Reduced postischemic expression of a glial glutamate transporter,GLT1, in the rat hippocampus

Perturbations of the synaptic handling of glutamate have been implicated in the pathogenesis of brain damage after transient ischemia. Notably, the ischemic episode is associated with an increased extracellular level of glutamate and an impaired metabolism of this amino acid in glial cells. Glutamate uptake is reduced during ischemia due to breakdown of the electrochemical ion gradients across neuronal and glial membranes. We have investigated, in the rat hippocampus, whether an ischemic event additionally causes a reduced expression of the glial glutamate transporter GLT1 (Pines et al. 1992) in the postischemic phase. Quantitative immunoblotting, using antibodies recognizing GLT1, revealed a 20% decrease in the hippocampal contents of the transporter protein, 6 h after an ischemic period lasting 20 min induced by four vessel occlusion. In situ hybridization histochemistry with ³⁵S labelled oligonucleotide probes or digoxigenin labelled riboprobes directed to GLT1 mRNA showed a decreased signal in the hippocampus, particularly in CA1. This reduction was more pronounced at 3 h than at 24 h after the ischemic event. We conclude that the levels of GLT1 mRNA and protein show a modest decrease in the postischemic phase. This could contribute to the delayed neuronal death typically seen in the hippocampal formation after transient ischemia.     

问号赖型钩端螺旋体重组质粒pDL121及其表达产物的初步研究

目的:探讨问号赖型钩端螺旋体重组质粒pDL121外源基因及其表达产物23 kDa蛋白的特点。方法:用6种内切酶对pDL121行酶谱分析,用Digoxin标记的pDL121外源基因片段作探针对不同种属的钩体进行杂交,用SDS-PAGE制备23 kDa蛋白,Western blot鉴定其免疫原性。结果:pDL121外源基因没有6种内切酶的酶切位点,重组探针与致病性钩体(serovar lai strain 017,serovar hebdomadis strain56610,serovar pomona strain 56608)有杂交信号,与非致病性钩体(serovar patoc strain Patoc I,serovar illini strain 3055)无杂交信号,亦不识别大肠杆菌。23 kDa兔抗血清可识别pDL121体外表达的23 kDa蛋白带和赖型钩体017株超声抗原成份;其抗体滴度为1/12800;用pDL121细菌裂解液主动免疫豚鼠,可使豚鼠抵抗强毒力株钩体攻击。结论:pDL121外源基因可能是赖型钩体的一个新基因;该重组探针能鉴别致病性钩体和非致病性钩体;23 kDa抗原有良好的免疫原性,可能是赖型钩体017株的保护性抗原。     

五味子甲素通过ATP结合盒转运子B1逆转胶质瘤干/祖细胞耐药性研究

目的探讨五味子甲素对胶质瘤干/祖细胞耐药性的影响及作用机制。方法自人胶质瘤细胞系SHG-44中分离培养胶质瘤干/祖细胞SHG-44s,予五味子甲素0、12.50、25.00和50.00μmol/L联合长春新碱400、800和1200 nmol/L,细胞活性检测试剂盒CCK-8细胞毒性实验检测SHG-44s细胞增殖活性,罗丹明123染色检测SHG-44s细胞泵出药物能力,实时聚合酶链反应(PCR)和Western blotting法检测SHG-44s细胞ATP结合盒转运子B1(ABCB1)基因转录和翻译能力。结果五味子甲素50μmol/L即可抑制SHG-44s细胞增殖活性(P=0.001,0.001,0.039),剔除这一浓度后无论长春新碱浓度为400、800或1200 nmol/L,联合应用五味子甲素均可抑制SHG-44s细胞增殖活性(长春新碱400 nmol/L组:P=0.007,0.001;长春新碱800 nmol/L组:P=0.001,0.000;长春新碱1200 nmol/L组:P=0.000,0.000)。倒置荧光显微镜观察,五味子甲素12.50μmol/L组和25.00μmol/L组SHG-44s细胞可见明显绿色荧光。流式细胞术显示,随着五味子甲素浓度的增加,SHG-44s细胞罗丹明123染色阳性细胞比例分别为10.40%、39.20%和45.20%。实时PCR法显示,五味子甲素12.50μmol/L组和25.00μmol/L组SHG-44s细胞ABCB1基因表达水平较0μmol/L组降低(P=0.027,0.006),尤以25.00μmol/L组显著(P=0.034)。Western blotting法显示,随着五味子甲素浓度的增加,SHG-44s细胞P-糖蛋白表达水平下降。结论五味子甲素通过抑制胶质瘤干/祖细胞表面已存在的ABCB1基因编码的P-糖蛋白泵出药物能力并降低ABCB1基因转录和翻译能力,逆转胶质瘤干/祖细胞耐药性。     

龈沟液中基质金属蛋白酶-9及基质金属蛋白酶组织抑制剂-1与牙周炎的关系

目的观察慢性牙周炎患者在牙周基础治疗前后龈沟液中基质金属蛋白酶-9（MMP-9）、基质金属蛋白酶组织抑制剂-1（TIMP-1）水平的变化。方法采用滤纸条牙周袋内取样法提取15例慢性牙周炎患者在牙周基础治疗前以及基础治疗结束后1个月的龈沟液样本，同时提取15例牙周健康者的龈沟液样本，采用免疫印迹法检测MMP-9与TIMP-1水平。结果慢性牙周炎患者治疗后龈沟液中MMP-9水平[（1．2±0．9）AU]较治疗前[（2．2±1．4）AU]显著下降，TIMP-1水平[（3．7±2．2）AU]较治疗前[（1.3±1．2）AU]显著升高，P〈0．05；MMP-9、TIMP-1水平分别与牙周病临床参数呈显著正相关、显著负相关。结论MMP-9、TIMP-1水平反映了牙周组织的破坏程度，可作为评价疗效的客观指标。     

Region-specific effects of acute and repeated restraint stress on the phosphorylation of mitogen-activated protein kinases

The mitogen-activated protein kinases (MAPKs) are a family of signal transduction mediators that regulate a host of cellular activities, including cell growth and proliferation, and differentiation and survival, via sequential phosphorylation and activation of a cassette of three protein kinases. MAPKs are also recruited when the brain undergoes synaptic plasticity and remodeling (e.g., during induction of long-term potentiation, learning and memory consolidation). The activities of some of these kinases are altered in response to various acute stimuli such as ischemic insult, visceral pain and electroconvulsive shock. In the present study we used immunoblotting techniques to examine the effects of acute and repeated restraint stress on the phosphorylation state of three MAPKs, the extracellular signal-regulated kinase Erk1/2, c-Jun-N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 MAPK, in different brain regions. A single exposure to 30 min of restraint stress-elevated phospho-Erk1/2 (P-Erk1/2) levels in all three brain regions examined (hippocampus, medial prefrontal cortex and cingulate cortex), but did not alter the phosphorylation pattern of the other two MAPKs in any region. In marked contrast, exposure to restraint for 11 days (30 min/day) reduced the levels of all three MAPKs, but only in the prefrontal cortex. The results are compared to the reported effects of acute and chronic stress on other biochemical and functional measures.     

银强化滴金免疫法检测丙肝病毒IgM的研究

目的建立一种检测血清丙肝病毒抗体IgM的方法。方法将丙肝病毒抗原点于硝基纤维膜上，应用Ф15nm的羊抗人IgM金标抗体与血清标本中的丙肝病毒IgM结合，再加银加强试剂，阳性者为棕黑色。结果整个试验过程需要10min，检测HCVAb／HCVRNA阳性临床标本，结果有良好的一致性，总符合率为96％。结论银强化滴金免疫法检测血清丙肝病毒抗体IgM，操作简单，试剂稳定、敏感、快速，适于基层。     

国产抗HCV酶联免疫诊断试剂质量分析

目的 了解国产抗HCV EIA试剂的质量情况。方法 用16例国产抗HCV EIA试剂盒对2种进口试剂对1426份来源于血站及医院的血清进行了检测,并对疑难样品用RIBA及PCR试剂进行了确证。对各种试剂的灵敏度,特异度及检测的抗体谱进行了分析。结果 目前国产试剂灵敏度不低于国外试剂,特异度尚有待于改进。国产试剂漏检及假阳性的主要原因为HCV NS3区抗原相对较弱,抗原纯度较低,用量不足,有待于改进。结论 国产抗HCV试剂灵敏度不低于国外试剂,特异度有待提高。     

ADAMTS13 phenotype in plasma from normal individuals and patients with thrombotic thrombocytopenic purpura

The activity of ADAMTS13, the von Willebrand factor cleaving protease, is deficient in patients with thrombotic thrombocytopenic purpura (TTP). In the present study, the phenotype of ADAMTS13 in TTP and in normal plasma was demonstrated by immunoblotting. Normal plasma (n = 20) revealed a single band at 190 kD under reducing conditions using a polyclonal antibody, and a single band at 150 kD under non-reducing conditions using a monoclonal antibody. ADAMTS13 was not detected in the plasma from patients with congenital TTP (n = 5) by either antibody, whereas patients with acquired TTP (n = 2) presented the normal phenotype. Following immunoadsorption of immunoglobulins, the ADAMTS13 band was removed from the plasma of the patients with acquired TTP, but not from that of normal individuals. This indicates that ADAMTS13 is complexed with immunoglobulin in these patients. The lack of ADAMTS13 expression in the plasma from patients with hereditary TTP may indicate defective synthesis, impaired cellular secretion, or enhanced degradation in the circulation. This study differentiated between normal and TTP plasma, as well as between congenital and acquired TTP. This method may, therefore, be used as a complement in the diagnosis of TTP.     

桥粒蛋白生化免疫特性研究

桥粒在含高浓度脲(0.5mol/L)的变性剂中(100℃)作用30分钟即能完全溶解.经SDS-PAGE分离获得7条高分子量(>67kd)蛋白带和一些小分子量角蛋白。这7条高分子量蛋白带是桥粒的特异组成蛋白,即桥粒蛋白Ⅰ和Ⅱ(Desmoplakin Ⅰ和Ⅱ),多肽3、4a、4b、5、6,它们的分子量分别为250kd和215kd、165kd、130kd、115kd、83kd及75kd.用制备电泳法1次可提取Desmoplakin Ⅰ 2～2.5mg,纯度达93.1％.等电聚焦电泳测定其等电点为6.8～7.2。氨基酸组成分析甘氨酸含量很高,免疫印迹表明能被Desmoplakin Ⅰ McAb识别.