Electroacupuncture in the repair of spinal cord injury:inhibiting the Notch signaling pathway and promoting neural stem cell proliferation

Electroacupuncture for the treatment of spinal cord injury has a good clinical curative effect, but the underlying mechanism is unclear. In our experiments, the spinal cord of adult Sprague-Dawley rats was clamped for 60 seconds. Dazhui(GV14) and Mingmen(GV4) acupoints of rats were subjected to electroacupuncture. Enzyme-linked immunosorbent assay revealed that the expression of serum inflammatory factors was apparently downregulated in rat models of spinal cord injury after electroacupuncture. Hematoxylin-eosin staining and immunohistochemistry results demonstrated that electroacupuncture contributed to the proliferation of neural stem cells in rat injured spinal cord, and suppressed their differentiation into astrocytes. Real-time quantitative PCR and western blot assays showed that electroacupuncture inhibited activation of the Notch signaling pathway induced by spinal cord injury. These findings indicate that electroacupuncture repaired the injured spinal cord by suppressing the Notch signaling pathway and promoting the proliferation of endogenous neural stem cells.     

Effect of electroacupuncture on the mRNA and protein expression of Rho-A and Rho-associated kinase Ⅱ in spinal cord injury rats

Electroacupuncture is beneficial for the recovery of spinal cord injury, but the underlying mechanism is unclear. The Rho/Rho-associated kinase(ROCK) signaling pathway regulates the actin cytoskeleton by controlling the adhesive and migratory behaviors of cells that could inhibit neurite regrowth after neural injury and consequently hinder the recovery from spinal cord injury. Therefore, we hypothesized electroacupuncture could affect the Rho/ROCK signaling pathway to promote the recovery of spinal cord injury. In our experiments, the spinal cord injury in adult Sprague-Dawley rats was caused by an impact device. Those rats were subjected to electroacupuncture at Yaoyangguan(GV3), Dazhui(GV14), Zusanli(ST36) and Ciliao(BL32) and/or monosialoganglioside treatment. Behavioral scores revealed that the hindlimb motor functions improved with those treatments. Real-time quantitative polymerase chain reaction, fluorescence in situ hybridization and western blot assay showed that electroacupuncture suppressed the m RNA and protein expression of Rho-A and Rho-associated kinase Ⅱ(ROCKⅡ) of injured spinal cord. Although monosialoganglioside promoted the recovery of hindlimb motor function, monosialoganglioside did not affect the expression of Rho-A and ROCKⅡ. However, electroacupuncture combined with monosialoganglioside did not further improve the motor function or suppress the expression of Rho-A and ROCKⅡ. Our data suggested that the electroacupuncture could specifically inhibit the activation of the Rho/ROCK signaling pathway thus partially contributing to the repair of injured spinal cord. Monosialoganglioside could promote the motor function but did not suppress expression of Rho A and ROCKⅡ. There was no synergistic effect of electroacupuncture combined with monosialoganglioside.     

缺陷腺病毒载体表达重复10次的Aβ3-10基因疫苗鼻黏膜免疫APPswe, PSEN1dE9转基因小鼠可诱导出Th2型免疫反应

Immunotherapy for Alzheimer’s disease (AD) is effective in improving cognitive function in transgenic mouse models of AD. Because the AN1792 [beta-amyloid (Aβ) 1-42] vaccine was halted because of T cell mediated meningoencephalitis, many scientists are searching for a novel vaccine to avoid the T cell mediated immune response caused by the Aβ1-42. Importantly, the time when the immunization is begun can influence the immune effect. In this study, an adenovirus vaccine was constructed containing 10 × Aβ3-10 repeats and gene adjuvant CpG DNA. Transgenic AD mice were immunized intranasally for 3 months. After 10 × Aβ3-10 vaccine immunization, high titers of anti-Aβ42 IgG1 predominant antibodies were induced. In spatial learning ability and probe tests, the 10 × Aβ3-10 immunized mice showed significantly improved memories compared to control mice. The 10 × Aβ3-10 vaccine resulted in a robust Th2 dominant humoral immune response and reduced learning deficits in AD mice. In addition, the 10 × Aβ3-10 vaccine might be more efficient if administered before Aβ aggregation at an early stage in the AD mouse brain. Thus, the adenovirus vector encoding 10 × Aβ3-10 is a promising vaccine for AD.     

载脂蛋白E在神经母细胞瘤细胞内表达对细胞生长的影响

目的观察人载脂蛋白E(apoE)在神经母细胞瘤细胞SK-N-SH内表达的分布情况及对细胞生长的影响。方法构建含apoEcDNA和绿色荧光蛋白(GFP)基因的真核表达载体,转染至SK-N-SH细胞,经G418结合荧光动态监测筛选稳定表达人apoE基因的细胞克隆,荧光显微镜下观察GFP-apoE融合蛋白在SK-N-SH细胞中的表达。采用MTT法和流式细胞术观察apoE3、apoE4对细胞增殖和细胞周期的影响。结果经酶切和DNA序列测定证实目的基因已插入重组质粒,经G418筛选得到的SK-N-SH细胞克隆中存在apoE基因表达,其表达分布与GFP不同,呈点状分布。转染apoE3的细胞增殖速度加快,G0G1期细胞比例降低,S期细胞增加;而转染apoE4的细胞则相反,表现为增殖速度减缓,G0G1期细胞比例增加,S期细胞减少。结论神经母细胞SK-N-SH细胞内apoE的表达对其生长具有表型特异性。通过建立稳定表达不同多态性的apoE的细胞克隆,为进一步研究apoE在神经细胞中的生物学功能以及apoE在阿尔茨海默病发病机制中的作用奠定了基础。     

真核表达载体pcDNA3.1-3a的构建及其在神经母细胞瘤SK-N-SH细胞中的表达

目的在pcDNA3.1真核表达载体中构建表达融合myc/his标签的甲基化酶表达载体pcDNA3.1-3a,并在神经母细胞瘤细胞株SK-N-SH细胞中进行验证,以期获得可表达甲基化酶催化结构域的融合基因。方法以甲基化酶的pcDNA4.0-GBD-3a-myc/his质粒为模板,通过PCR的方法扩增获得融合有myc/his标签序列的目的区段--甲基化酶催化结构域3a,将其克隆入真核表达载体pcDNA3.1;以EcoRⅠ、EcoRⅤ双酶切和PCR方法对构建的表达载体进行鉴定,并通过测序证明载体构建正确,同时检测甲基化酶3a在SK-N-SH细胞中的表达。结果通过PCR方法获得了含有甲基化酶催化结构域的真核表达载体pcDNA3.1-3a,并通过免疫印记方法验证了在神经母细胞瘤细胞株SK-N-SH细胞样品中用抗his标签抗体可以特异性识别目的蛋白。结论正确构建了甲基化酶表达载体pcDNA3.1-3a,其可在神经母细胞瘤细胞株SK-N-SH细胞中表达。     

共刺激分子OX40在急性脑梗死患者外周血调节性T细胞上的表达及其临床意义

目的探讨急性脑梗死患者外周血调节性T细胞(Treg)上共刺激分子OX40的表达情况及其临床意义。方法选择苏州大学附属第一医院神经内科自2019年4月至l2月收治的75例首次发病的急性脑梗死患者纳入病例组,选择同期招募的年龄、性别与病例组相匹配的36例健康志愿者纳入对照组。采用免疫荧光标记和流式细胞技术检测2组受试者肘静脉血中CD4+CD25+CD127^low Treg细胞上OX40的表达,并分析病例组的OX40^+Treg细胞比例与其美国国立卫生研究院卒中量表(NIHSS)评分、缺血半暗带体积、核心梗死灶体积、梗死灶体积的相关性,同时比较病例组的OX40^+Treg细胞比例在血管内治疗或静脉溶栓前后的变化。结果与对照组比较,病例组的外周血Treg细胞比例明显降低,OX40^+Treg细胞比例明显升高,差异均有统计学意义(P<0.05)。病例组的外周血OX40^+Treg细胞比例与其NIHSS评分呈正相关关系(rs=0.271,P=0.018),与其缺血半暗带体积、核心梗死灶体积、梗死灶体积亦均呈正相关关系(rs=0.435,P=0.000;rs=0.343,P=0.003;rs=0.245,P=0.034)。病例组中血管内治疗患者的术后第7天外周血OX40^+Treg细胞比例较术前明显降低,静脉溶栓患者的术后第3、7天外周血OX40^+Treg细胞比例较术前明显降低,差异均有统计学意义(P<0.05)。结论OX40在急性脑梗死患者外周血Treg细胞上异常表达,并与神经功能缺损、影像学表现及再灌注治疗密切相关。     

烟碱型乙酰胆碱受体在离体海马小胶质细胞上的表达与定位

目的 探讨烟碱型乙酰胆碱受体(α7-nAChR)在体外培养海马小胶质细胞上的表达和定位.方法 取新生SD大鼠的海马组织,进行胶质细胞的混合培养,然后分离纯化小胶质细胞,采用免疫荧光染色检测CD11b/c进行小胶质细胞的鉴定;采用RT-PCR和免疫荧光双标分别检测α7-nAChR在小胶质细胞中的表达和定位.结果 免疫荧光染色显示分离纯化的细胞表达小胶质细胞特异性抗体CD11b/c;RT-PCR能扩增出长度为450 bp的α7-nAChR目的 条带,免疫荧光双标检测显示小胶质细胞CD11b/c和α7-nAChR染色阳性,分别呈红色和绿色荧光,叠加后呈棕黄色,且细胞膜荧光信号较强.结论 α7-nAChR在体外培养的海马小胶质细胞中能正常表达,定位在细胞膜的功能性α7-nAChR较丰富.     

T-type calcium channel expression in cultured human neuroblastoma cells

Human neuroblastoma cells (SH-SY5Y) have similar structures and functions as neural cells and have been frequently used for cell culture studies of neural cell functions.Previous studies have revealed L-and N-type calcium channels in SH-SY5Y cells.However,the distribution of the low-voltage activated calcium channel (namely called T-type calcium channel,including Cav3.1,Cav3.2,and Cav3.3) in SH-SY5Y cells remains poorly understood.The present study detected mRNA and protein expres-sion of the T-type calcium...     

Low-voltage-activated calcium channels in human retinoblastoma cells

Retinoblastoma cells represent pluripotent neural-progenitor cells which, if induced to differentiate, express many features of mature human retinal neurons. Ca channel currents were recorded from isolated, undifferentiated human retinoblastoma Y79 cells in a bath solution containing 20 mM BaCl2 using whole-cell patch-clamp pipettes containing CsCl. The transient, macroscopic currents inactivated with a time constant of about 20 ms at -20 mV and had other properties similar to low-voltage-activated calcium channels described in other cell types: Activation curves fit by the Boltzmann relation had a midpoint of -32 mV and a slope factor of 6.8 mV (-80 mV holding potential) and inactivation curves had a midpoint of -40 mV and a slope factor of 3.7 mV. Non-stationary fluctuation analysis of the currents performed over a 2 kHz bandwidth indicated that each channel contributed 0.44 pA to the macroscopic transient current at -20 mV, suggesting a unitary conductance of about 7 pS.     

Formaldehyde increases intracellular calcium concentration in primary cultured hippocampal neurons partly through NMDA receptors and T-type calcium channels

Objective Formaldehyde at high concentrations is a contributor to air pollution.It is also an endogenous metabolic product in cells,and when beyond physiological concentrations,has pathological effects on neurons.Formaldehyde induces mis-folding and aggregation of neuronal tau protein,hippocampal neuronal apoptosis,cognitive impairment and loss of memory functions,as well as excitation of peripheral nociceptive neurons in cancer pain models.Intracellular calcium([Ca²⁺]_i) is an important intracellular messenger,and plays a key role in many pathological processes.The present study aimed to investigate the effect of formaldehyde on[Ca²⁺]_i and the possible involvement of N-methyl-Z)-aspartate receptors （NMDARs） and T-type Ca²⁺ channels on the cell membrane.Methods Using primary cultured hippocampal neurons as a model,changes of[Ca²⁺]_i in the presence of formaldehyde at a low concentration were detected by confocal laser scanning microscopy.Results Formaldehyde at 1 mmol/L approximately doubled[Ca²⁺]_i.（2R）-amino-5-phosphonopentanoate （AP5,25μmol/L,an NMDAR antagonist） and mibefradil(MIB,1 umol/L,a T-type Ca²⁺channel blocker),given 5 min after formaldehyde perfusion,each partly inhibited the formaldehyde-induced increase of[Ca²⁺]_i,and this inhibitory effect was reinforced by combined application of AP5 and MIB.When applied 3 min before formaldehyde perfusion,AP5 （even at 50μmol/L） did not inhibit the formaldehyde-induced increase of[Ca²⁺]_i but MIB（1 umol/L） significantly inhibited this increase by 70%.Conclusion These results suggest that formaldehyde at a low concentration increases[Ca²⁺]_i in cultured hippocampal neurons;NMDARs and T-type Ca²⁺ channels may be involved in this process.     

Functional analysis of Ca3.2 T-type calcium channel mutations linked to childhood absence epilepsy

PURPOSE: Childhood absence epilepsy (CAE) is an idiopathic form of seizure disorder that is believed to have a genetic basis. METHODS: We examined the biophysical consequences of seven mutations in the Ca(v)3.2 T-type calcium channel gene linked to CAE. RESULTS: Of the channel variants examined, one of the mutants, a replacement of glycine 848 in the domain II-S2 region with serine, resulted in significant slowing of the time courses of both activation and inactivation across a wide range of membrane potentials. These changes are consistent with increased channel activity in response to prolonged membrane depolarizations. CONCLUSIONS: Taken together, these findings suggest that such little changes in channel gating may contribute to the etiology of CAE.     

The alpha1I T-type calcium channel exhibits faster gating properties when overexpressed in neuroblastoma/glioma NG 108-15 cells

The recently cloned T-type calcium channel alpha1I (Cav3.3) displays atypically slow kinetics when compared to native T-channels. Possible explanations might involve alternative splicing of the alpha1I subunit, or the use of expression systems that do not provide a suitable environment (auxiliary subunit, phosphorylation, glycosylation...). In this study, two human alpha1I splice variants, the alpha1I-a and alpha1I-b isoforms that harbour distinct carboxy-terminal regions were studied using various expression systems. As the localization of the alpha1I subunit is primarily restricted to neuronal tissues, its functional expression was conducted in the neuroblastoma/glioma cell line NG 108-15, and the results compared to those obtained in HEK-293 cells and Xenopus oocytes. In Xenopus oocytes, both isoforms exhibited very slow current kinetics compared to those obtained in HEK-293 cells, but the alpha1I-b isoform generated faster currents than the alpha1I-a isoform. Both activation and inactivation kinetics of alpha1I currents were significantly faster in NG 108-15 cells, while deactivating tail currents were two times slower, compared to those obtained in HEK-293 cells. Moreover, the alpha1-b isoform showed significantly slower deactivation kinetics both in NG 1080-15 and in HEK-293 cells. Altogether, these data emphasize the advantage of combining several expression systems to reveal subtle differences in channel properties and further indicate that the major functional differences between both human alpha1I isoforms are related to current kinetics. More importantly, these data suggest that the expression of the alpha1I subunit in neuronal cells contributes to the "normalization" of current kinetics to the more classical, fast-gated T-type Ca2+ current.     

Inhibitory effect of calcium channel blockers on proliferation of human glioma cells in vitro

The effects of 2 specific calcium channel blockers, verapamil and nimodipine, on the proliferation of human glioma tumour cells were investigated in vitro. Tumour tissues for primary cell cultures were obtained bioptically from 3 patients with the histopathological diagnosis of glioblastoma. The [3H]-thymidine incorporation into glioma tumour cells DNA was used as a sensitive index of the cell proliferation. It was found that verapamil (10(-4)-10(-5) M) and nimodipine (10(-4)-10(-6) M) significantly inhibited the [3H]-thymidine uptake in a dose-related manner. The inhibitory effect of both calcium channel antagonists was reversed by simultaneous addition of calcium chloride (5 x 10(-3) M). These results indicate that verapamil and nimodipine may exert an antiproliferative effect on glioma cells growth acting through a blockade of specific voltage-dependent calcium channels.     

Morphine and methadone have different effects on calcium channel currents in neuroblastoma cells

Using patch-clamp technique, cellular calcium channel currents were studied on nine mouse neuroblastoma N1E115 cells. Both morphine and methadone decreased the T-type calcium currents in dose-dependent fashion. These effects were partially blocked by naloxane. On L-type calcium currents, morphine showed no effect. However, methadone inhibited the L-calcium currents in dose-dependent fashion. This effect was not antagonised by naloxone. Hence, the action of methadone on L-calcium channel is probably not associated with μ receptors.     

Potentiation of transmitter release from NMB human neuroblastoma cells by kappa-opioids is mediated by N-type voltage-dependent calcium channels

The selective kappa-opioid agonist trans-(+/-)-3, 4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl] benzenacetamidemethansulfonate (U50,488) potentiates both basal and depolarization-evoked [3H]dopamine release from NMB cells. The potentiation of dopamine release by U50,488 is mediated by N-type voltage-dependent calcium channels since it is blocked by omega-conotoxin, and is resistant to pertussis toxin (PTX)-treatment. When the stimulation of release by U50,488 is blocked by the N-channel antagonist omega-conotoxin, an inhibitory effect on dopamine release is revealed, suggesting that stimulatory and inhibitory effects of U50,488 are exerted in parallel.