红景天苷类似物MADP对谷氨酸诱导的大鼠海马神经元凋亡的保护作用

目的:观察新型红景天苷(salidroside,Sal)类似物4-甲氧基苯甲基-2-乙酰氨基2-脱氧-β-D-吡喃糖苷(4-methoxy-2-acetamido-2-deoxy-β-D-pyranoside,MADP)对谷氨酸(glutamate)损伤海马神经元的保护作用。方法:原代培养大鼠胚胎海马神经元,与浓度分别为60、120、240μmol/L的MADP或240μmol/L的Sal共同孵育24 h,加入125μmol/L谷氨酸损伤海马神经元15 min。使用相差显微镜观察海马神经元的形态变化;四甲基偶氮唑盐(methyl thiazolyl tetrazolium salt colorimetry,MTT)比色法测定细胞活力;甲基百里香酚蓝(methyl thymol blue,MTB)法测定细胞内游离的钙离子浓度;采用实时荧光定量PCR技术检测细胞凋亡相关基因Bcl-2,Bax,Caspase-3的表达水平。结果:MADP或Sal预处理24 h可改善谷氨酸损伤后神经元胞体的肿胀和突起的淡化,抑制胞内游离钙离子浓度的上升,提升Bcl-2/Bax mRNA的表达水平,降低Caspase-3 mRNA的表达水平。MADP对大鼠海马神经元的保护作用优于Sal。结论:与Sal相比较,MADP更好的发挥了拮抗谷氨酸对海马神经元损伤的作用,其机制可能与抑制钙离子内流,上调Bcl-2/Bax mRNA及下调Caspase-3 mRNA的表达水平相关。     

谷氨酸对大鼠海马兴奋毒性作用的形态学研究

目的：为明确谷氨酸对神经细胞有兴奋性和毒性作用。方法：选用具有较多N－甲基D－天门冬氨酸（NMDA）受体的海马作为研究对象,选用SD大鼠,给予L－谷氨酸钠盐（MSG）皮下注射,在不同的时间内观察大鼠的表现和其海马神经细胞受损的形态学的改变。结果：发现大鼠的早期行为表现较为兴奋,而后随时间的延长表现为迟钝。在光镜和电镜下,神经细胞早期为水肿,后期为水肿明显和细胞器的破坏直至细胞的因缩,损伤主要表现在CA1区。结论：提示边缘系统的海马结构易受谷氨酸的影响,同时也表明了谷氨酸对神经细胞的兴奋毒性作用,可为临床药物和食物添加剂的运用提供一定的依据。     

梓醇对谷氨酸诱导SD大鼠海马神经元损伤的保护作用

目的梓醇对谷氨酸诱导SD大鼠海马神经元损伤的影响。方法将培养7～9d的SD大鼠海马细胞进行NSE鉴定,然后随机分为:正常对照组;谷氨酸损伤组(Glu);谷氨酸损伤前予梓醇组(CAT+Glu)。采用倒置显微镜观察细胞形态学变化,噻唑蓝(MTT)法测定细胞存活率,比色法测定培养液中乳酸脱氢酶(LDH)含量,流式细胞术测定细胞凋亡率。结果与正常对照组比较,0.1mmol·L-1谷氨酸作用于海马神经元24h,出现明显细胞损伤,细胞存活率下降,培养液中LDH含量明显增加,细胞凋亡率增高(<0.01);0.2、1.0、5.0mg·L-1的梓醇可不同程度的改善谷氨酸损伤引起的神经细胞形态的改变,提高细胞存活力,减少LDH的漏出,降低细胞凋亡率,并呈一定的剂量依赖性。结论梓醇对谷氨酸诱导的海马神经细胞损伤有保护作用。     

急性脑缺血损伤大鼠海马神经元谷氨酸转运体的表达

目的：研究急性脑缺血损伤大鼠海马神经元谷氨酸转运体（EAAC1）的表达变化。 方法： 采用EAAC1反义寡核苷酸脑内注射，用插线法建立大鼠局灶性脑缺血模型(MCAO)。运用Western blot法和TTC染色观察缺血区EAAC1表达和梗塞体积；采用RT-PCR 和Western blot法，测定海马EAAC1 mRNA和蛋白在缺血1 h、6 h、24 h的变化。结果： 注射EAAC1反义寡核苷酸组大鼠梗塞体积［(105.67±8.70) mm3］显著小于正义组。缺血1 h大鼠海马EAAC1 mRNA表达（0.963±0.117）与假手术组（0.907±0.113）无明显差异，缺血6h、24h持续高于缺血1 h（分别为1.116±0.104和1.428±0.078）。而海马EAAC1蛋白表达（0.640±0.027）在缺血24 h高于假手术组，缺血1 h和6h EAAC1表达与假手术组比较无显著差异（分别为0.330±0.018、0.330±0.015）。结论： EAAC1可促进缺血脑损伤,在急性脑缺血病理过程中表达增加。     

Caspase-9和caspase-3在创伤后应激障碍大鼠海马神经元的表达及意义

目的:探讨caspase-9和caspase-3在创伤后应激障碍(PTSD)大鼠海马神经元中的表达及意义.方法:采用国际认定的无连续单一应激(SPS)方法刺激大鼠建立PTSD大鼠模型,取SPS刺激后1、4、7、14、28 d组和正常对照组.应用免疫组织化学、免疫荧光双标、激光共聚焦显微镜技术和免疫印迹法检测caspase-9和caspase-3蛋白的表达.结果:与正常对照组相比,caspase-9活性于SPS刺激后1 d升高,SPS刺激后7 d再次上调并达到高峰,之后逐渐下降.Caspase-3活性于SPS刺激后7 d达到高峰,之后逐渐下降.结论:caspase-9和caspase-3共同参与了PTSD大鼠海马神经元凋亡的调控.     

谷氨酸诱导的大鼠海马神经元兴奋毒性损伤中SNK/SPAR表达水平的变化

目的观察谷氨酸诱导的兴奋毒性损伤中血清诱导激酶(SNK)/树突棘相关的Rap特异性GTPase活化蛋白(SPAR)分子表达水平的变化。方法建立谷氨酸介导的大鼠海马神经元兴奋毒性损伤模型,RT-PCR方法检测SNK/SPARmRNA的表达;Western blot方法观察SNK/SPAR在蛋白质水平的表达;双重免疫荧光标记方法观察SNK/SPAR在神经元上的分布。结果100μmol/L谷氨酸刺激神经元10min后,1h内即可出现SNKmRNA表达水平升高,6h达高峰,随后逐渐下降,12h左右回到基线水平;3h可检测到SNK蛋白表达水平升高,36h达高峰,随后回降。SPAR表达水平的变化趋势与SNK相反。SNK与SPAR分布的变化主要体现在神经元的胞质和突起。结论SNK-SPAR途径可能参与了谷氨酸诱导的神经元兴奋毒性损伤过程。     

大鼠海马神经元PKCε特异性shRNA对神经元突起生长的影响

目的 构建shRNA特异性干扰海马神经元内PKCε,研究PKCε干扰后海马神经元突起长度的变化.方法 设计并构建PKCε的小分子干扰shRNA载体.分别在293T细胞中共转染PKCε和各干扰片段,Western blot验证干扰效果.将有干扰作用的载体转染海马神经元,用细胞免疫荧光法验证干扰内源PKCε的效果.激光共聚...     

ATP诱导大鼠原代培养海马神经元神经毒性及对GAP-43表达的影响

目的:观察不同浓度ATP对大鼠原代培养海马神经元的损伤作用及GAP-43表达的影响,并探讨其可能机制。方法:原代培养新生SD大鼠海马神经元至第8 d,MTT法检测不同浓度ATP作用下海马神经元的存活率,倒置相差显微镜观察海马神经元的形态学改变,免疫荧光细胞化学法观察损伤后海马神经元GAP-43的表达变化,钙离子荧光染料Flou-4/AM检测细胞内钙离子的浓度变化。结果:低浓度ATP对海马神经元无明显影响,高浓度ATP对海马神经元有毒性损伤作用,且呈剂量依赖性。P2X受体拮抗剂可阻断ATP介导的细胞毒性作用,但P2X7受体拮抗剂的阻断作用不明显。高浓度ATP作用海马神经元4 h后,GAP-43的表达水平明显上调,且表达部位发生改变,主要表达于胞体,突起表达减少甚至消失。高浓度ATP组荧光衰减速率明显减慢,说明高浓度ATP参与了海马神经元细胞内钙调节。结论:高浓度ATP对海马神经元有毒性损伤作用,损伤后GAP-43代偿性表达增多,且与细胞内钙离子浓度变化有关,P2X7受体可能不是介导ATP此作用的主要受体亚型。     

Lhx8基因沉默抑制NGF诱导的海马NSCs向胆碱能神经元分化的研究

目的:明确Lhx8在神经生长因子(nerve growth factors,NGF)促进海马神经干细胞向胆碱能神经元分化中的作用。方法:体外培养海马神经干细胞并应用NGF促进其向胆碱能神经元分化的过程中,将构建的Lhx8干扰慢病毒加入至培养液中,7 d后应用免疫荧光标记技术检测分化所得胆碱乙酰转移酶(choline acetyltransferase,Ch AT)和微管相关蛋白-2(microtubule-associated protein 2,MAP-2)双标阳性细胞。结果:在空白对照组中,仅检测到少量的Ch AT/MAP-2双标阳性细胞;在NGF组或是NGF联合阴性对照慢病毒组中则可检测到较多的Ch AT/MAP-2双标阳性细胞;在加入Lhx8干扰慢病毒的实验组中,分化所得的Ch AT/MAP-2双标阳性细胞数较NGF组或是NGF联合阴性慢病毒组明显减少。结论:Lhx8基因沉默抑制了NGF诱导的海马神经干细胞向胆碱能神经元的分化。     

白藜芦醇对氧化应激诱导的大鼠海马神经元损伤的影响

目的:研究不同剂量的白藜芦醇对大鼠海马神经元氧化损伤的影响。方法:通过体外原代培养获得大鼠海马神经元。实验分为对照(control,Con)组、三甲基氯化锡(trimethyltin chloride,TMT)组、溶剂对照(vehicle)组和白藜芦醇(resveratrol,Res)组。其中,Res设4个浓度:5μmol/L、10μmol/L、20μmol/L和40μmol/L;除对照组外,其它各组细胞均采用三甲基氯化锡(TMT)处理使细胞出现氧化应激状态。采用CCK-8法和荧光探针DCFH-DA法观察各组神经元的活力与细胞内活性氧(reactive oxygen species,ROS)含量的变化;TUNEL荧光染色法检测各组神经元的凋亡程度。结果:与TMT组相比,不同浓度的白藜芦醇均能提高神经元的活力,其中20μmol/L的作用最显著。20μmol/L白藜芦醇能抑制经TMT处理后神经元内的ROS水平以及凋亡程度的增加。结论:白藜芦醇具有抑制TMT诱导的大鼠海马神经元氧化损伤的作用。     

Raloxifene acutely reduces glutamate-induced intracellular calcium increase in cultured rat cortical neurons via inhibition of high-voltage-activated calcium current

There is increasing evidence indicating that estrogen replacement therapy produces neuroprotective actions but has undesirable side effects on the reproductive system. Raloxifene is a selective estrogen receptor modulator that exerts estrogen agonist action in the brain while acting as an estrogen antagonist in the reproductive system. In the present study, we investigated whether raloxifene affected the glutamate-induced calcium (Ca2+) overload in rat cultured cortical neurons. The bulk cytosolic intracellular Ca2+ level was measured by using confocal microscopy with fluorescent Ca2+ probe fluo3. Whole-cell recording technique was used to observe the effects of raloxifene on N-methyl-D-aspartate (NMDA)-evoked and voltage-activated Ca2+ currents in cultured cortical neurons. Pre-exposure of cortical neurons to raloxifene (0.5 microM-10 microM) for 3 min attenuated intracellular Ca2+ increase induced by application of glutamate (300 microM) for 1 min. The action of raloxifene was reversible after washout. ICI 182,780 and thapsigargin did not block the action of raloxifene. In whole-cell recording experiments, raloxifene (10 microM) significantly reduced the amplitude of the high-voltage-activated Ca2+ current but had no effect on NMDA-evoked Ca2+ current. The present study demonstrates that raloxifene acutely reduces glutamate-induced intracellular Ca2+ increase probably via inhibition of high-voltage-activated calcium channels.     

超氧阴离子自由基对大鼠肝卵圆细胞胞内自由钙浓度的影响

采用激光共聚焦扫描显微镜,观察黄嘌呤黄嘌呤氧化酶反应系统（Ｘ／ＸＯ）生成不同浓度的超氧阴离子自由基（Ｏ２）致培养的大鼠肝卵圆细胞（ＷＢ细胞）胞质内钙浓度的变化,结果发现：仅小剂量的Ｏ２引起胞质内钙浓度升高,约３０Ｓ后达到峰值,６０ｓ后恢复正常。部分细胞观察到数次钙浓度间断升高、幅度逐渐下降的现象。超氧化物歧化酶（ＳＯＤ）可抑制此变化,过氧化氢酶（ＣＡＴ）无效果,细胞在无钙液中观察仍出现钙峰,但受     

Contacts between medial and lateral perforant pathway fibers and parvalbumin expressing neurons in the subiculum of the rat

The entorhinal cortex (EC) projects via the perforant pathway to all subfields in the hippocampal formation. One can distinguish medial and lateral components in the pathway, originating in corresponding medial and lateral subdivisions of EC. We analyzed the innervation by medial and lateral perforant pathway fibers of parvalbumin-expressing neurons in the subiculum. A neuroanatomical tracer (biotinylated dextran amine, BDA) was stereotaxically injected in the medial or lateral entorhinal cortex, thus selectively labeling either perforant pathway component. Transport was allowed for 1 week. Transported BDA was detected with streptavidin-Alexa Fluor 488. Parvalbumin neurons were visualized via immunofluorescence histochemistry, using the fluorochrome Alexa Fluor 594. Via a random systematic sampling scheme using a two-channel, sequential-mode confocal laser scanning procedure, we obtained image series at high magnification from the molecular layer of the subiculum. Labeled entorhinal fibers and parvalbumin-expressing structures were three dimensionally (3D) reconstructed using computer software. Further computer analysis revealed that approximately 16% of the 3D objects ('boutons') of BDA-labeled fibers was engaged in contacts with parvalbumin-immunostained dendrites in the subiculum. Both medial and lateral perforant pathway fibers and their boutons formed such appositions. Contacts are suggestive for synapses. We found no significant differences between the medial and lateral components in the relative numbers of contacts. Thus, the medial and lateral subdivisions of the entorhinal cortex similarly tune the firing of principal neurons in the subiculum by way of parvalbumin positive interneurons in their respective terminal zones.     

吡咯喹啉醌对D-半乳糖致衰老大鼠海马神经元的影响

目的观察吡咯喹啉醌(PQQ)对D-半乳糖(D-gal)致大鼠海马神经细胞衰老的影响。方法用大剂量D-gal致大鼠脑老化模型,侧脑室注射PQQ,50 d后行脑组织切片H-E染色和尼氏染色,观察海马神经元的形态学变化,流式细胞仪检测细胞的凋亡率;用TBA法和Fenton反应测海马组织自由基含量;W estern b lot观察C-FOS蛋白的表达。结果D-gal处理组鼠海马神经元胞体变小,尼氏小体的吸光度值(A)降低,细胞凋亡率和自由基含量增加,C-FOS蛋白的表达降低;而同时PQQ处理后,与对照组相比海马神经元的胞体变大,尼氏小体的A值增加,自由基含量和神经元的凋亡率无明显增加,PQQ剌激了C-FOS蛋白的表达。结论PQQ能延缓D-gal引起的大鼠海马神经细胞衰老。     

白果内酯对糖氧剥夺小鼠原代海马神经元内钙离子浓度的影响

目的:观察白果内酯对糖氧剥夺(OGD)后小鼠原代海马神经元内游离钙离子浓度的影响及机制探究。方法:分离和培养小鼠原代海马神经元,实验分为对照组(control)、糖氧剥夺组(OGD)和白果内酯组(OGD+bilobalide),对照组予以常规培养小鼠原代海马神经元,糖氧剥夺组利用氧葡萄糖剥夺/再恢复模型模拟小鼠原代海马神经元的缺血再灌注损伤,白果内酯组在氧葡萄糖剥夺后再恢复前应用20μmol/L的白果内酯。利用流式细胞仪检测各组小鼠原代海马神经元内游离的钙离子浓度,利用Western Blot技术检测各组小鼠原代海马神经元Cav1. 2蛋白的表达,最后利用膜片钳技术检测各组小鼠原代海马神经元L型电压依赖型钙离子通道的功能。结果:与糖氧剥夺组相比,白果内酯组小鼠原代海马神经元游离钙离子浓度降低(P <0. 05)、Cav1. 2蛋白表达量减少(P <0. 05)、L型电压依赖性钙离子通道功能减弱(P <0. 05),但仍未恢复到对照组水平(P <0. 05)。结论:白果内酯可能通过减少小鼠原代海马神经元Cav1. 2表达和L型电压依赖性钙离子通道的功能降低细胞内游...     

Excessive intracellular Ca2+ inhibits glutamate-induced Na(+)-K+ pump activation in rat hippocampal neurons.

1. The effects of increased intracellular Ca2+ concentration ([Ca2+]i) on Na(+)-K+ pump activity in CA1 pyramidal neurons of rat hippocampal slices were investigated. The postglutamate hyperpolarization (PGH), which follows glutamate (GLU)-induced depolarization (GD), was used as an index of Na(+)-K+ pump activity, as was a ratio of PGH area to the preceding GD area (PGH ratio). 2. Perfusion of slices with saline containing Ca2+ ionophore (A23187, 10 microM) inhibited the PGH without producing apparent signs of cell deterioration. A 60-100% (85 +/- 15%, mean +/- SD) reduction in the PGH ratio occurred after 20-50 min of A23187 superfusion in 12 of 18 neurons tested. Complete abolition of the PGH occurred in 8 of these 12 cells exposed to A23187 for 30-120 min. 3. Application of A23187 in Ca(2+)-free/high-Mg2+ solution did not abolish the PGH, although small (less than 50%; 37 +/- 10%) reductions in the PGH ratio were observed after perfusion of 50 min or longer in five neurons tested. 4. Intracellular injection of the Ca2+ chelator bis-(o-amino-phenoxy)-N,N,N',N'-tetraacetic acid (BAPTA, 300-400 mM) blocked inhibition of the PGH by A23187. After 50 min of perfusion with Ca2+ ionophore, no reduction of the PGH ratio was observed in five neurons tested. 5. Rundown of the PGH without apparent change in membrane properties was observed in three neurons that were stable for greater than 2-3 h, allowing repetitive GLU applications. 6. Block of the PGH produced by a Na(+)-K(+)-adenosinetriphosphatase (ATPase) inhibitor (strophanthidin) prolonged the duration of GDs because of a delay in repolarization.(ABSTRACT TRUNCATED AT 250 WORDS)     

低氧缺血海马神经元内Ca~(2 )动态变化的LSCM测定

测定脑组织细胞内 Ｃａ~(2 )浓度是评价缺血引起神经元损伤的重要指标。本研究用激光扫描共聚焦显微镜（ＬＳＣＭ）和Ｆｌｕｏ－３／ＡＭ荧光探针标记技术,从单个活细胞水平检测缺血刺激诱发的海马神经元内Ｃａ２＋瞬间动态变化。结果显示低氧使胞内Ｃａ２＋浓度显著升高。谷氨酸引起Ｃａ２＋浓度升高缓慢但持续时间长。缺血对Ｃａ２＋浓度影响不显著。撤除葡萄糖则引起胞内Ｃａ２＋迅速降低。实验结果表明不同缺血刺激因素引起的钙振荡各异。用ＬＳＣＭ对活细胞内部非侵入光学断层扫描,能清晰记录到这些瞬态钙值变化。     

Extensive nuclear localization of alpha-synuclein in normal rat brain neurons revealed by a novel monoclonal antibody

Synuclein was initially named for its localization in both presynaptic nerve terminals and portions of nuclear envelope. However, subsequent studies only confirmed the presynaptic localization of this protein in the brain; its nuclear localization in the neurons remained elusive. Here, two new monoclonal antibodies against alpha-synuclein (alpha-SYN) were produced. Epitope mapping using phage peptide display showed that the epitopes of the two antibodies were localized in two distinct specific sequences of the C-terminal domain of alpha-SYN. One antibody named 3D5 recognized amino acids 115-121 of alpha-SYN and the other antibody named 2E3 identified the amino acids 134-138 of the protein. Western blot analysis demonstrated that both 2E3 and 3D5 detected a 19 kD protein from rat and human brain homogenates, which was identical to the molecular size of recombinant alpha-SYN. However, immunohistochemical staining on normal adult rat brain sections showed that the two antibodies revealed distinct patterns of subcellular localization of alpha-SYN immunoreactivity. Both 3D5 and 2E3 detected the presynaptic alpha-SYN but only 3D5 detected the nuclear alpha-SYN. The nuclear localization of alpha-SYN was further confirmed by Western blot analysis in isolated nuclear fraction where the same size of alpha-SYN was detected, and by immunoelectron microscopy using colloidal gold probes where gold particles were specifically localized in portions of peri- and intra-nucleus. The nuclear positive neurons were distributed extensively in almost all the brain regions. This is the first report well characterizing the extensive localization of alpha-SYN in the neuronal nuclei throughout the brain in normal conditions. This finding indicates an important physiological function of this molecule in the nuclei of brain neurons, which deserves further investigations.     

制备单克隆抗体检测PES1的表达

目的:制备PES1单克隆抗体(mAb)并用所制备的抗体检测PES1在多种肿瘤细胞中的表达及其在成年大鼠不同组织中的表达分布。方法:纯化GST-PES1(1-322aa)融合蛋白后注入小鼠进行免疫,经过细胞融合、筛选以及用Western blot进行特异性鉴定后获得mAb。再用制备的mAb检测PES1在不同的人肿瘤细胞和大鼠不同组织中的表达。结果:所制备的PES1 mAb能特异地识别PES1。用所制备的mAb检测发现,在所检测的人乳腺癌、卵巢癌、肝癌以及肺癌等细胞中均有不同程度的表达;PES1类似物pescadillo在大鼠的乳腺和卵巢组织中表达,但在其他组织中没有被检测到。结论:成功地制备了PES1的mAb。PES1可能与肿瘤的发生发展有一定的关系;由于PES1明显表达于雌激素重要的靶器官,所以可能参与雌激素信号通路。     

Immunohistochemical study of monoclonal antibody MGD-1 in gastric carcinoma

This paper reports a pathological and immunohistochemical study of gastric carcinoma for immunoreactivity with a monoclonal antibody. MGD-1, raised against cells from an adenocarcinoma of stomach. Fifty-four of 61 gastric carcinomas (89%) were positive for MGD-1. Metastatic gastric carcinoma in local nodes was positive in all 11 such cases. Out of 40 examples of chronic atrophic gastritis, only three, with mild dysplasia, were positive (7.5%). Forty cases with normal gastric mucosa were negative. The MGD-1 detection-rate of well- and poorly-differentiated gastric carcinoma was 85% and 93% respectively. The metastatic cells and cells infiltrating the submucosa and muscular layer were more frequently positive and showed stronger staining with MGD-1 than those in mucosa. These results show that MGD-1 possesses a high degree of specificity for gastric carcinoma and could be used diagnostically.