脊髓半横断损伤后大鼠皮神经元细胞骨架蛋白基因表达的变化

目的 探讨神经再生过程中细胞骨架蛋白的作用机制,了解中枢和外周神经系统神经元应答神经损伤和再生的差异。方法 用原位分子杂交方法观察了大鼠T10－11脊髓半横断损伤后1、3、5、7、10、14、21、28、56d皮层感觉运动区皮层神经元中α－管蛋白和3个神经丝蛋白亚基mRNA的表达变化。结果 脊髓半横断损伤后损伤侧皮层感觉运动区皮层神经元中α-管蛋白、神经丝－L、－M、－H mRNA表达明显下调。α－管蛋白mRNA表达水平在损伤后1d降低,而神经丝亚基mRNA表达水平直到下一个时间点（损伤后3d）才下调,至损伤后56d均无恢复趋势。结论 皮层神经元损伤后α－管蛋白mRNA的表达被抑制,提示皮层神经元和外周神经元应答损伤的信号不同。     

脊髓半横断损伤后大鼠皮层神经元细胞骨架蛋白基因表达的变化

目的　探讨神经再生过程中细胞骨架蛋白的作用机制 ,了解中枢和外周神经系统神经元应答神经损伤和再生的差异。　方法　用原位分子杂交方法观察了大鼠T10～ 11脊髓半横断损伤后 1、3、5、7、10、14、2 1、2 8、5 6d皮层感觉运动区皮层神经元中α 管蛋白和 3个神经丝蛋白亚基mRNA的表达变化。　结果　脊髓半横断损伤后损伤侧皮层感觉运动区皮层神经元中α 管蛋白、神经丝 L、 M、 HmRNA表达明显下调。α 管蛋白mRNA表达水平在损伤后 1d降低 ,而神经丝亚基mRNA表达水平直到下一个时间点 (损伤后 3d)才下调 ,至损伤后 5 6d均无恢复趋势。结论　皮层神经元损伤后α 管蛋白mRNA的表达被抑制 ,提示皮层神经元和外周神经元应答损伤的信号不同     

MEF2C调控大鼠脊髓背根节感觉神经元P物质和低分子量神经丝微管蛋白的表达

目的:研究肌细胞增强因子2C(MEF2C)在大鼠脊髓背根神经节神经元细胞内的表达及其与P物质和低分子量神经丝微管蛋白合成的关系。方法:分离培养背根神经节神经元细胞,然后暴露于不同浓度的神经生长因子下24h,最后用实时聚合酶链反应技术检测P物质与低分子量神经丝微管蛋白基因在背根神经节神经元细胞内的表达。通过化学转染方法将合成的3条siRNA-MEF2C分别转染PC12细胞株,并用实时聚合酶链反应技术筛选干扰效率最高的siRNA。采用化学转染方法干扰背根神经节神经元细胞内MEF2C的表达,在高浓度神经生长因子刺激背根神经节神经元细胞后采用实时聚合酶链反应技术检测干扰后背根神经节神经元细胞内P物质与低分子量神经丝微管蛋白基因的表达。结果:P物质及低分子量神经丝微管蛋白基因表达随刺激用神经生长因子浓度的升高而升高。使用化学转染方法成功地干扰背根神经节神经元细胞内MEF2C的表达,MEF2C较对照组下降52%,同时没有检测到对cAMP反应元件结合蛋白表达的影响。实验组背根神经节神经元细胞内P物质在RNA水平较对照组下降了39%,而低分子量神经丝微管蛋白在RNA水平较对照组下降了62%。结论:神经生长因子促进大鼠脊髓背根节感觉神经元内P物质与低分子量神经丝微管蛋白的合成。大鼠背根神经节神经元细胞内P物质及低分子量神经丝微管蛋白基因表达受MEF2C调控。     

神经损伤与再生中神经元GAP-43mRNA表达的原位杂交研究

目的：研究周围神经损伤过程中生长相关蛋白（GAP-43）mRNA表达的变化规律。方法：建立大鼠坐骨神经中段钳夹损伤模型，用原位杂交技术对大鼠的腰髓和背根神经节的GAP-43mRNA表达进行观察。结果：大鼠坐骨神经损伤2d后，腰髓腹角运动神经元和背根节感觉神经元可检测到GAP-43mRNA杂交信号；术后4、7和14d明显增强；术后30d减弱，60d已恢复正常。结论：周围神经损伤诱导神经元胞体GAP-43mRNA表达显著增强，表明GAP-43在神经再生过程中起重要作用。     

脊髓半横断损伤后皮层脊髓神经元细胞骨架蛋白基因表达的变化

为了解中枢神经系统神经元对轴突损伤的应答特点,进一步探讨神经损伤修复的细胞分子机制,我们在脊髓半横断动物模型上观察了皮层感觉运动区脊髓皮层神经元中α-Tub、NF-L、NF-M和NF-HmRNA的表达变化。损伤后1天皮质脊髓神经元中α-管蛋白mRNA明显降低,至损伤后2个月仍处于较低水平。管蛋白mRNA表达的下调与坐骨神经、面神经损伤后外周神经元的变化结果截然相反。因此,神经损伤后管蛋白的上调与有效再生关系密切。损伤后3天～2月皮层脊髓神经元内NF-L、-M和-HmRNA的含量明显降低。虽然外周和中枢神经系统轴突损伤后均引起神经丝m…     

信息分子与面神经再生

面神经的损伤修复是一个复杂的过程,神经再生主要是轴突的再生,前提是神经元细胞体的存活。神经元细胞体的调控和再生微环境的营养和导向对受损神经的再生及再生轴突的延伸起重要作用。随着神经生物学及现代免疫学的发展,研究发现许多与细胞信号传导有关的分子如：神经营养因子类、细胞因子类及粘附分子参与了面神经损伤及其修复的过程,并发挥了重要的生物学功能。     

神经生长因子mRNA在大鼠脊髓和脊神经节的表达——原位杂交研究

我们以 SD大鼠坐骨神经为材料 ,在 NGF- c DNA文库建立的基础上 ,人工合成神经生长因子引物 ,并用 PCR地高辛标记法标记 NGF探针 ,采用原位分子杂交组织化学方法 ( ISHH) ,观察 NGF- m RNA神经生长基因表达细胞在大鼠腰段脊髓和脊神经节内的分布。结果发现在大鼠坐骨神经损伤模型腰段脊髓横切面的前角、侧角及腰背根神经节均有 NGF基因的表达细胞 ,蓝色反应物弥散性分布于胞浆内 ,呈细小颗粒状或长柱状。损伤侧要强于未损伤侧 ,并对其杂交信号进行定量分析 ,结果显示在大鼠坐骨神经损伤模型术后第 5天、第 10天及第 15天 ,脊髓前角运动神经元 ,侧角交感神经元、背根节感觉神经元内的杂交信号增强 ,表明损伤的早、中期 NGF- m RNA表达量增加。讨论了神经再生的理化因素     

坐骨神经源性弥散因子对成年青蛙再生轴突的定向诱导作用

目的：探讨损伤后周围神经产生的活性因子对活体成年青蛙断神经再生轴突的定向趋化诱导作用。方法：通过微型渗透压泵以不同的角度和距离朝被切断的支配左皮胸肌的神经末端提供坐骨神经条件性培养基（conditioned medium,CM）300-100kD组分,形成浓度梯度。28－32d后,取出胸部组织行免疫组织化学染色示再生的皮胸肌神经轴突,并分析再生轴突定向生长的导向值（directional value）。结果：再生的皮胸肌神经轴突明显地朝向微型泵管口CM浓度梯度高处生长,导向值为0．84,对照组再生的神经轴突呈无定向生长,导向值为0．04。两组间有显著性差异（P＜0．001）。管口与神经断端间距离和夹角的大小变化对导向值无显著性影响。结论：损伤后的周围神经可产生分子量介入30－100kD可弥散活性因子,对活体成年青蛙断神经的再生轴突有定向诱导作用。     

神经生长相关蛋白B一50（GAP一43）与神经再生和发育的关系

神经生长相关蛋白Ｂ一５０（ＧＡＰ一４３）与神经再生和发育的关系张艳萍（中国医学科学院基础医学研究所北京１００００５）神经元受损伤后能够再生或者退化，都受着许多内在和外在因素的调控。目前受到注意的是，在神经元发育、分化和再生期间，有一类与轴突生长相伴随...     

C型Niemann-Pick病小鼠神经元变性与细胞骨架蛋白异常磷酸化

为探讨导致神经元病理改变的可能机制以进行干预治疗,本研究采用组织学、免疫组织化学、免疫荧光组织化学及免疫印记等技术对C型Niemann-Pick病(NPC)小鼠(npc-1小鼠)的神经病理、细胞骨架蛋白及细胞周期M期标志进行了研究。结果显示:在npc-1小鼠脑干、小脑白质、基底神经节及大脑白质有诸多神经元含脂质沉积并有球状神经轴突形成。这些异常的神经轴突内含有大量的异常磷酸化的细胞骨架蛋白tau及神经丝蛋白NF。M期标志物的存在表明有丝分裂激酶活化参与这一病理结构的形成过程。本文结果提示:神经元骨架蛋白的异常磷酸化对npc-1小鼠神经病理的形成过程有重要作用,有丝分裂激酶异常活化可能导致这些骨架蛋白的异常磷酸化。针对这一发现可设计干预方案来阻止神经元变性过程。     

Growth-associated proteins and regeneration-induced gene expression in the aging neuron

Axonal elongation and sprouting during regeneration are retarded with aging but the etiology of this is unclear. We investigated whether this age-associated decline is related to a decline in expression of three different growth-associated proteins (GAPs): alpha(1)-tubulin, neurofilament (NF) light subunit (NF-L) and GAP-43. Northern analysis was performed on L4-L5 dorsal root ganglia (DRG) of young (3 months) and aged (23 months) rats following a sciatic nerve crush and compared to their age-matched controls. The results show that initial mRNA levels of alpha(1)-tubulin and NF-L in the control aged rat DRG were half those of the control young adults, whereas expression of GAP-43 was unchanged. Two weeks after axotomy, the expression of alpha(1)-tubulin and GAP-43 in the aged DRG was induced to the same levels as in the axotomized young adult, and the expression of NF-L decreased proportionately in both age groups. These results indicate that certain neuronal mRNAs, such as alpha(1)-tubulin and NF-L may be maintained at lower levels in aging DRG neurons, whereas others, such as GAP-43 appear to be unaltered. However, during regeneration, the aging DRG neuron appears capable of inducing alpha(1)-tubulin, NF-L and GAP-43 as well as the young adult.     

GDNF在坐骨神经损伤后大鼠L4—6背根节神经元的表达及变化

目的 观察坐骨神经夹伤后不同时间点,胶质细胞源性神经营养因子（ＧＤＮＦ）在成年大鼠Ｌ４～６背根节（ＤＲＧ）中的分布,探讨ＧＤＮＦ对感觉神经元损伤的反应。方法 成年大鼠右侧坐骨神经夹伤后,分别取伤后１、５、７、１０、１４、２８和５６ｄＬ４～６背根节,行抗ＧＤＮＦ多克隆抗体免疫组织化学ＡＢＣ法染色,之后对染色结果进行图像分析。结果 ＧＤＮＦ免疫反应存在于坐骨神经夹伤后１、５、７、１０、１４、２８和５６ｄ成年大鼠Ｌ４～６背根节神经元中,图像分析结果表明,伤后１、３、５和７ｄ的损伤侧背根节神经元的平均光密度大于对照侧背根节神经元。结论 大鼠坐骨神经夹伤后,ＧＤＮＦ在背根节Ｌ４～６感觉神经元中有一定量的变化,且伤后１周内ＧＤＮＦ在伤侧背根节神经元中的表达增强。     

Aminergic receptors in astrogliotic plaques from patients with multiple sclerosis

The expression of capsaicin (VR1) receptor by A-fiber primary afferent neurons has been investigated by double immunohistochemical staining with VR1 and 200 kD neurofilament (NF200; an A-fiber marker) antibodies, and by VR1 immunohistochemical staining in combination with cholera toxin B subunit (CTB; also an A-fiber marker) retrograde tracing. Approximately 30% of the VR1-positive dorsal root ganglion (DRG) neurons were NF200-positive. Intra-sciatic nerve injection of CTB labeled over 30% of the VR1-positive neurons in the L5 DRG. Size frequency distribution analysis revealed that these VR1 and NF200, or VR1 and CTB, double-labeled neurons were predominantly small and medium sized. These results suggest that capsaicin receptors are likely to be expressed by Adelta-fiber neurons as well as C-fiber neurons.     

Post-traumatic survival in different subpopulations of sensory neurons

An expression of high molecular component of neurofilament triplet NF200 (marker of neurons forming A-fibers) and binding of isolectin B4 (IB4) was examined immunohistochemically in LIV-LV dorsal root ganglia (DRG) neurons after ligation or transection of the sciatic nerve in rat. NF200 immunoreactivity was detected in 15% of all neurons in DRG of intact rats. Ligation of sciatic nerve caused a two-fold decrease in number of NF200-positive neurons by 90th day after nerve injury, however in animals treated with peripheral nerve regeneration stimulator xymedon the number of surviving NF200-positive neurons was increased by 50.7% as compared with control group (nerve ligation without treatment). In DRG of intact rats 23.6% of neurons showed IB4 binding. Of the DRG neurons 2.6% were labeled by IB4 at 30th day after ligation of the nerve. At 90th day after ligation no IB4-positive neurons were revealed in DRG of untreated rats, while xymedon treatment was shown to result in more than 8-fold increase in the number of surviving IB4-positive neurons. IB4-positive neurons have greater probability of entering the posttraumatic apoptosis. After nerve ligation the survival of NF200- and IB4-positive neurons was less than that one following nerve transection, suggesting that axon lengthening could be a the factor supporting neuronal survival. Pyrimidine derivative xymedon promoted the survival of neurons in both subpopulations with predominant effect on IB4-positive neurons.     

Neurofilament immunoreactivity in populations of rat primary afferent neurons: A quantitative study of phosphorylated and non-phosphorylated subunits

Summary Neurofilament subunits in rat dorsal root ganglion (DRG) neurons were examined using five antibodies: NFH, RT97 and NFHP recognise the 200 kDa subunit (NF200); NFH recognises both phosphorylated and non-phosphorylated forms of NF200 whereas RT97 and NFHP are specific for the phosphorylated and non-phosphorylated forms respectively; 155 and anti-68 kD recognise the 155 kDa and 68 kDa subunits respectively. All the antibodies apart from NFHP distinguished between the two populations of neurons corresponding to the light (L) and small dark (SD) cell types as previously shown for RT97. This demonstrates that L and SD neurons contain different levels of neurofilament and that the ability to discriminate between them is not unique to the antibody RT97. It is also evident that DRG neurons contain neurofilament composed of all three subunits. Since NFH and RT97, but not NFHP, distinguished between the two populations, it appears that it is the presence of the phosphorylated form of NF200 that provides the basis for discrimination between the two cell types. After dephosphorylation of the neurofilament, NFHP also discriminated between the two populations, indicating that there is more NF200 regardless of phosphorylation state in the L neurons.Observations made from unfixed DRGs indicate that all neurons contain some neurofilament and the neurofilament rich and neurofilament poor populations were also apparent.The use of colchicine apparently caused a small increase in neurofilament levels in at least some perikarya, presumably due to its blocking effect on axoplasmic transport. This caused some SD neurons to become neurofilament rich.We conclude that L neurons contain more neurofilament than SD neurons since both cell types contain non-phosphorylated NF200, but the L neurons also contain a much greater amount of the phosphorylated form.     

Increase of galanin mRNA in lumbar dorsal root ganglion neurons of adult rats after partial sciatic nerve ligation

Partial sciatic nerve ligation (PSNL) is a widely used model for the study of neuropathic pain. However, there is little information on neuropeptide expression in primary sensory neurons after PSNL. We examined galanin (GAL) mRNA expression in L4 and L5 dorsal root ganglion (DRG) neurons of adult rats after PSNL. We found that 4 and 14 days after PSNL the percentages of GAL mRNA positive neurons were significantly increased in the ipsilateral DRG compared to the contralateral side. Using combined retrograde fluorescent dye tracing and in situ hybridization, we found that 47% of the injured neurons and 10% of the spared neurons were GAL mRNA positive. Since only 2-3% of neurons in the contralateral uninjured DRG were GAL mRNA positive, PSNL induced up-regulation of GAL mRNA in both injured and spared DRG neurons.     

Nerve growth factor stimulates synthesis of calcitonin gene-related peptide in dorsal root ganglion cells during sensory regeneration in capsaicin-treated rats

Administration of human recombinant nerve growth factor (rhNGF) into one hindpaw of capsaicin-treated rats can locally facilitate the regeneration of calcitonin gene-related peptide (CGRP)-containing primary sensory neurons (Schicho, R., Skofitsch, G., Donnerer, J., 1999. Brain Res. 815, 60-69). In this study we used in situ hybridization histochemistry (ISH) to determine synthesis of CGRP mRNA in lumbar L4 dorsal root ganglion (DRG) cells during NGF-induced regeneration. Whereas 8 days after the capsaicin treatment alone (50 mg/kg s.c.) CGRP mRNA expression in DRG cells was reduced to 40-60% of control levels, the additional intraplantar injections of rhNGF (5 x 4 microg) during this time period were able to raise CGRP mRNA expression again. The increase in CGRP expression was seen ipsi- and contralaterally and it was more pronounced in small- and medium-sized (about 110% of control levels), than in large-sized CGRP-producing cells (70% of controls). The percentage of the CGRP-expressing neurons in capsaicinized and in capsaicin + NGF-treated animals stayed unaltered. In conclusion, the present results demonstrate that NGF-induced regeneration of capsaicin-lesioned sensory afferents is accompanied by an elevated production of CGRPmRNA mainly in small- and medium-sized DRG cells.     

中药神经再生素作用于背根神经节细胞过程中基因的表达变化

目的 探讨中药神经再生素(NRF)作用的分子生物学机制。方法 采用半定量PCR方法，观察和比较NRF组、NGF组和空白对照组中生长相关蛋白43(GAP-43)、低分子量神经丝蛋白(NF-L)、翻译延伸因子2A3-2(EF-Ts，RRAJ5161)和锌指样蛋白DDP2(F196315)基因水平的表达变化。结果 在NRF作用于离体培养的DRG细胞过程中，GAP-43、NF-L、2A3-2和DDP2基因表达在不同时间呈不同程度的上调。结论 NRF促神经生长的作用与NGF相似，可作用于蛋白翻译水平，促进神经细胞的生长；作用于细胞骨架蛋白水平，维持神经细胞的形态和神经细胞正常生理活动；作用于神经细胞特异的蛋白水平，具有维持细胞生存及促进神经细胞突起生长的作用；还可以作用于反式因子调控水平，影响相关基因的表达，促进神经元存活和神经突起延伸。     

针刺对去部分背根猫脊髓和背根节NGF及NGF mRNA的影响

用免疫组织化学和地高辛标记 c RNA探针原位分子杂交技术 ,观察了针刺对成年备用背根猫 (切断一侧 L1～ 5 、L7～ S2节段脊髓背根 ,保留 L6 背根 )脊髓 板层、背核和背根节内神经生长因子和神经生长因子 m RNA的影响。结果证明 ,针刺促进了备用根背根节的神经生长因子基因的表达 ,使备用背根节内神经生长因子、神经生长因子 m RNA阳性神经元数量明显增多 ,并且针刺时间越长促进作用也越强 ;而对脊髓 板层与背核则无明显影响。本实验结果提示 ,针刺对备用根猫脊髓侧支生芽具有促进作用 ,这种作用主要体现在通过增加背根节神经生长因子基因的表达和神经生长因子的合成来实现的