甲氨蝶呤对早期神经胚基因表达的影响

目的:利用甲氨蝶呤(methotrexate,MTX)干预孕鼠,探讨MTX对早期神经胚基因表达的影响。方法:用MTX(4.5 mg/kg体重)干预孕鼠,通过NimbleGene表达谱芯片、Real time-PCR及免疫组化等方法进行差异表达基因的筛选和验证。结果:MTX处理后神经管畸形(NTDs)发生率为32.1%。表达谱芯片筛选出166个差异表达基因,其中4个凋亡相关基因(Endog,Trp53,Casp3,Bax)均表现为上调(fold change1.5,P0.05),3个增殖相关基因(Ptch1,Pla2g4a,Foxg1)均表现为下调(fold change0.67,P0.05);NTDs胚胎神经上皮Caspase-3表达显著升高(P0.05),phospho-histone H3(pH3)表达显著降低(P0.05)。结论:MTX影响了早期神经胚的基因表达,尤其是引起了凋亡、增殖相关基因表达的异常,这可能在叶酸缺乏引起NTDs发生的相关机制之一。     

白介素-6保护小脑颗粒神经元抗谷氨酸的神经毒性作用

目的：探讨白介素-6（IL-6）对谷氨酸诱导的神经元损伤的防治作用及其作用机制。方法：用IL-6慢性预处理培养的小脑颗粒神经元,然后后用谷氨酸急性刺激小脑颗粒神经元。用噻唑兰（MTT）比色法和末端脱氧核苷酸转移酶介导的原位缺口末端标记（TUNEL）法分别观察神经元的功能和凋亡的变化;用激光扫描共聚焦显微镜（LSCM）和逆转录聚合酶链式反应（RT—PCR）法分别检测神经元内Ca^2＋浓度的动态变化和IL-6信号转导蛋白gp130 mRNA的表达。结果：IL-6（2．5、5和10ng／ml）慢性预处理培养的小脑颗粒神经元,可浓度依赖性地改善谷氨酸诱导的神经元活性降低;并可明显减少谷氨酸诱导的神经元凋亡;还可显著抑制谷氨酸激发的神经元内Ca^2＋超载。此外。经IL-6慢性预处理的小脑颗粒神经元表达gp130mRNA明显低于未经IL-6预处理的神经元。结论：IL-6能保护神经元抵抗由谷氨酸诱导的兴奋毒性作用,IL-6的这种神经保护机制可能与它抑制神经元内Ca^2＋超载密切相关,而且可能由gp130细胞内信号转导途径介导。     

凋亡和周期基因芯片研究As2O3对NB4细胞凋亡相关基因表达谱的影响

目的:利用细胞凋亡和周期基因芯片研究As₂O₃作用前后NB4细胞基因表达谱的差异性,寻找As₂O₃诱导NB4细胞凋亡的相关基因并分析其可能机制。方法:流式细胞仪检测细胞凋亡率,抽提对照及诱导组细胞的mRNA,通过逆转录将As₂O₃处理前后的NB4细胞cDNA进行生物素标记,用含269个目的基因的细胞凋亡和周期基因芯片进行杂交,GEArray软件分析,筛选出As₂O₃诱导前后表达有差异的基因。芯片结果用荧光定量聚合酶链反应(realtimepolymerasechainreaction)进行验证。结果:筛选出As₂O₃作用前后表达有差异的基因共100条(占芯片基因总数的37.2%),其中97条(97/100,97%)基因表达上调,3条(3/100,3%)基因表达下调。表达上调的基因主要包括肿瘤坏死因子配体和受体家族、bcl2家族、半胱氨酸家族、DNA损伤检测和P53途径以及细胞分裂周期蛋白和激酶等基因。结论:As₂O₃主要通过上调促凋亡基因表达来诱导NB4细胞凋亡,其中TNFSF15、Apaf1、Caspase3和p16等基因可能参与As₂O₃诱导的NB4细胞凋亡,As₂O₃诱导NB4细胞凋亡的可能机制主要涉及TNF途径、线粒体途径、Caspase途径、细胞周期抑制途径和P53途径等。     

免疫性损伤小鼠肝脏的基因表达谱变化

采用卡介苗和脂多糖联合诱导的方法建立小鼠免疫性肝损伤模型, 分别提取正常对照组与免疫性肝损伤组小鼠的肝脏总RNA, 经反转录用Cy3-dUTP和Cy5-dUTP分别标记制备对照组与模型组来源的cDNA探针, 并将cDNA探针与小鼠基因表达谱芯片杂交, 杂交结果经芯片扫描仪扫描并用相关软件进行分析. 结果表明, 与对照组相比, 免疫性肝损伤组有293条基因发生了差异表达, 其中188条基因表达量明显上调, 另外105条基因表达量明显下调. 通过对一些关键差异表达基因的生物学功能分析表明, 卡介苗与脂多糖联合诱导的小鼠免疫性肝损伤的发生、发展过程与肝细胞的免疫反应、细胞合成与代谢、细胞凋亡及转运等过程密切相关, 这对进一步阐明免疫性肝损伤高度相关的基因表达调控网络, 进而阐明免疫性肝损伤的病理机制具有十分重要的作用.     

谷氨酸在心肺脑复苏中的影响

心跳呼吸骤停及心肺脑复苏(CPCR)的过程均导致机体发生缺血/再灌注损伤(I瓜)等复杂的病理生理变化.脑的缺血再灌注损伤是一个快速的级联反应,包括能量代谢障碍、氧自由基的生成增多、兴奋性氨基酸释放增加、炎性细胞因子释放增加等.这些环节紧密连接,互为因果,形成恶性循环,最终导致神经元的凋亡或坏死.谷氨酸是大脑中一种关键的兴奋性神经递质,它的过多释放在神经元损伤发展过程中起着重要作用.随着细胞外高浓度谷氨酸的结合,N-甲基-D-天冬氨酸受体(NMDAR)开放,大量Ca2+内流,导致细胞内钙超载,是引起脑损伤的共同通路.本文就神经兴奋毒性的研究进行综述.     

应用寡核苷酸芯片筛选维甲酸诱导神经母细胞瘤细胞系SH-SY5Y分化成神经元过程中的差异表达基因

目的:应用寡核苷酸芯片筛选维甲酸(RA)诱导神经母细胞瘤细胞系SH-SY5Y分化成神经元过程中的差异表达基因。方法:从人胎脑及不同类型神经系统肿瘤组织中获取目的基因,查询相应基因mRNA序列,设计并合成探针,制备了含218种基因的神经功能相关的寡核苷酸芯片。应用RA诱导SH-SY5Y8d分化成成熟神经元,提取对照组和实验组每天的总RNA,通过逆转录荧光标记cDNA探针并与芯片杂交,洗片后扫描获取图像,数据分析获得差异表达基因,并通过RT-PCR进行验证。结果:发现13种基因表达上调,没有得到下调基因。RT-PCR验证结果基本与芯片结果一致。结论:SH-SY5Y经RA诱导分化成神经元存在一些差异表达的基因,寡核苷酸芯片技术可为研究SH-SY5Y诱导分化成神经元的分子作用机理提供技术平台。     

腺苷A2A受体在小鼠颅脑创伤与外周组织损伤模型中的作用差异

本文旨在探索腺苷A2A受体在颅脑创伤、皮肤创伤及放射损伤复合创伤中的作用差异.分别观察和检测野生型小鼠、A2A受体基因敲除小鼠以及给予A2A受体激动剂CGS21680治疗的小鼠在皮肤创伤、放射损伤复合创伤后的伤口愈合时间以及颅脑创伤后的神经功能缺损情况、伤侧皮层脑含水量、脑脊液中谷氨酸浓度.结果表明,CGS21680促进外周组织伤口愈合,却加重颅脑创伤模型的神经功能损害,这与其促进谷氨酸释放有关.相反,A2A受体基因敲除显著延迟小鼠皮肤创伤及放射损伤复合创伤模型的伤口愈合,而在颅脑创伤模型中通过抑制谷氨酸释放产生保护效应.本研究初步证实,A2A受体激活促进谷氨酸大量释放可能是其在中枢损伤与外周损伤产生作用差异的机理之一,这为将来临床应用A2A受体激动剂减轻外周损伤,而用A2A受体拈抗剂减轻颅脑损伤提供了一定的实验依据.     

高温诱导金黄仓鼠胚神经上皮细胞基因的差异表达

以mRNA差异显示法研究高温作用后金黄仓鼠胚神经上皮细胞基因的差异表达。结果表明,高温处理后第16h,从鼠胚神经上皮细胞基因表达中检出两条良好重复性差异序列。该差异序列为未知序列,属新EST,提示高温可能诱导金黄仓鼠胚神经上皮细胞基因表达发生变化。     

cDNA微阵列分析人apoE4转基因鼠肾脏基因表达谱的变化

目的研究人apoE4转基因鼠肾脏的基因表达谱变化.方法分别提取人apoE4转基因鼠和正常C57BL/6J小鼠的肾脏总RNA,经逆转录合成cDNA探针后分别与鼠cDNA表达点阵杂交,再用ESTblot软件进行分析,并用Northern印迹证明基因表达的改变.结果人apoE4转基因鼠肾脏中有38个基因的mRNA表达升高,22个基因的mRNA表达降低.其中血浆谷胱甘肽过氧化物酶前体、视黄酸γ受体和白介素5受体等基因的表达明显增加.B-raf原癌基因、促红细胞生成素受体、整联蛋白α4的基因表达显著降低.Northern杂交证明转基因鼠肾脏的c-Jun基因表达升高.结论人apoE4转基因鼠肾脏的c-Jun、血浆谷胱甘肽过氧化物酶前体、白介素5受体等基因的表达增加;促红细胞生成素受体、整联蛋白α4等基因的表达减少.     

肾上腺髓质素降低培养海马神经元胞内游离钙离子浓度

经荧光探针Fluo 3-AM标记细胞内游离钙后,用激光共聚焦显微镜检测肾上腺髓质素(adrenomedullin,ADM)对原代培养大鼠海马神经元内游离钙浓度([Ca^2 ]1)的影响。实验结果如下：(1)ADM(0．01-1．0μmol／L)浓度依赖性地降低细胞内钙浓度。(2)降钙素基因相关肽受体阻断剂(calcitonin gene-related peptide,CGRP8-37)预处理可部分抑制ADM的效应。(3)ADM可显著抑制高钾引起的[Ca^2 ]1增加。(4)ADM可显著抑制三磷酸肌醇(inositol 1,4,5-trisphosphate,IP3)引起的内钙释放,而对兰尼定(ryanodine)引起的内钙释放无显著影响。以上结果提示,ADM降低培养海马神经元内游离钙浓度,此作用与其抑制IP,引起的内钙释放有关,ADM对静息状态下的Ca^2 内流无影响,但可显著抑制高钾引起的Ca^2 内流,CGRP受体介导了ADM的上述效应。     

The regulation and role of neuronal gap junctions during neuronal injury

In the mammalian CNS, excessive release of glutamate and overactivation of glutamate receptors are responsible for the secondary (delayed) neuronal death following neuronal injury, including ischemia, traumatic brain injury (TBI) and epilepsy. The coupling of neurons by gap junctions (electrical synapses) increases during neuronal injury. In a recent study with the use of in vivo and in vitro models of cortical ischemia in mice, we have demonstrated that the ischemic increase in neuronal gap junction coupling is regulated by glutamate via group II metabotropic glutamate receptors (mGluR). Specifically, we found that activation of group II mGluRs increases background levels of neuronal gap junction coupling and expression of connexin 36 (Cx36; neuronal gap junction protein), whereas inactivation of group II mGluRs prevents the ischemia-mediated increases in the coupling and Cx36 expression. Using the analysis of neuronal death, we also established that inactivation of group II mGluRs or genetic elimination of Cx36 both dramatically reduce ischemic neuronal death in vitro and in vivo. Similar results were obtained using in vitro models of TBI and epilepsy. Our study demonstrated that mechanisms for the injury-mediated increase in neuronal gap junction coupling are part of the mechanisms for glutamate-dependent neuronal death.     

The regulation and role of neuronal gap junctions during neuronal injury

In the mammalian CNS, excessive release of glutamate and overactivation of glutamate receptors are responsible for the secondary (delayed) neuronal death following neuronal injury, including ischemia, traumatic brain injury (TBI) and epilepsy. The coupling of neurons by gap junctions (electrical synapses) increases during neuronal injury. In a recent study with the use of in vivo and in vitro models of cortical ischemia in mice, we have demonstrated that the ischemic increase in neuronal gap junction coupling is regulated by glutamate via group II metabotropic glutamate receptors (mGluR). Specifically, we found that activation of group II mGluRs increases background levels of neuronal gap junction coupling and expression of connexin 36 (Cx36; neuronal gap junction protein), whereas inactivation of group II mGluRs prevents the ischemia-mediated increases in the coupling and Cx36 expression. Using the analysis of neuronal death, we also established that inactivation of group II mGluRs or genetic elimination of Cx36 both dramatically reduce ischemic neuronal death in vitro and in vivo. Similar results were obtained using in vitro models of TBI and epilepsy. Our study demonstrated that mechanisms for the injury-mediated increase in neuronal gap junction coupling are part of the mechanisms for glutamate-dependent neuronal death.     

Activation of calcium/calmodulin-dependent protein kinase IV in long term potentiation in the rat hippocampal CA1 region

The importance of well characterized calcium/calmodulin-dependent protein kinase (CaMK) II in hippocampal long term potentiation (LTP) is widely well established; however, several CaMKs other than CaMKII are not yet clearly characterized and understood. Here we report the activation of CaMKIV, which is phosphorylated by CaMK kinase and localized predominantly in neuronal nuclei, and its functional role as a cyclic AMP-responsive element-binding protein (CREB) kinase in high frequency stimulation (HFS)-induced LTP in the rat hippocampal CA1 region. CaMKIV was transiently activated in neuronal nuclei after HFS, and the activation returned to the basal level within 30 min. Phosphorylation of CREB, which is a CaMKIV substrate, and expression of c-Fos protein, which is regulated by CREB, increased during LTP. This increase was inhibited mainly by CaMK inhibitors and also by an inhibitor for mitogen-activated protein kinase cascade, although to a lesser extent. Our results suggest that CaMKIV functions as a CREB kinase and controls CREB-regulated gene expression during HFS-induced LTP in the rat hippocampal CA1 region.     

Epidermal growth factor induces oxidative neuronal injury in cortical culture

Recently, we have demonstrated that certain neurotrophic factors can induce oxidative neuronal necrosis by acting at the cognate tyrosine kinase-linked receptors. Epidermal growth factor (EGF) has neurotrophic effects via the tyrosine kinase-linked EGF receptor (EGFR), but its neurotoxic potential has not been studied. Here, we examined this possibility in mouse cortical culture. Exposure of cortical cultures to 1-100 ng/ml EGF induced gradually developing neuronal death, which was complete in 48-72 h; no injury to astrocytes was noted. Electron microscopic findings of EGF-induced neuronal death were consistent with necrosis; severe mitochondrial swelling and disruption of cytoplasmic membrane occurred, whereas nuclei appeared relatively intact. The EGF-induced neuronal death was accompanied by increased free radical generation and blocked by the anti-oxidant Trolox. Suggesting mediation by the EGFR, an EGFR tyrosine kinase-specific inhibitor, C56, attenuated EGF-induced neuronal death. In addition, inhibitors of extracellular signal-regulated protein kinase 1/2 (Erk-1/2) (PD98056), protein kinase A (H89), and protein kinase C (GF109203X) blocked EGF-induced neuronal death. A p38 mitogen-activated protein kinase inhibitor (SB203580) or glutamate antagonists (MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione) showed no protective effect. The present results suggest that prolonged activation of the EGFR may trigger oxidative neuronal injury in central neurons.     

Haloperidol-induced neuronal apoptosis: role of p38 and c-Jun-NH(2)-terminal protein kinase

We examined patterns and mechanisms of cell death induced by haloperidol. Cortical cell cultures exposed to 10-100 microM: haloperidol for 24 h underwent neuronal death without injuring glia. The degenerating neurons showed hallmarks of apoptosis, featuring cell body shrinkage, nuclear chromatin condensation and aggregation, nuclear membrane disintegration with intact plasma membrane, and prominent internucleosomal DNA fragmentation. Neither glutamate antagonists nor antioxidants prevented the haloperidol-induced neuronal apoptosis. The c-Jun-NH(2)-terminal protein kinase and p38 mitogen-activated protein kinase were activated within 1 h and were sustained over the next 3 h following exposure of cortical neurons to 30 microM haloperidol. Haloperidol-induced neuronal apoptosis was partially attenuated by 10-30 microM PD169316, a selective inhibitor of p38 mitogen-activated protein kinase. Inclusion of 1 microg/ml cycloheximide, a protein synthesis inhibitor, or 100 ng/ml insulin prevented activation of both kinases and subsequent neuronal death. The present study demonstrates that cortical neurons exposed to haloperidol undergo apoptosis depending on activation of p38 mitogen-activated protein kinase and c-Jun-NH(2)-terminal protein kinase sensitive to cycloheximide and insulin.     

SIK2 is a key regulator for neuronal survival after ischemia via TORC1-CREB

The cAMP responsive element-binding protein (CREB) functions in a broad array of biological and pathophysiological processes. We found that salt-inducible kinase 2 (SIK2) was abundantly expressed in neurons and suppressed CREB-mediated gene expression after oxygen-glucose deprivation (OGD). OGD induced the degradation of SIK2 protein concomitantly with the dephosphorylation of the CREB-specific coactivator transducer of regulated CREB activity 1 (TORC1), resulting in the activation of CREB and its downstream gene targets. Ca(2+)/calmodulin-dependent protein kinase I/IV are capable of phosphorylating SIK2 at Thr484, resulting in SIK2 degradation in cortical neurons. Neuronal survival after OGD was significantly increased in neurons isolated from sik2(-/-) mice, and ischemic neuronal injury was significantly reduced in the brains of sik2(-)(/-) mice subjected to transient focal ischemia. These findings suggest that SIK2 plays critical roles in neuronal survival, is modulated by CaMK I/IV, and regulates CREB via TORC1.     

Oxygen or glucose deprivation-induced neuronal injury in cortical cell cultures is reduced by tetanus toxin.

We examined glutamate-mediated neurotoxicity in cortical cell cultures pretreated with 1-5 micrograms/ml tetanus toxin to attenuate the Ca(2+)-dependent release of neurotransmitters. Efficacy of the tetanus toxin pretreatment was suggested by blockade of electrical burst activity induced by Mg2+ removal and by reduction of glutamate efflux induced by high K+. Tetanus toxin reduced neuronal injury produced by brief exposure to elevated extracellular K+ or to glutamate, situations in which release of endogenous excitatory neurotransmitter is likely to play a role. Furthermore, although glutamate efflux evoked by anoxic conditions may occur largely via Ca(2+)-independent transport, tetanus toxin attenuated both glutamate efflux and neuronal injury following combined oxygen and glucose deprivation. With prolonged exposure periods, the neuroprotective efficacy of tetanus toxin was comparable to that of NMDA receptor antagonists. Presynaptic inhibition of Ca(2+)-dependent glutamate release may be a valuable approach to attenuating hypoxic-ischemic brain injury.     

利用基因芯片检测转基因作物

选用常用的两种报告基因、两种抗性基因、两种启动子序列和两种终止子序列为探针,将其PCR扩增产物用MicroGrid Ⅱ型全自动点样仪按矩阵排列点样于包埋有氨基的载玻片上,制备成转基因作物检测型基因芯片。利用该芯片对4种转基因水稻、木瓜、大豆、玉米进行检测,结果表明,该芯片能对转基因作物做出快速、准确的检测。 Abstract:Some selected available sequences of reporter genes,resistant genes,promoters and terminators are amplified by PCR for the probes of transgenic crop detection gene chip.These probes are arrayed at definite density and printed on the surface of amino-slides by bioRobot MicroGrid Ⅱ.Results showed that gene chip worked quickly and correctly,when transgenic rice,pawpaw,maize and soybean were applied.     

Neuroprotective Effect of Hypothermia in Cortical Cultures Exposed to Oxygen-Glucose Deprivation or Excitatory Amino Acids

Abstract: We examined the effect of moderate hypothermia (30°C) on neuronal injury in murine cortical cell cultures. Lowering the temperature during and after a period of oxygen-glucose deprivation reduced both the release of glutamate to the bathing medium and accompanying neuronal degeneration. Hypothermia immediately after brief exposure to high concentrations of NMDA or glutamate also reduced the resulting neuronal degeneration. This protective effect was not eliminated when MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione were added immediately after washout of the exogenously added excitotoxin, suggesting that it was mediated by actions additional to reduction of endogenous late glutamate release. Hypothermia applied only during exposure to NMDA or glutamate, whether brief or prolonged, did not reduce subsequent cytosolic calcium accumulation or neuronal degeneration, suggesting that the postsynaptic induction of NMDA receptor-mediated excitotoxicity is not sensitive to temperature reduction. However, hypothermia during prolonged S -α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or kainate exposure did reduce neuronal degeneration.