谷氨酸受体拮抗剂对大鼠实验性脑缺血的保护作用

目的探讨谷氨酸受体拮抗剂MKS01对大鼠实验I生脑缺血的保护作用。方法健康雄性SD大鼠30只,完全随机分为假手术组、模型组、局灶性脑缺血MK801组,每组10只。模型组和MK801组复制局灶性脑缺血大鼠模型,观察谷氨酸受体拮抗剂MK801对大鼠脑组织病理变化和动物行为评分。结果与假手术组相比,模型组大鼠在缺血24h后表现出明显的神经行为障碍,动物行为评分为（7．20±2．92）分;MK801组为（5．71±1．38）分。2组分别与假手术组对比及模型组与MK801组间对比,差异均有统计学意义（均P〈0．01）。模型组大鼠脑缺血24h后,脑组织出现明显缺血区,缺血区占全脑（27．59±3．74）％;MK801组脑缺血区域占全脑（22．67±6．67）％。MK801组脑缺血面积与模型组相比,以及MK801组、模型组与假手术组相比,差异有统计学意义（P〈0．05或P〈0．01）。结论谷氨酸受体拮抗剂MK801静脉注射给药可明显改善脑缺血大鼠的行为障碍,缩小缺血区面积,表明谷氨酸受体拮抗剂MK801对实验性大鼠脑缺血具有保护作用。     

谷氨酸及其受体与阿片类身体依赖性

阿片类物质具有极强的身体依赖性,其戒断后的痛苦体验是一种负性强化作用,是产生强迫觅药行为的内在驱动力［１］。阿片类物质身体依赖性的机制尚不十分明确,早期的研究主要通过对阿片依赖动物进行脑内核团定位毁损或注入阿片受体拮抗剂后观察对戒断症状影响来探寻身体戒断的神经解剖通路。大量研究发现,蓝斑核（ＬＣ）对戒断症状的表达具有极其显著的影响［２］。人们围绕ＬＣ进行了一系列电生理、神经递质改变及分子生物学研究后发现,ＬＣ内去甲肾上腺素能（ＮＡ）神经元的高兴奋性,谷氨酸（Ｇｌｕ）水平增高与身体戒断症状表达之间具有密…     

谷氨酸及其受体与帕金森氏病

谷氨酸及其受体与帕金森氏病张春芬伦学庆１（济宁医学院药理教研室、１附属医院神经外科，济宁２７２１１３）１９９６－０６－２６收稿，１９９７－０４－０４修回作者简介：张春芬，女，３０岁，硕士，副教授中国图书分类号Ｒ７４５．７；Ｒ９７１；Ｒ９７７．４帕金森...     

谷氨酸受体与全身麻醉机制研究进展

谷氨酸(Glu)是哺乳动物脑内主要的兴奋性氨基酸,Glu受体是其结合靶点,其在脑内分布广泛,其中大脑皮质和海马的Glu受体密度最高。中枢Glu受体至少可分为5种类型,分别命名为N-甲基-D-天门冬氨酸(N-methyl-D-aspartale,NMDA)受体、α-氨基-3-羟基-5-甲基-4-异恶唑丙酸(α-amino-3-hydroxy-5-methyl-4-isox-azole-propionate,AMPA)受体、海人藻酸(kainic acid,KA)受体、代谢型Glu受体(metabotropic glutamete receptors,mGluR)和L-2-氨基-4-磷酸丁酸(L-AP4)受体。其中NMDA、AMPA、KA受体都是由Glu门控的阳离子通道,属离子型Glu受体;非NMDA受体mGluR和L-AP4受体均属于G蛋白偶联的Glu受体。自1846年莫顿演     

N-甲基-D-天冬氨酸型谷氨酸受体的调节与氰化物中枢毒作用

氰化物可通过间接与直接两条途径激活(N-甲基-D-天冬氨酸型)NMDA受体，首先氰化物引起的能量障碍能通过促使细胞外谷氨酸(Glu)浓度增高和细胞内Ca^2 稳态失调，间接地激活NMDA受体，其次氰化物可能作为NMDA受体直接的调节剂，通过调节NMDA受体中NR1或NR2亚型等半胱氨酸残基的氧化还原位点，提高NMDA受体的活性，随着间接和直接NMDA受体的激活，产生一系列由NMDA受体介导的中枢毒作用，最终导致神经元细胞损伤、凋亡及死亡。由此认为NMDA受体的激活在氰化物诱导的神经损伤机制中可能起关键的作用。     

N-甲基-D-天冬氨酸受体与抑郁障碍

越来越多研究表明,除单胺神经递质外,谷氨酸及相关受体在抑郁症病因中也扮演了重要的角色,特别是N-甲基-D-天冬氨酸(NMDA)受体,有证据表明NMDA受体的过度激活是抑郁障碍的病理生理机制之一。本文总结了谷氨酸及相关受体与抑郁障碍的关系,对NMDA受体成为潜在抗抑郁药靶点做一简要讨论。     

代谢型谷氨酸受体与应激损伤

代谢型谷氨酸受体 (mGluRs)在应激性损伤中的作用日益受到重视 ,它可参与GC水平的调节 ,影响谷氨酸神经毒性作用和突触可塑性 (LTP、LTD)的诱导 ,由此表明mGluRs在应激性损伤中可能占有重要的地位。由于不同类型的mGluRs具有不同的作用 ,机制较为复杂 ,因此 ,今后还需进一步加强mGluRs与应激性损伤关系的研究     

抑郁症与谷氨酸传导

对抑郁症病因病理学的传统解释主要集中于中枢神经单胺类递质的功能失调,然而,新的研究表明,除单胺神经递质外,谷氨酸等也扮演了重要的角色。本文首先介绍了谷氨酸受体的分型,然后就抗抑郁药引起的谷氨酸受体的生化、药理学的改变,以及谷氨酸功能失调在临床和动物模型上的表现予以综述,以增进研究者对这个领域的关注。     

艾灸减轻新生小鼠缺氧缺血性脑病的作用及其机制研究

目的　研究艾灸减轻新生小鼠缺氧缺血性脑病（HIE）的作用及其机制。方法　55只出生7 d的ICR小鼠按照随机数字表法分为假手术组（Sham组）、模型组（HI组）和艾灸组（MOX组）。HI组和MOX组采用右侧颈总动脉结扎联合缺氧法制备HIE模型,MOX组建模后艾灸刺激大椎穴,35 min/次,1次/d,连续治疗3 d。处理结束后麻醉取脑,DAPI染色观察脑组织形态结构,TUNEL染色检测脑组织细胞凋亡;免疫荧光染色检测脑组织caspase-9表达;放射免疫分析法测定脑组织caspase-3含量。结果　与Sham组相比,HI组小鼠脑组织排列疏松,脑组织凋亡细胞数和caspase-9表达显著增多（P＜0.05）;与HI组相比,MOX组小鼠脑组织排列较致密,脑组织凋亡细胞数、caspase-9和caspase-3表达显著减少（P＜0.05）。结论　艾灸可能通过降低脑组织caspase-9和caspase-3的表达来减少细胞凋亡,从而减轻新生小鼠缺氧缺血性脑损伤。     

Caspase 8和9在肿瘤局部微环境形成早期与调控蛋白Twist1的关联性

目的:研究Caspase 8、9在肿瘤局部微环境早期线形程序性坏死(LPPCN)及血管生成拟态(VM)形成中对上皮-间充质转变(EMT)调控蛋白Twist1表达的影响。方法:将96只C57BL小鼠随机分为Caspase 8抑制剂组、Caspase 9抑制剂组、Caspase 8、9抑制剂联合用药组(联合组)和对照组,制备小鼠荷瘤模型。采用免疫组织化学法检测EMT调控蛋白Twist1在LPPCN和VM周围第1~4层肿瘤细胞的表达水平。结果:4组LPPCN周围第1~4层肿瘤细胞Twist1核阳性染色指数差异有统计学意义(P<0.05或P<0.01),且呈递减趋势;对于同层肿瘤细胞,联合组和对照组的细胞核Twist1阳性染色指数比较差别有统计学意义(均P<0.01)。LPPCN、VM周围第1~4层肿瘤细胞、同一层不同组的肿瘤细胞浆Twist1阳性染色指数差异均无统计学意义(均P>0.05);VM周围第1~4层肿瘤细胞、同一层不同组的肿瘤细胞核Twist1阳性染色指数差异均无统计学意义(均P>0.05)。结论:Caspase 8、9介导的凋亡机制启动与EMT发生存在关联性。     

Titanium dioxide nanoparticles cause apoptosis in BEAS-2B cells through the caspase 8/t-Bid-independent mitochondrial pathway

To understand the underlying mechanism for apoptosis induced by titanium dioxide nanoparticles (TNP), human airway epithelial cell line was cultured to investigate the relevant apoptosis pathways. Our results showed that the levels of reactive oxygen species and morphological apoptosis increased in a dose-dependent manner whereas cell viability decreased in a similar manner in response to TNP exposure in the BEAS-2B cells. The activities of caspase 3 and PARP were also increased in parallel to the morphological apoptosis. Levels of caspase 9 increased significantly whereas there were no detectable changes in caspase 8 and t-Bid in the TNP treated cells. Caspase 9 inhibition blocked the TNP-induced activation of caspase 3 significantly. The levels of bax, cytochrome C, p53 and bcl-2 also changed reflecting the activation of intrinsic apoptosis pathway. Our results provide solid evidence that apoptosis in BEAS-2B cells exposed to TNP occurred via a mitochondrial apoptosis pathway independent of caspase 8/t-Bid pathway.     

Opposing roles for caspase and calpain death proteases in L-glutamate-induced oxidative neurotoxicity

Oxidative glutamate toxicity in HT22 murine hippocampal cells is a model for neuronal death by oxidative stress. We have investigated the role of proteases in HT22 cell oxidative glutamate toxicity. l-glutamate-induced toxicity was characterized by cell and nuclear shrinkage and chromatin condensation, yet occurred in the absence of either DNA fragmentation or mitochondrial cytochrome c release. Pretreatment with the selective caspase inhibitors either benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (pan-caspase), N-acetyl-Leu-Glu-His-Asp-aldehyde (caspase 9) or N-acetyl-Ile-Glu-Thr-Asp-aldehyde (caspase 8), significantly increased l-glutamate-induced cell death with a corresponding increase in observed nuclear shrinkage and chromatin condensation. This enhancement of glutamate toxicity correlated with an increase in l-glutamate-dependent production of reactive oxygen species (ROS) as a result of caspase inhibition. Pretreating the cells with N-acetyl-l-cysteine prevented ROS production, cell shrinkage and cell death from l-glutamate as well as that associated with the presence of the pan-caspase inhibitor. In contrast, the caspase-3/-7 inhibitor N-acetyl-Asp-Glu-Val-Asp aldehyde was without significant effect. However, pretreating the cells with the calpain inhibitor N-acetyl-Leu-Leu-Nle-CHO, but not the cathepsin B inhibitor CA-074, prevented cell death. The cytotoxic role of calpains was confirmed further by: 1) cytotoxic dependency on intracellular Ca²⁺ increase, 2) increased cleavage of the calpain substrate Suc-Leu-Leu-Val-Tyr-AMC and 3) immunoblot detection of the calpain-selective 145 kDa α-fodrin cleavage fragment. We conclude that oxidative l-glutamate toxicity in HT22 cells is mediated via calpain activation, whereas inhibition of caspases-8 and -9 may exacerbate l-glutamate-induced oxidative neuronal damage through increased oxidative stress.     

A modified RBL-2H3 mediator release assay for the detection of polyclonal IgE antibody

RBL-2H3 mediator release assay, developed for specific IgE screening studies, was not as sensitive as passive cutaneous anaphylaxis (PCA) assay in the polyclonal antibody detection. In the present investigation, the detection sensitivity of RBL-2H3 assay was elevated by modifying the experiment protocols from choosing the proper releasing medium and optimizing the sensitization manner. The polyclonal antibody was generated from Brown Norway (BN) rats exposed to Ovalbumin (OVA). In contrast to Tyrode buffer A, RBL-2H3 cells cultured in DMEM had a lower spontaneous secretion and a higher response to antigen stimulation, both of which could help to increase the detection sensitivity. The rat sera used in the sensitization process should be diluted appropriately to avoid the proliferation-promoting effect on RBL-2H3 cells. The results of the kinetics of sensitization showed that prolonging the sensitization time and then reculturing the cells in IgE free medium for a further 24?h after the removal of rat sera could reach a marked increase in the degree of sensitization. The highest anti-OVA antibody titer detected by the modified RBL-2H3 assay was 4096, while PCA assay was 1024. These data provide evidence that the modified RBL-2H3 mediator release assay has a promising prospect in the determination of the biologic activity of polyclonal antibody.     

纳豆激酶基因在毕赤酵母中的表达纯化及抗体制备

目的实现纳豆激酶(NK)在巴斯德毕赤酵母中的分泌表达,以纯化产物为抗原制备兔抗NK血清。为建立双抗夹心酶联免疫吸附反应法测定生物体内NK含量,进一步研究NK在体内代谢与功能奠定基础。方法将NK基因克隆到毕赤酵母表达载体pHBM905A中,鉴定后的重组质粒用SalⅠ酶切线性化后电转化到毕赤酵母宿主菌GS115中,甲醇诱导表达,经盐析和Millipore超滤膜过滤分离纯化表达产物。纤维蛋白法检测纯化产物的活性,并以纯化产物为抗原免疫新西兰大白兔制备兔抗NK血清。结果SDS-PAGE结果显示NK成功表达,其相对分子质量(Mr)约为27 000,纤维蛋白平板实验显示表达产物具有较好的纤溶活性。酶联免疫法测得多克隆抗体效价约为1∶8 000,Western blot结果显示在Mr27 000附近有1条特异带出现。结论NK在巴斯德毕赤酵母中实现了分泌表达,表达产物具有较好的纤溶活性和免疫原性。     

Functional interaction between adenosine A2A and group III metabotropic glutamate receptors to reduce parkinsonian symptoms in rats

Non-dopaminergic drugs acting either on adenosine A2A or metabotropic glutamate (mGlu) receptors reduce motor impairment in animal models of Parkinson's disease (PD), suggesting a possible functional interaction between these receptors to regulate basal ganglia function. The present study therefore tested the behavioural effects of compounds acting selectively on A2A or on specific mGlu receptor subtypes, alone or in combination, in rodent models of PD. Acute administration of the adenosine A2A receptor antagonists CSC or MSX-3 at the highest doses tested (5 and 1.25mg/kg, respectively) significantly reduces haloperidol-induced catalepsy. Furthermore, the anticataleptic effect of MSX-3 was enhanced by a 3-week treatment. Acute administration of the selective group III mGlu agonist ACPT-I produces potent anticataleptic effects and prolongs time on rotarod of 6-OHDA-lesioned rats. In contrast, acute or chronic administration of MPEP (mGlu5 receptor antagonist) has no anticataleptic action. Furthermore, the acute co-administration of ACPT-I 1mg/kg, but not 5mg/kg, with CSC markedly reduces catalepsy. Opposite effects are observed after a 3-week co-administration. The co-administration of ACPT-I with MSX-3 has anticataleptic effects both after acute or chronic treatment. In contrast, acute combination of subthreshold doses of CSC and MPEP has no effect. After a 3-week treatment, however, the combination of CSC and MPEP was found to reduce haloperidol-induced catalepsy. Altogether, these results show for the first time that systemic activation of group III mGlu receptors with ACPT-I provides benefits in parkinsonian rats and underlie a possible interaction with A2A receptors to regulate basal ganglia motor function.     

Ni3S2 uptake by lung cells and its interaction with plasma membranes

The uptake and biological transformation of Ni3S2 was studied on guinea pig alveolar macrophages (GPAM) in primary culture. Two different pathways are observed: (i) phagocytosis of alpha Ni3S2 crystals and subsequent degradation to minute particles, which are recovered bound to the membranes of phagocytic vacuoles and to lysosomal membranes. These degradation products contain sulphur in very reduced quantities, as revealed by energy-dispersive spectrometry (EDS). (ii) Extracellular degradation to regular round particles (0.1-0.2 microns) and irregular minute particles (10-30 nm). Round particles may enter the cell by pinocytosis and are characterized by the loss of sulphur. Minute particles are bound preferentially to cell membranes, to cytoplasmic organelles, such as endoplasmic reticulum, mitochondria and peroxysomes, to liposomes and to the euchromatinic part of nuclei. EDS analyses in these particles revealed the substitution of sulphur by phosphorus. This observation suggests the formation of an Ni-P complex with the phosphate groups of membranous and liposomal phospholipids and of the euchromatinic part of DNA or RNA. Steady-state fluorescence polarization analysis were carried out on GPAM and, for comparative purposes, on human embryonic pulmonary epithelial cells (L132 cell line). In both cell types they revealed a significant increase of membrane fluidity, induced either by desaturation of aliphatic chains or directly by the cleavage of fatty acid chains.     

艰难梭菌毒素C蛋白的克隆表达和抗体制备

目的克隆艰难梭菌毒素C(tcdC)基因并构建TcdC蛋白的原核表达载体,制备多克隆抗体。方法从艰难梭菌标准株(ATCC43255)基因组DNA中扩增获得tcdC基因的部分片段,连接到原核表达载体并转化到大肠杆菌中,诱导表达GST-TcdC/L和IL1-TcdC/L融合蛋白,前者用作免疫抗原,后者用作检测抗原。采用SDS-PAGE法鉴定融合蛋白的表达,并通过Ni柱纯化IL1-TcdC/L融合蛋白,Q柱纯化GST-TcdC/L融合蛋白。将纯化的GST-TcdC/L融合蛋白于兔背部皮内多点注射,每只1 mg,4周1次,共3次。最后1次免疫后2周,采血分离血清。用IL1-TcdC/L蛋白包被酶联板,采用ELISA法检测多抗血清效价;Western蛋白印迹法检测多抗特异性,结果 SDS-PAGE结果表明,所表达的2个融合蛋白与预期分子质量一致。ELISA结果显示,制备的TcdC多克隆抗体效价>1:6.4×10~4。Western蛋白印迹实验显示,制备的TcdC/L多克隆抗体能特异性识别艰难梭菌中的TcdC蛋白。结论成功克隆了艰难梭菌tcdC/L基因并进行了原核表达,成功制备了其多克隆抗体,为后续研究tcdC基因在艰难梭菌致病过程中的作用及机制奠定了基础。     

Signal transduction events elicited by natural products: Role of MAPK and caspase pathways in homeostatic response and induction of apoptosis

Many natural products elicit diverse pharmacological effects. Using two classes of potential chemopreventive compounds, the phenolic compounds and the isothiocyanates, we review the potential utility of two signaling events, the mitogen-activated protein kinases (MAPKs) and the ICE/Ced-3 proteases (caspases) stimulated by these agents in mammalian cell lines. Studies with phenolic antioxidants (BHA, tBHQ), and natural products (flavonoids; EGCG, ECG, and isothiocyanates; PEITC, sulforaphane), provided important insights into the signaling pathways induced by these compounds. At low concentrations, these chemicals may activate the MAPK (ERK2, JNK1, p38) leading to gene expression of survival genes (c-Fos, c-Jun) and defensive genes (Phase II detoxifying enzymes; GST, QR) resulting in survival and protective mechanisms (homeostasis response). Increasing the concentrations of these compounds will additionally activate the caspase pathway, leading to apoptosis (potential cytotoxicity). Further increment to suprapharmacological concentrations will lead to nonspecific necrotic cell death. The wider and narrow concentration ranges between the activation of MAPK/gene induction and caspases/cell death exhibited by phenolic compounds and isothiocyanates, respectively, in mammalian cells, may reflect their respective therapeutic windowsin vivo. Consequently, the studies of signaling pathways elicited by natural products will advance our understanding of their efficacy and safety, of which many may become important therapeutic drugs of the future.     

Sorbitol-induced apoptosis of human leukemia is mediated by caspase activation and cytochrome <Emphasis Type="Italic">c</Emphasis> release

It has been reported that sorbitol induces apoptosis in several cancer cell lines. However, the molecular mechanism underlying the sorbitol-induced apoptotic process is not yet clearly understood. In the present study, the intracellular signaling pathways of sorbitol-induced apoptosis in human K562 cells were investigated using both morphological analysis and DNA fragmentation technique. In this study, we demonstrated that sorbitol-induced apoptosis in human K562 cells is a concentration- and time-dependent manner. This sorbitol-induced apoptosis in human K562 cells was also accompanied by the up-regulation of Bax, and down-regulation of p-Bcl-2, but no effect on the levels of Bcl-X(L). Moreover, the sorbitol treatment resulted in a significant reduction of mitochondria membrane potential, increase in the release of mitochondrial cytochrome c (cyt c), and activation of caspase 3. Furthermore, treatment with caspase 3 inhibitor (z-DEVD-fmk) was capable of preventing the sorbitol-induced caspase 3 activity and cell death. These results clearly demonstrate that the induction of apoptosis by sorbitol involves multiple cellular/molecular pathways and strongly suggest that pro- and anti-apoptotic Bcl-2 family proteins, mitochondrial membrane potential, mitochondrial cyt c, and caspase 3, they all participate in sorbitol-induced apoptotic process in human K562 cells.