大鼠下丘脑室旁核小胶质细胞对次声的反应

目的探讨次声作用后大鼠下丘脑室旁核小胶质细胞与星形胶质细胞的变化及其相互关系。方法将SD大鼠反复暴露于声压级16Hz130dB的次声环境中。用抗大鼠Ⅲ型补体受体标志物（0X42）和抗胶质细胞原纤维酸性蛋白（GFAP）的免疫组化方法，观察次声作用后即刻，7d，14d大鼠下丘脑室旁核小胶质细胞与星形胶质细胞的变化及其相互关系。结果正常大鼠下丘脑室旁核的小胶质细胞和星形胶质细胞的数量较少，一般为静息性形态，胞体小，突起细长，染色浅淡。次声作用后大鼠下丘脑小胶质细胞被激活，胞体变大，突起短粗，染色深，7d以后逐渐减弱；次声作用后第7d起星形胶质细胞变多，胞体变大，突起变粗，染色深，第14d达到高潮；小胶质细胞和星形胶质细胞之间的关系密切。结论次声作用后小胶质细胞比星形胶质细胞早被激活；两者的关系密切。     

8Hz次声对大鼠海马和颞叶皮层RyR的表达影响

目的研究8 Hz,90 dB、100 dB、130 dB的次声对SD大鼠海马及颞叶皮层兰尼定受体(RyR)表达的影响. 方法雄性SD大鼠随机分为正常对照组、8 Hz,90 dB、100 dB、130 dB的1、7、14、21、28、35、42 d组共28组.最后一次作用结束后立即取脑组织并进行RyR免疫组织化学染色,光学显微镜下观察海马及颞叶皮层RyR表达的变化. 结果次声作用组大鼠脑组织海马及颞叶皮层RyR表达均较对照组减少, 8 Hz, 90 dB组、100 dB组以21 d减少最为明显,而130 dB组变化较为复杂,有2个峰值,14 d和42 d,其中42 d阳性表达最少(均 P <0.01),且21 d时,100 dB组的表达减少较90 dB组为少,90 dB组和100 dB组阳性表达在21 d后均有所回升. 结论 8 Hz,90 dB、100 dB和130 dB次声可引起大鼠海马及颞叶皮层RyR表达减少,其变化规律与次声作用参数有关.     

GFAP和Fos蛋白在戊四氮致痫大鼠前脑中的表达变化

目的 研究大鼠在戊四氮导致癫痫发作时前脑内星形胶质细胞和神经元的形态学反应及其相互关系。方法 应用免疫组织化学单标记法分别显示前脑内GFAP和Fos蛋白表达的时间规律，并用免疫组织化学双重标记显示GFAP和Fos蛋白表达的相互关系。结果 在戊四氮导致大鼠癫痫发作早期，前脑的星形胶质细胞被激活，细胞体积增大，突起粗大，GFAP表达阳性，随着存活时间的变化，星形胶质细胞的反应经历先逐渐升高后降低的过程。被激活的星形胶质细胞和神经元表达Fos蛋白阳性，也呈现逐渐升高又降低的变化；另外，GFAP阳性星形胶质细胞和Fos阳性神经元在前脑主要分布在大脑皮层、海马、杏仁核等部位，二者的分布特征基本一致。结论 星形胶质细胞可能和神经元一起参与了戊四氮所致癫痫发作的变化。     

红藻氨酸致痫大鼠海马Fos和GFAP的共同表达

目的 研究红藻氨酸（kainic acid,KA)诱导大鼠癫痫发作后海马（hippocampus,HI)内神经元和星形胶质细胞的时空效应性反应变化。方法 大鼠侧脑室内注射KA，用抗即刻早期基因Fos蛋白和抗胶质原纤维酸性蛋白（GFAP）的双重免疫荧光组织化学方法结合激光共聚焦显微镜技术，显示痫性发作后HI同一部位内反应性神经元与星形胶质细胞的分布。结果 KA诱导大鼠癫痫发作，HI内的Fos阳性神经元和GFAP阳性星形胶质细胞明显增多。两分布范围基本一致，且癫痫诱发30min后GFAP开始增多，1h达高峰；1h后Fos阳性产物开始增多；2h达高峰；部分Fos阳性神经元周围有GFAP免疫反应产物包绕，显示反应性神经元（Fos阳性）与反应性星形胶质细胞（GFAP阳性）之间关系密切。结论 HI内的神经元和星形胶质细胞与癫痫发作直接相关且存在相互关系。可能共同参与癫痫的发生及其调节。     

大鼠前脑缺血再灌注后GFAP、S-100表达的变化

目的 探讨胶质纤维酸性蛋白（GFAP）和S-100蛋白在大鼠前脑缺血再灌注后反应性星形胶质细胞的活化情况.方法 利用免疫组织化学方法检测前脑缺血再灌注模型的细胞活化情况.结果 脑缺血再灌注后第1d,顶叶皮层和海马可见少量GFAP阳性细胞表达 脑缺血再灌注第3d及第5d后GFAP阳性表达明显增加,并与对照组比较有统计学意义（P＜0.01）.S-100蛋白在脑缺血再灌注后第1d即有增加,并随着时间延长表达明显增强,各时间点与对照组有显著性差异（P＜0.01）.结论 脑缺血再灌注后GFAP、S-100蛋白表达增加,说明反应性星形胶质细胞的活化参与了脑缺血损伤后神经元的修复过程.     

红藻氨酸诱发大鼠复杂部分性癫痫发作的海马神经元和星形胶质细胞反应及相互关系

目的：观察大鼠癫痫发作后海鸟内神经元与星形胶质细胞反应变化的时空效应及相互关系。方法：以红藻氨酸诱发的大鼠复杂部分性癫痂发作为模型，利用免疫组织化学法，在原位显示癫痫发作后15、30、60、90、120、180min6个时间点海马神经元Fos蛋白及星形胶质细胞内胶质原纤维酸性蛋白（GFAP）的表达变化、相互关系及分布规律。结果：致痫后15min海马内GFAP表达开始增多，60min达高峰。Fos阳性神经元在癞痴诱发后30min开始出现，120min达高峰。海马内GFAP阳性细胞与Fos阳性神经元分布规律基本一致。结论：在癫痫病理状态下，海马内星形胶质细胞的反应略早于神经元，两者之间分布呈平行关系，它们之间可能存在着复杂的信息通讯，以复合体的形式其同对各种病理生理刺激作出反应。     

小鼠次声刺激后海马内bcl-2mRNA表达的变化

目的研究小鼠接受不同声压级次声刺激后海马内bcl-2mRNA表达的改变。方法BALB／C小鼠暴露于16Hz，声压为90dB和130dB次声。每天作用2h，分别作用1、7、14、21和28d后，采用原位杂交方法观察小鼠海马内bcl-2mRNA表达的改变。结果90dB和130dB次声作用后，海马区域内bcl-2mRNA的表达明显增多；130dB次声作用组反应比90dB次声作用组强；在相同声压级强度的次声作用下，随着作用次数的增加，海马内bcl-2mRNA的杂交阳性反应产物增多。结论海马对次声作用敏感，次声可以通过改变海马内bcl-2mRNA的含量造成脑损伤，这是次声导致脑损害的重要因素之一。次声的这一作用效应与声压级和作用时间有关。     

次声对成年大鼠齿状回神经前体细胞增殖的影响

目的研究次声对成年大鼠海马齿状回神经前体细胞增殖的影响。方法大鼠随机等分为正常对照组、假次声组和次声组（每组16只）。次声组暴露于8Hz、130dB次声环境7d（2h／d），暴露结束后第1、3、7、14d处死，采用抗5-溴脱氧尿嘧啶尿苷（BrdU）免疫组化方法，观察齿状回BrdU阳性细胞数的变化。结果次声作用结束后第1d，齿状回BrdU阳性细胞数与假次声组和正常对照组相比均无统计学差异；第3d及第7d，BrdU阳性细胞数减少（P〈0．05），第14d恢复正常水平。结论8Hz、130dB次声可抑制正常成年大鼠海马神经前体细胞增殖，可能与次声引起大鼠脑内微环境改变有关。     

不同浓度人参皂甙Rd对次声性脑损害的保护

目的观察次声对大鼠记忆功能的影响及不同剂量人参皂甙Rd对其脑损害的治疗作用。方法通过Y型电迷宫训练将成绩相近的SD大鼠随机分为5个组，除正常对照组以外均接受16Hz 130dB的次声作用gh／d。3个药物组在次声作用前3d开始分别给予不同剂量（30mg／kg、10mg／kg、2mg／kg）的人参皂甙Rd。次声作用7d后再次评定每组大鼠的迷宫成绩，并用单链DNA（Single-stranded DNA，ssDNA）免疫标记法检测海马内ssDNA（凋亡细胞）数。结果与正常对照组相比单纯次声组大鼠学习记忆功能下降、标记的ssDNA阳细胞数明显增多（P〈0．05）；与单纯次声组相比人参皂甙（30mg/kg、10mg／kg组）有明显的保护作用，减轻了学习记忆功能下降，ssDNA阳性细胞数明显减少（P〈0．05）。结论16Hz 130dB次声可引发大鼠海马损伤、细胞凋亡、记忆功能减退，人参皂甙可明显减轻这些损害。     

次声对大鼠海马5-HT、5-HTR、RyR表达及恢复的研究

目的研究大鼠海马5-羟色胺(5-HT)、5-羟色胺受体(5-HTR)、兰尼定受体(RyR)表达在8 Hz、130 dB的次声作用后1周和2周时的恢复情况. 方法大鼠暴露于8 Hz、130 dB次声1、7、14、21、28、35、42 d后置安静环境观察1周或2周,取脑组织并进行免疫组织化学染色,光学显微镜下观察海马5-HT、5-HTR、RyR表达. 结果次声作用组大鼠脑组织海马5-HT、5-HTR、RyR表达均较对照组减少,最低值分别出现于21 d、28 d和42 d(P<0.01).次声停止作用后,5-HT、5-HTR、RyR表达均逐渐回升,且随时间延长大部分恢复到正常对照水平.结论 8 Hz、 130 dB次声可引起大鼠海马5-HT、5-HTR、RyR表达减少,其变化规律与观察指标有关;次声引起的5-HT、5-HTR、RyR表达减少在停止次声作用后可逐渐恢复正常.     

Autonomous activity of CaMKII is only transiently increased following the induction of long-term potentiation in the rat hippocampus

A major role has been postulated for a maintained increase in the autonomous activity of CaMKII in the expression of long-term potentiation (LTP). However, attempts to inhibit the expression of LTP with CaMKII inhibitors have yielded inconsistent results. Here we compare the changes in CaMKII autonomous activity and phosphorylation at Thr286 of alphaCaMKII in rat hippocampal slices using chemical or tetanic stimulation to produce either LTP or short-term potentiation (STP). Tetanus-induced LTP in area CA1 requires CaMKII activation and Thr286 phosphorylation of alphaCaMKII, but we did not observe an increase in autonomous activity. Next we induced LTP by 10 min exposure to 25 mM tetraethyl-ammonium (TEA) or 5 min exposure to 41 mM potassium (K) after pretreatment with calyculin A. Exposure to K alone produced STP. These protocols allowed us to monitor temporal changes in autonomous activity during and after exposure to the potentiating chemical stimulus. In chemically induced LTP, autonomous activity was maximally increased within 30 s whereas this increase was significantly delayed in STP. However, in both LTP and STP the two-fold increase in autonomous activity measured immediately after stimulation was short-lived, returning to baseline within 2-5 min after re-exposure to normal ACSF. In LTP, but not in STP, the phosphorylation of alphaCaMKII at Thr286 persisted for at least 60 min after stimulation. These results confirm that LTP is associated with a maintained increase in autophosphorylation at Thr286 but indicate that a persistent increase in the autonomous activity of CaMKII is not required for the expression of LTP.     

In Vitro Phosphorylation in Isolated Horizontal Cells of the Fish Retina: Effects of the State of Light Adaptation

Horizontal cells, which are second-order neurons of the vertebrate retina, exhibit synaptic plasticity governed by light and dark adaptation. We have investigated the alterations in the protein phosphorylation patterns of isolated carp ( Cyprinus carpio ) horizontal cells in relation to their state of light adaptation by using an in vitro phosphorylation assay and compared the resulting data with protein synthesis patterns of the whole retina. Phosphoproteins and [³⁵S]methionine-labelled proteins were analysed by one- and two-dimensional gel electrophoresis followed by autoradiography. The state of light adaptation significantly affected the in vitro phosphorylation of horizontal cell proteins with molecular weights of 68, 56/58, 47, 28 and 15 kDa, but had no effect on the protein synthesis of retinal proteins. In the light the most prominent increase of ³²P incorporation was observed in the 47 kDa protein. The biochemical properties of this protein closely resembled those of the growth-associated GAP-48, found in the fish retina. In addition, the phosphorylation of horizontal cell homogenates in the presence of protein kinase activators such as cyclic AMP, calcium, calmodulin and phospholipids revealed that horizontal cells of the fish retina contain cyclic AMP-, calcium/calmodulin- and calcium/phospholipid-dependent protein kinase activity resulting in the phosphorylation of several horizontal cell proteins, including the phosphoproteins which were affected by the state of light adaptation.     

Bcl-2和Bax蛋白在人脑星形细胞瘤中的表达及意义

目的　研究 Ｂｃｌ２ 和 Ｂａｘ 蛋白与人脑星形细胞瘤发生的关系。方法　收集人脑星形细胞瘤标本５８ 例, 行 Ｓ Ｐ 染色。结果　 Ｂｃｌ２ 在星形细胞瘤中阳性表达率为５６９ ％ , 且表达率随肿瘤恶性程度的增加而增高。 Ｂａｘ 的表达率为５５２ ％ , 显著高于对照组。 Ｂｃｌ２ 表达阳性的肿瘤中 Ｂａｘ 蛋白表达率（６６７ ％ ） 显著高于 Ｂｃｌ２ 表达阴性的肿瘤（４００ ％ ） 。结论　 Ｂｃｌ２ 和 Ｂａｘ 蛋白的表达与星形细胞瘤的发生和发展有较密切的关系, 且它们在星形细胞瘤的发生中可能存在相互作用的关系。     

Proteins of peripheral myelin are associated with glycosphingolipid/cholesterol-enriched membranes

A characteristic feature of the vertebrate nervous system is the ensheathment of axons by myelin, a multilamellar membrane specialization produced by polarized glial cells. Although the main protein and lipid components of the myelin sheath are well characterized, relatively little is known about the mechanisms of their intracellular distribution to the respective sites of assembly within the myelin sheath. To analyze whether peripheral myelin protein trafficking is mediated by glycosphingolipid/cholesterol-enriched membranes (GEMs), we studied the association of established myelin proteins, peripheral myelin protein 22 (PMP22), protein zero (P0), plasmolipin, and myelin basic protein (MBP), with these membrane microdomains. To examine the association of the selected peripheral myelin proteins with detergent-insoluble GEMs, purified myelin from sciatic nerve of adult rat was extracted with Triton X-100 at 4 degrees C and 37 degrees C and, in additional experiments, was pretreated with the cholesterol chelator methyl-beta-cyclodextrin. The material was then centrifuged to equilibrium in sucrose gradients, and fractions were analyzed by Western blotting. Here we demonstrate for the first time that PMP22, P0, and plasmolipin prepared from purified peripheral myelin are associated with GEMs. To characterize whether the association of these proteins is a specialized feature of myelinating Schwann cells, we studied the distribution of PMP22, P0, and plasmolipin in transiently transfected HeLa cells. These experiments confirm the specific association of these proteins with GEMs in both neural and nonneural cell types.     

人脑星形细胞肿瘤中FHIT、PCNA蛋白表达关系的研究

目的研究人脑星形细胞肿瘤中FHIT、PCNA蛋白的差异表达，探讨它们与病理分级之间的关系。方法应用免疫组化SP法检测了50例星形细胞肿瘤中不同级别的FHIT、PCNA蛋白的表达水平，以10例非肿瘤脑组织作对照。结果非肿瘤脑组织FHIT、PCNA蛋白阳性表达率分别为100％，0％，星形细胞肿瘤中FHIT、PCNA蛋白阳性表达率分别为40％，86％；尽管在星形细胞肿瘤中FHIT、PCNA蛋白表达总体差异有统计学意义（P〈0．05），但Ⅲ级与Ⅳ级比较差异无统计学意义（P〉0．05）；经统计学分析FHIT、PCNA蛋白呈显著负相关（P〈0．01）。结论FHIT、PCNA蛋白的表达可能与星形细胞肿瘤的恶性程度有关，星形细胞肿瘤中FHIT、PCNA蛋白表达之间的显著负相关，提示FHIT蛋白可能对细胞增殖具有负性调控作用。     

Testis–brain RNA-binding protein (Translin) is primarily expressed in neurons of the mouse brain

The subcellular location(s) of the DNA- and RNA-binding protein, Testis–Brain RNA-Binding Protein (TB-RBP)/Translin in mouse brain has been determined in paraffin sections by immunocytochemistry with an affinity purified antibody to mouse recombinant TB-RBP. Nuclear staining was frequently seen in neurons throughout the brain, but no TB-RBP/Translin was detected in many of the neurons in superficial layers of the cerebral cortex and in some cells of the cerebellum. Cytoplasmic staining extending into the dendrites was seen in large neurons such as pyramidal neurons in Layer 5 of the cortex and magnocellular neurons of the hypothalamus or the brainstem raphe.     

Membrane binding and anticoagulant properties of protein S natural variants

Introduction

Protein S (PS) is a vitamin K-dependent plasma glycoprotein with a key role in the control of coagulation pathway on phospholipid membranes. We compared anticoagulant and membrane binding properties of PS altered by natural mutations (N217S, DelI203D204) affecting the epidermal growth factor like-domain 4 (EGF4) and causing PS deficiency.

Materials and methods

Binding of recombinant, immunopurified PS (rPS) to several conformation-specific antibodies, to C4BP and to phospholipid liposomes was investigated by ELISA. PS binding to cells was analysed by flow cytometry. PS inhibitory activities were studied in plasma and purified systems.

Results and conclusions

Conformational changes produced by mutations were revealed by mapping with calcium-dependent antibodies. The immunopurified recombinant mutants (rPS) showed at 200-800nM concentration reduced inhibition of coagulation (rPS217S, 10.2-17.3%; rPSDelI203D204, 5.8-8.9% of rPSwt) in FXa 1-stage clotting assay with APC. In thrombin generation assays the inhibition of ETP was reduced to 51.6% (rPS217S) and 24.1% (rPSDelI203D204) of rPSwt. A slightly shortened lag time (minutes) was also observed (rPS217S, 2.58; rPSDelI203D204, 2.33; rPSwt, 3.17; PS deficient plasma, 2.17).In flow cytometry analysis both mutants efficiently bound apoptotic cells in adhesion or in suspension. The affinity for phosphatidylserine-rich vesicles (apparent Kd: rPSwt 27.7 ± 1.6 nM, rPS217S 146.0 ± 16.1 nM and rPSDelI203D204 234.1 ± 28.1 nM) was substantially increased by membrane oxidation (10.9 ± 0.6, 38.2 ± 3.5 and 81.4 ± 6.0 nM), which resulted in a virtually normal binding capacity of mutants at physiological PS concentration.These properties help to define the molecular bases of PS deficiency, and provide further elements for PS-mediated bridging of coagulation and inflammation.     

Selective extraction, solubilization, and reversed-phase high-performance liquid chromatography separation of the main proteins from myelin using tetrahydrofuran/water mixtures

The number of solvents capable of dissolving myelin and proteolipid protein (PLP) and of being used as a mobile phase for the separation of myelin proteins by reversed-phase high-performance liquid chromatography (RP-HPLC) is very limited. In a thorough study, we found that aqueous tetrahydrofuran (THF) fulfilled such a requirement. The maximal amount of protein extracted corresponded to a THF/water ratio of 4:1 v/v and a polarity index of 5.16. This mixture dissolved a purified PLP preparation completely, 60% of proteins from fresh myelin, and 20% of white matter total proteins. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of those extracts, followed by densitometric analysis, showed that the amount and type of proteins dissolved depended on the polarity (i.e., the content of water) of the solvent mixture used. This selective effect was greater for basic protein in myelin preparations. Crude extracts highly enriched in basic protein can be prepared. In addition, the solvent system THF/water proved to be very useful as a mobile phase in RP-HPLC for separating myelin proteins. Using a C3 column and a linear gradient from 30% to 100% THF in water, both containing 0.1% trifluoroacetic acid (TFA), we separated completely the three main protein fractions of central nervous system (CNS) myelin in a short period of time. The high solubility power of THF/water mixtures prolonged greatly the life of the column.     

朊蛋白在神经系统作用机制研究进展

细胞型朊蛋白(PrP~C)作为一种跨膜糖蛋白在哺乳动物中广泛存在。基因敲除的研究显示PrP~C在神经系统的活动中的关键作用包括周围神经髓鞘的形成以及对神经毒素刺激的保护。PrP~C在不同的细胞类型中也有不同的生物学作用。如PrP~C模块化结构、多种结合伴侣以及与脂质筏的密切关联的特性,使其具有组装多组分复合物的能力,从而触发不同的信号通路,调节细胞分化。PrP~C在大脑中参与的病理性作用仍然没有一致的定论,其错误折叠产生的异构体PrP~(SC)是朊病毒疾病的主要致病因素。但有证据指出PrP~C在朊病毒疾病中发挥的致病作用独立于羊瘙痒病朊蛋白亚型(PrP~(SC)),在朊病毒感染过程中,朊病毒疾病的临床和神经病理症状与大脑中PrP~C而不是PrP~(SC)的表达水平成正比。另外,PrP~C可能还是一种与神经退行性病变相关的蛋白,参与β淀粉样蛋白(Aβ)等聚集性蛋白的神经毒素信号转导,还充当α-突触核蛋白的细胞受体,促进其在细胞吸收以及大脑中传播。虽然朊病毒的研究已经取得很大的进展,但PrP~C在大脑中的作用仍然没有明确,因此探索PrP~C在细胞中作用具有十分重要的意义。