能的保护作用。方法选择胎龄为15d的SD大鼠为研究对象,获取DRG神经元并进行体外分散培养,培养48h后,随机分为对照组、谷氨酸损伤组和谷氨酸损伤+GM1保护组,继续培养12h。终止培养后,观察各组神经元的形态结构改变,用MTT法鉴定细胞的存活率。结果对照组DRG神经元细胞贴壁呈单层散在分布,少部分出现细胞聚集现象,突起较长且互相交织形成网状。谷氨酸损伤组DRG神经元细胞聚集现象明显,神经元突起变短、断裂甚至消失。谷氨酸损伤+GM1保护组DRG神经元细胞部分呈簇状聚集,部分呈单个散在分布,突起仍然相互交织。MTT结果显示谷氨酸损伤+GM1保护组细胞存活率高于谷氨酸损伤组。结论神经节苷脂可以影响损伤胎鼠DRG神经元的形态改变,对胎鼠背根神经节神经元具有一定的保护作用。     

坐骨神经损伤后脊神经节神经元凋亡的形态学观察

周围神经损伤后，神经修复困难的原因之一是一定数量的神经元胞体死亡。运用NGF可保护感觉神经元胞体免于死亡，但对于细胞死亡的性质目前尚未肯定，因而对NGF的保护机理也就不清楚。本研究通过对大鼠坐骨神经切断再吻合后，观察其脊神经节感觉神经元的胞体形态变化，认为神经元死亡的主要形式是细胞凋亡(apoptosis)。     

Divalent cation dependent inactivation of the high-voltage-activated Ca-channel current in chick sensory neurons

We have used the whole-cell clamp technique to investigate inactivation of the -conotoxin sensitive high-voltage-activated Ca-channel current (HVA current [2]) carried either by Ca, Ba or Sr (2.5 mM) in chick sensory neurons. At a low internal EGTA concentration (0.1 mM), Ca-channel currents clearly inactivated irrespective of the species of divalent cation carrying the current. During 150 ms pulses, current inactivated to 0.57, 0.67 and 0.75 of the peak current in Ca, Ba and Sr solution, respectively. Time constants of inactivation (26±10 ms and 280±50 ms, mean±S.D., in Ba) were largely independent of the membrane potential. Double-pulse experiments showed that the amount of inactivation left by a pre-pulse was proportional to the amplitude of the current evoked by the pre-pulse. No inactivation was induced by an outward current elicited by a strong depolarization to ü60 mV. With an internal EGTA concentration of 20 mM, the amount of inactivation was significantly smaller. In conclusion, the inactivation of the HVA Ca-channel currents during current flow depends mostly on the entry of divalent cations irrespective of their species.     

Single-channel current/voltage relationships of two kinds of Na+ channel in vertebrate sensory neurons

The electrical signals of nerve and muscle are fundamentally dependent on the voltage-gated Na⁺ channel, which is responsible for the rising phase of the action potential. At least two kinds of Na⁺ channel are expressed in the membrane of frog dorsal root ganglion (DRG) cells: Na⁺ channels with fast kinetics that are blocked by tetrodotoxin (TTX) at high affinity, and Na⁺ channels with slower kinetics that are insensitive to TTX. Recordings of single-channel currents from frog DRG cells, under conditions favoring Na⁺ as the charge carrier, reveal two distinct amplitudes of single-channel events. With 300 mM external Na⁺, single-channel events that can be measured in the presence of 1 M TTX have a slope conductance 7.5 pS. In the absence of TTX, events with a slope conductance of 14.9 pS dominate. Ensemble averages of the smaller single-channel events display the slower kinetics characteristic of the macroscopic TTX-insensitive Na⁺ currents, and ensemble averages of the larger events display the faster kinetics characteristic of the TTX-sensitive currents. The results are consistent with the idea that the toxin-binding site is sufficiently close to the pore to influence ion permeation.     

血竭及其成分龙血素B影响背根神经节细胞钠离子通道的计算机模拟研究

应用膜片钳技术，观察中药血竭及其成分龙血素B对背根神经节细胞河豚毒素敏感型钠通道电流的影响。以Hodgkin—Huxley模型拟合实验资料，进行参数估计，并对药物作用前后的细胞膜动作电位进行计算机模拟。结果表明，m^3h模型能较好地拟合河豚毒素敏感型钠通道电导；药物作用于钠通道后，半激活电压均向去极化方向偏移；药物作用后的细胞产生动作电位的阈强度提高。说明血竭及其成分龙血素B并非类似河豚毒素完全抑制河豚毒素敏感型钠通道电流。血竭可能通过其成分龙血素B影响通道的激活过程阻滞动作电位的产生，干预痛觉信息的中枢传人而产生镇痛效应。     

Stimulatory Effects of NMDA on Intracellular Ca~(2 ) Nonlinear Kinetic Model in Rat Dorsal Root Ganglion Neurons

INTRODUCTION　　 The N- methyl- D- aspartate receptor( NMDAR) is a highly Ca2 permeable,lig-and- gated ion channel in neurons and a member of the ionotropic glutamate receptorfamily,Itis the famous agonistof the NMDA receptor,the latter being named afte…     

K+ Channels and Their Modulation by 5-HT in Drosophila Photoreceptors: A Modelling Study

In order to clarify the role of inactivating and noninactivating K⁺ conductances in nonspiking neurons, we developed an isopotential model of the Drosophila photoreceptor membrane based on Hodgkin-Huxley-type equations. The model includes voltage dependent potassium conductances, the shaker (g _KA) and the delayed rectifier (g _Ks). The model parameters were derived from published results by Hardie and coworkers and nearly identical model was used also in our previous work (J. E. Niven, M. Vähäsöyrinki, M. Kauranen, R. C. Hardie, M. Juusola, and M. Weckström. The Contribution of shaker K⁺ channels to the information capacity of Drosophila photoreceptors. Nature. 421:630–634, 2003). The model explains how the two types of channels function together to define the voltage dependent properties of the photoreceptor membrane. Additionally the model enables us to run simulations of conditions which are difficult to achieve in patch clamp, like prolonged membrane depolarizations by light adaptation. Effects of the activation of the delayed rectifier type conductance were found to be in accordance with published experimental work but the inactivation of the shaker channels, in addition to its importance in the determination of the resting potential, produced voltage amplification over equivalent passive membrane under dark adapted conditions. This phenomenon was not present in light adapted conditions. The modulation of the voltage dependence of the conductances as reported by serotonin (5-HT) caused the shaker to act essentially like the delayed rectifier conductance.     

Substance P in Dorsal Root Ganglion Neurons in Young and Adult Rats,after Nociceptive Stimulation during the Neonatal Period

The nervous system is highly plastic during the neonatal period, being sensitive to noxious stimuli, which may cause short‐ and long‐term pain responsivity changes. Understanding plasticity in peripheral pain pathways is crucial, particularly when the nervous system is still under development and remodeling process. Substance P (SP) is widely used as a marker for peripheral neurons with unmyelinated and small myelinated fibers. We investigated the number of SP immunoreactive neurons in the dorsal root ganglion (DRG) of male and female Wistar rats, 15 and 180 days after nociceptive stimulation during the neonatal period. Right and left 5th lumbar (L5) DRG were incubated in rabbit polyclonal anti‐substance P primary followed by biotinylated donkey anti‐rabbit secondary antibodies. Reaction was revealed with a nickel‐diaminobenzidine solution. Labeled neurons were counted and compared between ages, genders and groups. Gender differences were present in both ages, with the number of SP‐positive DRG neurons being larger in 15‐days‐old males on both sides. After 180 days, males showed a larger number of SP‐positive neurons than females only on the nociceptive stimulated side. An increased number of SP‐positive neurons in the DRG on the stimulated side was present in females, immediately after nociceptive stimulation, but not after 180 days. In conclusion, neonatal noxious stimulation caused a permanent increase in SP‐positive DRG neurons in males that was not observed in females, suggesting that differences in pain processing/responsivity between genders could be related to morphological alterations of the nervous system. Anat Rec, 301:849–861, 2018. © 2017 Wiley Periodicals, Inc.