异丙酚对大鼠海马神经元NMDA受体通道电流的影响

目的 研究异丙酚对大鼠海马神经元N-甲基-D-天冬氨酸（NMDA）受体通道电流的影响。方法 体外培养新生Wistar大鼠海马神经元8—12d，采用全细胞膜片钳技术和压力喷射给药方式，钳制电压为-80mV，记录3、48μg∥ml异丙酚对100μmol／LNMDA诱发NMDA受体通道电流的影响；然后用7-氨基丁酸（GABA）受体拮抗剂荷包牡丹碱100μmol／L阻断GABA．受体，观察3、48μg／ml异丙酚对NMDA受体通道电流的影响。结果 3、48μg／ml异丙酚抑制自发兴奋性突触后电流并直接激动GABA．受体，使Cl．内流，产生外向超极化的GABA电流，从而间接抑制NMDA受体通道电流。用荷包牡丹碱100μmol／L阻断GABA．受体后，3、48μg∥ml异丙酚仍可抑制100μmol／LNMDA诱发的NMDA受体通道电流（P〈0．05）。结论 异丙酚通过激活GABA．受体影响NMDA受体通道电流，对NMDA受体通道也有直接抑制作用。     

膜相关性鸟苷酸激酶与脑缺血

膜相关性鸟苷酸激酶（membrane associated guanylate kinase,MAGUK）是一类突触后致密蛋白（postsynaptic density,PSD）,定位于突触后膜的突触后致密区,包括PSD-95、SAP-102、PSD-93和SAP-97.MAGUK家族成员含有多个蛋白结合位点,这些特殊的结合位点负责介导多种信号的转导.MAGUK参与中枢神经系统疾病发生和发展机制,已成为神经科学研究的热点.文章简要综述了MAGUK在缺血性脑损伤中的作用.     

缺血预处理介导神经保护的分子机制

大量动物实验证实,缺血预处理可产生强大的器官保护作用,但动物实验向临床试验转化的进展和结果不尽如人意.对缺血预处理介导的神经保护的分子机制进行研究,寻找可转化到临床的安全且有效的预处理诱导方式,对于提高卒中和手术患者神经组织对缺血缺氧的耐受力,实现安全和有效的神经保护具有重要意义.文章从预处理活化受体、线粒体、转录因子和蛋白激酶等方面对缺血预处理介导神经保护的分子机制进行了综述.     

家兔延髓孤束核神经元NMDA受体和非NMDA受体在急性缺氧中的作用

目的探讨成年家兔延髓孤束核（NTS）神经元上N-甲基-D-天冬氨酸（NMDA）受体和非NMDA受体在急性缺氧引起的外周化学感受器兴奋性传导中的作用。方法通过显微注射技术向家兔NTS内注射NMDA受体拮抗剂2-氨基-5-磷酸戊酸（AP-5）和（或）非NMDA受体拮抗剂6．7-二硝基喹诺林-2．3-二酮（DNQX），观察其对急性缺氧时膈神经放电变化的影响。结果应用AP-5或DNQX对缺氧引起的呼吸增强均产生了抑制效应，两种受体拮抗剂之间有交互作用。结论哺乳动物NTS神经元上的NMDA受体和非NMDA受体在介导急性缺氧时外周化学感受性信号向中枢的传递中均发挥作用，NMDA受体尤为重要。两种受体有协同性作用。     

Effects of In Vitro Ethanol and Fetal Ethanol Exposure on Glutathione Stimulation of N-Methyl-D-Aspartate Receptor Function

The present studies investigated the effects of glutathione (GSH; γ-glutamylcysteinylglycine) and its oxidized form (GSSG) on neuronal N -methyl-D-aspartate (NMDA) receptor activation in both acute and chronic preparations of ethanol exposure. It was demonstrated using fura-2-loaded dissociated brain cells from newborn rat pups that both GSH and GSSG (0–4 mM) produced concentration-dependent increases in intracellular calcium similar to those produced by NMDA and other agonists of the NMDA receptor. GSH-stimulated calcium entry was not inhibited by low intoxicating concentrations of ethanol, which contrasts with ethanol's typical inhibitory effect on NMDA-stimulated receptor activation. Behavioral studies in adult rats demonstrated that ethanol-induced sleep times were significantly decreased when 10 μl of GSSG (20 mM) were administered intracere-broventricularly approximately 5 min before an intraperitoneal injection of 20% (w/v) ethanol (3 g/kg). These findings suggest that the less potent effect of ethanol on GSH-stimulated calcium entry as well as the reduction in ethanol-induced sleep times may be related to the presence of glycine in the peptide. The glycine found in GSH may activate the glycine site and block or reduce ethanol's action on this site. It appears that although GSH may play an important role in the activation of the NMDA receptor, this action does not involve a process that is sensitive to acute ethanol exposure. In contrast, when rat pups were chronically exposed to ethanol via prenatal exposure before the fura-2 preparation, increases in NMDA- and GSH-stimulated calcium entry were significantly decreased relative to those in pair-fed and ad libitum-fed controls. Thus, chronic in utero exposure to ethanol may alter the NMDA-receptor complex, such that calcium entry mediated by NMDA or GSH activation is significantly reduced.     

C末端缺失NR2B亚单位表达载体的构建及其在NMDA受体装配研究中的应用

目的：研究NMDA受体NR2B亚单位细胞内C末端，在NR1—1a／NR2B亚型NMDA受体装配和表面表达中的作用。方法：构建C末端不同缺失和GFP标记的NR2B亚单位表达载体，单独转染或与NR1亚单位共转染到HEK293细胞，用抗GFP多克隆抗体和Cy3荧光素交联的二抗作活细胞表面受体染色。结果：成功构建了8个C末端不同缺失的GFP—NR2B表达质粒(GFP—NR2B△1～△8)。将GFP—NR1—1a，GFP—NR2B及GFP—NR2B△1～△8分别单独转染HEK293细胞，不能获得迭细胞膜表面的表达。而将这些质粒分别与NR1—1a共转染293细胞，发现GFP—NR2B及C末端部分缺失的GFP—NR2B△1～△6，均能与NR1—1a一起表达于细胞膜表面，而仅留有PDZ结合基序的GFP—NR2B△7和C末端全长缺失的GFP—NR2B△8，则不能与NR1—1a一起表达于细胞膜表面。结论：NR1—1a与NR2B的共表达和装配，是形成NR1—1a／NR2B亚型NMDA受体并表达于细胞表面的必要条件。NR2B与NR1—1a装配时，NR2B对NR1—1a C1盒中内质网滞留基序的遮蔽和阻滞作用，并不依赖于NR2BC末端某一特定的区域，相反可能仅要求有一定长度的NR2BC末端。     

炎症后内脏高敏感大鼠前扣带回皮质NR2A、NR2B表达变化

背景:研究显示N-甲基-D-天冬氨酸(NMDA)受体参与了伤害性信号的传递,中枢前扣带回皮质(ACC)在内脏高敏感大鼠内脏疼痛反应的调节中起重要作用,该作用是由NMDA受体活性增强介导的。目的:检测炎症后内脏痛觉过敏大鼠ACC区域NMDA受体亚基NR2A、NR2B的表达变化,探讨两者在炎症后内脏高敏感形成中的作用。方法:以去氧胆酸结肠灌注建立炎症后内脏痛觉过敏大鼠模型。造模3周后观察实验组和对照组结肠组织病理学改变,以对结直肠扩张(CRD)的内脏运动反射(VMR)幅值为指标评价内脏痛敏感性的变化,以免疫荧光法和蛋白质印迹法检测ACC区域NR2A、NR2B表达情况。结果:实验组和对照组大鼠结肠组织均未见明显病理学改变。CRD压力为60 mm Hg(伤害性刺激)时,实验组VMR幅值显著高于对照组(P0.05)。与对照组相比,实验组ACC区域NR2A、NR2B荧光强度和蛋白表达量均显著上调(P0.05)。结论:ACC区域NMDA受体亚基NR2A、NR2B表达上调在炎症后内脏高敏感的形成中发挥重要作用。     

黄芩苷对脊髓缺血神经保护作用的研究

目的探讨黄芩苷对脊髓缺血神经保护作用机制的研究。方法建立新西兰大白兔脊髓缺血模型,分为模型组、高剂量组、低剂量组及假手术组。采用H-E染色、原位末端转移酶标记技术(TUNEL)、免疫组织化学、逆转录反应系统(RT-PCR)等技术检测N-甲基-D-天门冬氨酸受体(NMDAR)、诱导型一氧化氮合酶(iN-OS)、半胱氨酸天冬氨酸蛋白酶3(Caspase-3)的表达水平,观察不同剂量黄芩苷对脊髓缺血的神经保护作用。结果模型组脊髓正常结构完全消失,低剂量组结构较完整,假手术组脊髓结构正常。高剂量组缺血损害情况介于低剂量组于假手术组之间。凋亡指数、表达NMDAR、iNOS、Caspase-3的阳性细胞百分比及相应mRNA表达水平:模型组最高,低剂量组、高剂量组、假手术组则依次降低,差别具有统计学意义(P<0.05)。结论黄芩苷能抑制神经细胞凋亡,对脊髓缺血具有神经保护作用。     

Effects of low and high doses of selective sigma ligands: further evidence suggesting the existence of different subtypes of sigma receptors

Several high affinity sigma (σ) ligands, such as DTG, JO-1784, (+)-pentazocine, BD-737 and L-687,384, administered at low doses act as agonists by potentiating N-methyl-D-aspartate (NMDA)-induced activation of pyramidal neurons in the CA₃ region of the rat dorsal hippocampus. This potentiation is dose-dependent at doses between 1 and 1000 μg/kg, IV but bell-shaped dose-response curves are obtained. Other σ ligands like haloperidol, BMY-14802, (+)3-PPP and NE-100 administered at low doses act as σ antagonists, since they do not modify the NMDA response but suppress the potentiation of the NMDA response induced by σ agonists. Because high doses of the σ agonists do not potentiate the NMDA response, the present experiments were undertaken to assess if, at high doses, these σ ligands could also act as σ antagonists and suppress the potentiation induced by low doses of σ agonists. High doses of DTG, JO-1784, BD-737, and L-687,384, administered acutely, had an effect similar to that of low doses of haloperidol, by suppressing and preventing the potentiation induced by low doses of DTG, JO-1784, BD-737, L-687,384 and (+)-pentazocine. High doses of (+)-pentazocine suppressed the effect of a low dose of (+)-pentazocine but did not affect the potentiation induced by a low dose of the other σ agonists. The potentiation induced by a low dose of a σ₁ agonist was not further increased by the subsequent administration of another low dose of a σ₁ agonist. All together, these results strongly suggest that more than two subtypes of σ receptors exist in the CNS. Received: 1 April 1996 / Final version: 14 August 1996     

Tat-LK15运载NR2B siRNA治疗慢性炎性疼痛的效果

目的 探讨大鼠鞘内注射细胞穿透肽Tat-LK15介导NR2B siRNA治疗大鼠慢性炎性疼痛的可行性.方法 健康雄性SD大鼠72只,6周龄,体质量180~200 g,采用随机数字表法分为4组(n=18):空白对照组(C组)、慢性炎性痛模型组(CFA组)、Tat-LK15/NR2B siRNA组(siTat-LK15组)和Tat-LK15/阴性对照siRNA组(ncTat-LK15组).除C组不给予任何处理外,其余大鼠右后趾皮下注射完全弗氏佐剂(CFA)制作慢性炎性痛模型.CFA组、siTat-LK15组及ncTat-LK15组于模型制备后第1、3、5天分别鞘内注射生理盐水、Tat-LK15/NR2B siRNA及Tat-LK15/阴性对照siRNA 10μL.每组取12只大鼠于造模前1 d及鞘内注射后3、7、14、21 d时测定机械缩足反应阈(PWMT)和热缩足反应潜伏期(PWTL).每组取6只大鼠于鞘内注射后7 d处死后取脊髓,检测鞘内注射对大鼠脊髓NR2B蛋白表达的影响.结果 鞘内注射后7 d,siTat-LK15组大鼠脊髓NR2B蛋白表达下调.鞘内注射后3、7、14及21 d,siTat-LK15组PWMT与PWTL与CFA组相比明显升高,差异有统计学意义(P<0.01),而ncTat-LK15组PWMT与PWTL较CFA组相比差异无统计学意义(P>0.05).结论 鞘内注射Tat-LK15/NR2B siRNA可有效减轻CFA所致大鼠慢性炎性疼痛.