周期素依赖性激酶抑制剂对神经元损伤的保护作用

周期素依赖性激酶抑制剂(cyclin-dependent kinases inhibitors,CDKIs)分为INK4(inhibitor of CDK4,INK4)和KIP(kinase inhibition protein,KIP)两大家庭,主要通过视网膜母细胞瘤蛋白(retinoblastoma protein,pRb)或p53两条途径对细胞周期起调控作用。在脑缺血再灌注损伤后周期素依赖性激酶(cyclin-dependent kinases,cDKs)所调控的信号通路被激活,细胞周期紊乱可促使神经元凋亡,CDKIs对神经元损伤具有保护作用。     

7蛋白酶体抑制剂对星形胶质细胞周期素D1和周期素依赖性激酶4表达的影响

目的：研究蛋白酶体抑制对体外培养的星形胶质细胞周期素Dl（cyclinD1）和周期素依赖性激酶4（CDK4）表达的影响。方法：SD乳鼠皮质星形胶质细胞原代培养,并纯化鉴定;予不同浓度（2和4μmol·L^-1）的蛋白酶体抑制剂（lactacystin）对第二代星形胶质细胞进行短期（12h）急性干预处理,应用免疫荧光及Westernblot检测星形胶质细胞cyclinD1和CDK4表达的水平。结果：纯化传代的皮质星形胶质细胞经胶质纤维酸性蛋白（GFAP）免疫荧光鉴定,其阳性率可达99％;lactacystin2和4μmol·L^-1可诱导星形胶质细胞cyclinDl和CDK4表达的下降,与对照组相比差异有显著统计学意义（P〈0．01）。结论：一定程度蛋白酶体活性抑制可诱导培养的星形胶质细胞cyclinD1和CDK4表达的减少,从而影响胶质细胞细胞周期,促进胶质细胞分化。提示蛋白酶体功能障碍后可能通过影响胶质细胞细胞周期来参与阿尔茨海默病的病理改变。     

出生5 d大鼠海马神经元缺氧耐受性研究

目的建立出生5d大鼠海马神经元原代培养方法，并研究该神经元的缺氧耐受性。方法改进出生5d大鼠海马神经元原代培养方法，分离海马时分为充氧组与对照组，培养过程中倒置显微镜和细胞核因子（NF）免疫组织化学法观察细胞生长和形态。原代培养7d后，分别对出生5d大鼠海马神经元和出生1d大鼠海马神经元进行12h、24h的缺氧处理（0％02、5％C02、95％N2），MTT法检测细胞存活情况。结果（1）充氧组中培养的出生5d大鼠海马神经元生长状态要优于对照组中培养的神经元。（2）缺氧12h出生1d以及5d大鼠海马神经元存活率分别为（81．5±2．3）％和（89，4±2，2）％（P〈0．01）；缺氧24h二者存活率分别为（75．0±2．8）％和（85．0±2．2）％（P〈0．01）。5d海马神经元缺氧耐受能力优于1d大鼠海马神经元。结论海马神经元分离培养过程中充氧有利于神经元的原代培养，出生5d大鼠海马神经元缺氧耐受力强于出生1d大鼠海马神经元。     

抑郁大鼠模型额叶及海马区Rho激酶表达变化的初步研究

目的：观察抑郁大鼠模型急性期额叶及海马区Rho激酶（ROCK）的表达变化,探讨Rho／ROCK通路是否与抑郁症形成有关。方法：建立Wistar大鼠抑郁症模型;在建模成功后24h迅速断头取前脑,在冰浴上分离出双侧前额叶及海马脑组织,应用Westernblot分别测定大鼠额叶及海马脑组织内ROCKⅠ及ROCKⅡ含量。结果：与正常对照组比,抑郁模型大鼠左侧和右侧前额叶ROCKⅠ表达均显著增高（分别P〈0．01,P〈0．05）,以左侧为著;ROCKⅡ表达亦有显著增高（均P〈0．05）。抑郁模型大鼠左右侧海马区域ROCKI表达显著增高（分NP〈0．05,P〈0．01）,以右侧明显;ROCKⅡ表达亦有显著增高（分别P〈0．05,P〈0．01）。结论：ROCKⅠ及ROCKⅡ可能参与了抑郁症形成的病理过程。     

脾酪氨酸激酶在不同等级胶质瘤中的表达及其与周期素1水平的相关性

目的 探讨人脑胶质瘤中脾酪氨酸激酶(Syk)的表达及其与周期索1(CyclinDl)水平的相关性.方法 收集贵州航天医院神经外科自2005年1月至2010年1月间手术切除并经病理证实的脑胶质瘤标本46例,其中Ⅰ级13例,Ⅱ级9例,Ⅲ级5例,Ⅳ级胶质母细胞瘤19例.另取5例因脑创伤行内减压术患者的正常脑组织标本作为对照,RT-PCR检测脑组织标本Syk mRNA、CyclinD1 mRNA的表达并分析二者的相关性.结果 与正常脑组织比较,Ⅰ、Ⅱ、Ⅲ、Ⅳ级脑胶质细胞瘤标本Syk mRNA表达较低,CyclinDl mRNA表达较高,差异有统计学意义(P＜0.05),而且胶质瘤的病理级别越高,Syk mRNA的表达越低,CyclinDl mRNA表达较高,差异均有统计学意义(P＜0.05);胶质瘤中Syk与CyclinDl的表达呈负相关关系(r=-0.832,P=0.000).结论 Syk在脑胶质瘤中表达较低或缺失,提示其可能具有抑癌基因功能,其机制可能与下调胶质瘤中CyclinD1的表达有关.     

多氯联苯对大鼠海马c-fos表达的研究

多氯联苯 (polychlorinated biphenyl,PCB)结构类似于甾体类激素能竞争该类激素的受体发挥一定生物功能。此研究通过 PCB是否对大鼠海马结构和功能产生影响 ,以了解 PCB对学习、记忆功能的影响。1　材料和方法　选用 Wistar雌性大鼠 2 4只 ,随机分为 4组。分别饲喂含质量分数 1× 1 0 1 2 、1× 1 0 1 1 、1× 1 0 1 0 PCB饲料及正常饲料。1个后进行交配、受孕、分娩。断乳后的仔鼠分别选取体重近似的雄性大鼠 1 0只 ,对应饲喂相应的饲料 ,3个月后断头处死 ,分离海马并以 4g/ L多聚甲醛固定待测。海马标本常规 HE染色 ,免疫组化 ABC…     

Caspase-3在戊四氮点燃大鼠海马组织中的表达

目的：研究Caspase-3在戊四氮（pentylenetetrazole,PTZ）急性点燃大鼠海马组织中的表达情况。方法：通过腹腔注射戊四氮建立急性点燃大鼠模型，运用逆转录聚合酶链式反应技术分析Caspase-3 mRNA在癫痫发作后的表达情况。结果：癫痫发作后海马组织Caspase-3 mRNA的表达水平早期即出现升高（8h组与对照组相比，P＜0．05），且持续较长一段时间（5d组与对照组相比，P＜0．05）。结论：Caspase-3可能参与了癫痫后海马神经元的凋亡过程。     

糖尿病大鼠海马endophilin-A1和dynamin-1的表达研究

目的研究糖尿病大鼠海马组织endophilin-A1和dynamin-1的表达,以期进一步研究其与糖尿病认知功能损害的关系。方法 14只雄性SD大鼠随机分为两组:正常对照(NC)组和糖尿病(DM)组。腹腔注射链脲佐菌素55mg.kg-1制备糖尿病模型,5周后断头取材,分别进行如下实验:①Western Blot法检测海马组织endophilin-A1和dynamin-1的表达;②免疫组化法观察海马区dynamin-1的表达。用SPSS17.0软件作独立样本t检验。结果①Endophilin-A1和dynamin-1在两组大鼠海马组织中均有表达,其中DM组(0.66±0.12,p=0.004)endophilin-A1的aD值明显低于NC组(0.97±0.07),Dynamin-1在两组中的aD值差别无统计学意义(p=0.841);②免疫组化法显示海马CA3区、DG区dynamin1在各组中均有表达,差异无统计学意义(p=0.114,p=0.783)。结论糖尿病大鼠海马组织endophilin-A1表达下调,而dynamin-1表达未发现明显改变,值得进一步研究以探讨二者与糖尿病突触功能损害的关系。     

海马神经元体外培养的实验研究

海马是中枢神经系统的重要结构,与许多变性疾病有关。我们应用细胞培养技术,研究了新生大鼠海马神经元的体外生长情况及神经营养因子对它的促生长作用,现报告如下。一、材料与方法1.材料:新生48h的Wistar大鼠由山东大学动物实验中心提供;碱性成纤维细胞生长因子(basic fibroblast growth     

氯化钴诱导大鼠海马神经元低氧对LDHA表达影响的研究

目的观察氯化钴(CoCl_2)诱导神经元低氧模型中乳酸脱氢酶A(LDHA)及凋亡相关蛋白Bax、Bcl-2的表达变化,进一步探讨LDHA可能发挥的作用。方法对新生大鼠海马神经元进行原代培养,通过免疫荧光法进行细胞鉴定。利用CoCl2构建神经元低氧模型,在培养至第6天的细胞培养液中加入不同量的CoCl_2,使其浓度分别达到0μmol·L~(-1),50μmol·L~(-1),100μmol·L~(-1),200μmol·L~(-1)。CoCl_2作用48h后提取细胞蛋白,通过蛋白印迹法(Wb)测定各组细胞LDHA及Bcl-2相关X蛋白(Bax)、B细胞淋巴瘤/白血病-2基因(Bcl-2)的表达量。结果 LDHA表达量随CoCl_2浓度增加而增加,差异有统计学意义(P <0.05);实验组Bax表达量较对照组(P <0.01),但各实验组间比较差异无统计学意义(P> 0.05);Bcl-2和Bcl-2/Bax各组间差异均无统计学意义(P> 0.05)。结论 CoCl_2可诱导神经元Bax表达上调,诱导神经元低氧、凋亡。CoCl_2可诱导神经元LDHA表达上调,在一定范围内,随CoCl2浓度增加,LDHA表达增多。轻度低氧情况下,神经元可能通过上调LDHA表达而减少凋亡。     

癫痫持续发作对大鼠海马神经元的影响

目的 研究实验性癫痫大鼠在癫痫发作持续不同时间对海马神经元的影响。方法24只SD大鼠随机分成4组：诱发火鼠瘢痫持续状态（status cpilepticus，SE）〈10、10～30、〉30min组及正常对照组。于电镜下观察各组海马神经元超微结构变化；采用免疫组化方法检测凋亡相关基因bcl-2、bax的蛋白表达及通过流式细胞技术检测细胞凋亡情况。结果SE〈10min组海马神经元所受影响不大．SE10～30min组海马神经元具有明显凋亡特征．SE〉30min组多数海马神经元呈坏死性改变。结论大鼠SE对海马神经元损伤有凋亡和坏死两种不同形式．这与癫痫发作的持续时间密切相关。     

Direct Cellular Peptidomics of Supraoptic Magnocellular and Hippocampal Neurons in Low-Density Cocultures

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Differential Regulation of Preprotachykinin-A mRNA Expression in Striatum by Excitation of Hippocampal Neurons

In this report we have studied the influence of hippocampal neurons on neuropeptide mRNA expression in both dorsal and ventral striatum in the rat. Intrahippocampal unilateral kainic acid injections were performed in control animals and in animals with a unilateral 6-hydroxydopamine-induced dopamine deafferentation of the striatum. In situ hybridization combined with quantitative image analysis was used to study the expression of preprotachykinin A mRNA encoding the neuropeptides substance P and neurokinin A. The 6-hydroxydopamine-induced lesion caused a decrease of preprotachykinin A mRNA levels in the ipsilateral dorsal striatum and in both sides of the ventral striatum. In normal rats, the intrahippocampal kainic acid injection caused a twofold increase in preprotachykinin A mRNA in the limbic parts of the striatum, which are innervated by the hippocampus. No effect of the kainic acid injection was seen in the lateral parts of the dorsal striatum, a region which does not appear to be innvervated by the hippocampus. Animals with a 6-hydroxydopamine lesion showed a similar kainic acid-mediated increase in preprotachykinin A mRNA in parts of the ventral striatum. In the dopamine-lesioned dorsal striatum and ventral striatum the decreased preprotachykinin A mRNA levels were normalized by the intrahippocampal kainic acid injection. These results show that kainic acid-mediated excitation of hippocampal neurons causes a dopamine-independent induction of preprotachykinin A mRNA expression in parts of the ventral striatum, and reverses the dopamine deafferentation-induced decrease of preprotachykinin A mRNA in both dorsal and ventral striatum. Combined, our results suggest that hippocampal neurons can regulate preprotachykinin A mRNA expression in both the ventral and the dorsal striatum.     

Electrophysiological Interactions Between 5-Hydroxytryptamine and Thyrotropin Releasing Hormone on Rat Hippocampal CA1 Neurons

Intracellular recording from CA1 neurons of the rat hippocampal slice preparation was used to examine the possibility of functional interactions between 5-hydroxytryptamine (5-HT) and thyrotropin releasing hormone (TRH), which act as cotransmitters in other areas of the central nervous system. 5-HT (30 μM) elicited complex effects consisting of biphasic changes in membrane potential and a strong depression of the afterhyperpolarization (AHP) following a spike burst. TRH (10 μM) did not alter membrane potential or input conductance but it produced a partial block of the AHP. Under single-electrode voltage clamp, 5-HT and TRH both reduced the amplitude of voltage-activated total K⁺ currents. When the two substances were co-applied, their actions were occluded. The voltage-activated K⁺ current remaining in Ca²⁺-free solution lost its sensitivity to 5-HT and TRH, suggesting that the K⁺ current modulated by TRH and 5-HT was Ca²⁺-dependent, although TRH itself did not depress high-threshold voltage-activated Ca²⁺ currents. When a relatively small concentration (5 μM) of 5-HT was co-applied with an equimolar amount of TRH, the degree of block of the spike AHP was the sum of the two individual effects of these drugs. It is suggested that in hippocampal pyramidal cells 5-HT and TRH influenced neuronal excitability by depressing a Ca²⁺-dependent K⁺ current, a phenomenon perhaps mediated through a common intracellular second messenger pathway.     

一种改良的胚胎大鼠颞叶海马区神经元的培养方法

目的:探索胚胎大鼠颞叶海马区神经元的改良培养方法。方法:在无血清培养基础上,采用改良的分离纯化法获取单细胞悬液,接种后在不同阶段通过加入不同配方的培养基进行培养,并进行免疫组化鉴定。结果:用该方法培养的颞叶海马区神经元细胞存活率高,生长状态良好,且βⅢ-tublin免疫染色为阳性,神经元细胞纯度可达90%以上。结论:改良分离、结合分阶段的不同培养条件是一种简单、高效的颞叶海马区神经元的纯化培养方法。     

Activation of mGluR5 Induces Rapid and Long-Lasting Protein Kinase D Phosphorylation in Hippocampal Neurons

Metabotropic glutamate receptors (mGluRs), including mGluR5, play a central role in regulating the strength and plasticity of synaptic connections in the brain. However, the signaling pathways that connect mGluRs to their downstream effectors are not yet fully understood. Here, we report that stimulation of mGluR5 in hippocampal cultures and slices results in phosphorylation of protein kinase D (PKD) at the autophosphorylation site Ser-916. This phosphorylation event occurs within 30 s of stimulation, persists for at least 24 h, and is dependent on activation of phospholipase C and protein kinase C. Our data suggest that activation of PKD may represent a novel signaling pathway linking mGluR5 to its downstream targets. These findings have important implications for the study of the molecular mechanisms underlying mGluR-dependent synaptic plasticity.     

Hippocampal neuron loss in epilepsy and after experimental seizures

In this study, the hippocampus and its relationship to epilepsy is reviewed from a neuropathological point of view. The variability found in previous studies regarding the extent of the damage to neurons makes interpretation difficult. The aim of this study was to clarify the neuron loss by quantitation. The neuron density in the different H-fields of the pyramidal band and in the granule cell area has been determined throughout the hippocampi by means of light microscope and an ocular grid. The brains of 20 patients with epilepsy were studied. The type of epilepsy was verified from the case history and encephalographic recordings. The distribution of neuron loss was evaluated by statistical analyses of variance applied to the different means and related to the clinical assessments. The normal neuronal distribution was evaluated in 29 persons without any known cerebral disorders. In experimental studies, the changes in neuron density in relation to seizures were determined. 8 rats were studied after electroconvulsive seizures, elicited 3 times daily for 50 days. 10 animals served as controls. 20 gerbils of a seizure-sensitive strain with different types of seizures, 5 in each group, were the object of the second experimental study which also included a group of 5 gerbils of the seizure-resistant type. The aim of this part of the study was to illustrate the damage to the neurons in relation to different seizure types. The main result of the study of patients with epilepsy was that neuron loss occurs in all areas investigated. The severely affected areas were field H₃ and the granule cells. The neurons of field H₁ seemed to be damaged earlier in life, possibly due to hypoxia accompanying generalized convulsions. The study of the normal neuronal distribution in the hippocampus revealed a neuronal reduction of 20% in old age. This ageing phenomenon was taken into account when evaluating the pattern of neuron loss in the patients with epilepsy. No specific pattern of neuron loss characterized the different types of epilepsy. Neuron loss was related to generalized convulsions and increased throughout life with the duration of the disorder. The experimental studies of rats revealed that no neuron loss occurred as a result of generalized convulsions alone. In contrast, the pyramidal neurons of fields H₂ and H₃ were reduced in the gerbils with frequent generalized convulsions. However, the number of seizures was low, in comparison with the number of generalized convulsions in rats. The hypothesis is put forward that the neuron loss in the gerbils in this area, known for its high bursting activity, is due to seizure-activity which has been shown by others to occur interictally. It was thus suggested that seizure-activity even without clinical seizures might be responsible for neuron loss in the hippocampus also in man, in relation to epilepsy of a certain intensity and duration. The consequences of this with regard to prevention of the development of severe epilepsy after single and even minor seizures and the choice of treatment in the individual cases are considered and future projects are mentioned.     

Human Hippocampal Neurons Track Moments in a Sequence of Events

An indispensable feature of episodic memory is our ability to temporally piece together different elements of an experience into a coherent memory. Hippocampal time cells—neurons that represent temporal information—may play a critical role in this process. Although these cells have been repeatedly found in rodents, it is still unclear to what extent similar temporal selectivity exists in the human hippocampus. Here, we show that temporal context modulates the firing activity of human hippocampal neurons during structured temporal experiences. We recorded neuronal activity in the human brain while patients of either sex learned predictable sequences of pictures. We report that human time cells fire at successive moments in this task. Furthermore, time cells also signaled inherently changing temporal contexts during empty 10 s gap periods between trials while participants waited for the task to resume. Finally, population activity allowed for decoding temporal epoch identity, both during sequence learning and during the gap periods. These findings suggest that human hippocampal neurons could play an essential role in temporally organizing distinct moments of an experience in episodic memory.SIGNIFICANCE STATEMENT Episodic memory refers to our ability to remember the what, where, and when of a past experience. Representing time is an important component of this form of memory. Here, we show that neurons in the human hippocampus represent temporal information. This temporal signature was observed both when participants were actively engaged in a memory task, as well as during 10-s-long gaps when they were asked to wait before performing the task. Furthermore, the activity of the population of hippocampal cells allowed for decoding one temporal epoch from another. These results suggest a robust representation of time in the human hippocampus.