丙二醛对大鼠海马神经元结构的破坏和钙离子稳态的影响

脂质过氧化中间产物丙二醛(Malondialdehyde,MDA)在生物体内表现了广泛的生物毒性,MDA也是机体过度训练和运动性疲劳的重要生理指标.采用光学显微镜和透射式电子显微镜观察不同浓度MDA作用后海马神经元形态和超微结构的变化,并采用荧光分光光度法测定原代培养的海马神经元中Ca2+-ATPase活性的变化和胞质游离钙离子水平的变化,探讨MDA对海马神经元形态和结构上的破坏及神经元钙离子稳态的影响.在光镜下可观察到MDA作用下神经元突触变短,胞体肿胀,出现细胞死亡或凋亡的形态特征;在电镜下可观察到线粒体结构的明显破坏,内膜上的嵴颗粒减少或消失;同时MDA还通过抑制质膜Ca2+-ATPase的活性和其它的途径,破坏神经元胞质游离Ca2+稳态.结果表明,MDA可通过破坏海马神经元的结构和影响胞质中钙离子稳态,破坏神经元的生理功能,在机体运动性中枢疲劳形成中可能发挥重要作用.     

持续癫痫样放电导致海马神经元钙离子动力学变化的研究

目的：研究低镁介质致痫的培养海马神经元癫痫模型中神经元内游离钙离子（[Ca^2＋]i）的时空分布及其动力学改变,以探讨钙离子在癫痫发病过程中的作用。方法：联合应用共聚焦激光扫描显微镜和膜片钳,运用较高时间分辨率动态观察培养海马神经元癫痫模型[Ca^2＋]i和电生理变化,以及化学门控钙离子通道阻滞剂的影响。结果：致痫后海马神经元胞浆和核内游离钙离子迅速上升到（612±65）nmol／L和（620±69）nmol／L水平,NMDA受体阻断剂MK-801（10μmol／L）和非NMDA受体阻断剂NBQX（10μmol／L）可使[Ca^2＋]i的升高明显减少;升高的[Ca^2＋]i恢复有明显的延迟现象,90min和150min癫痫样放电后[Ca^2＋]i恢复的时间分别为（114．8±5．2）和（135．0±22．7）（P〈0．05）。结论：持续的癫痫样放电可导致海马神经元细胞内钙超载,这个效应可被MK-801阻断,化学门控钙离子通道也参与了细胞外Ca^2＋内流的过程。     

CD226单克隆抗体诱导人脐静脉内皮细胞胞质钙离子浓度的变化

为观察CD226单克隆抗体(mAb)对培养人脐静脉内皮细胞(HUVECs)胞质钙离子变化的影响,我们用Fluo-3作为钙指示剂,用激光共聚焦显微镜观测不同状态下CD226 mAb作用后HUVECs胞质钙离子[Ca2 ]i的变化。结果发现：(1)用Hanks液平衡,CD226 mAb作用后HUVECs[Ca2 ]i水平缓慢升高后回到原位;加入二抗(羊抗鼠IgG)交联后[Ca2 ]i水平有较大幅度的升高,随后回到原位,与此同时,细胞外液中[Ca2 ]。水平有一定程度的下降;(2)用D-Hanks液平衡,CD226 mAb作用后HUVECs[Ca2 ]i水平无显著变化,加入二抗发生交联作用后,[Ca2 ]：水平有较大幅度的下降;(3)用EGTA预处理后,CD226 mAb及其二抗交联对HUVECs[Ca2 ]i变化无显著影响。以上结果提示,CD226mAb及其二抗交联可诱导不同状态的HUVECs胞质钙离子水平发生不同程度的变化,从而参与一系列的生理和病理过程。     

吗啡对培养海马神经元钙离子作用的机制研究

目的：研究吗啡对海马神经元[Ca^2 ]i影响的机制,为探索吗啡成瘾的神经生物学机制与可能的治疗途径。方法：荧光探针Fluo-4标记细胞内游离钙后,用激光共聚焦显微镜检测吗啡对大鼠原代培养海马神经元[Ca^2 ]i的影响。结果：吗啡急性刺激引起海马神经元[Ca^2 ]i升高,CTOP不能阻断吗啡引起的细胞内[Ca^2 ]i增加,而naltrindole能阻断吗啡引起的细胞内[Ca^2 ]i反应;Thapsigargin预处理阻断吗啡诱导的细胞内[Ca^2 ]i增加,Verapamil预处理不能完全抑制吗啡引起的细胞内[Ca^2 ]i增加;吗啡长时程作用后,海马神经元[Ca^2 ]i升高,加入纳络酮急性戒断后,不能阻断吗啡引起的细胞内[Ca^2 ]i升高,反而引起[Ca^2 ]i异常升高。结论：吗啡急性刺激引起的海马神经元内游离钙增加主要来源于δ2阿片受体介导的IP3敏感的钙库释放。     

电磁辐射对原代培养海马神经元的损伤效应及其机制探讨

研究X带高功率微波、S带高功率微波及电磁脉冲辐射对原代培养海马神经元的损伤效应并探讨其机制。通过体外培养原代海马神经元,建立电磁波辐照细胞模型。采用Annexin V-PI双标记、流式细胞术检测细胞凋亡与坏死,原子力显微镜检测细胞膜表面形态,Fluo-3-AM荧光探针负载、激光扫描共聚焦显微镜测定胞内[Ca2 ]i。结果表明,辐射后海马神经元凋亡与坏死均增加,其中坏死增加明显;细胞膜表面粗糙度加大,膜穿孔增多;胞内[Ca2 ]i明显升高。且以上变化均以X带高功率微波组最重,S带高功率微波组次之,电磁脉冲组最轻。提示细胞膜穿孔增多,膜通透性增加,导致胞外Ca2 内流增加,甚至胞内钙超载是辐射致海马神经元凋亡与坏死的机制之一;三种电磁辐射对海马神经元的损伤程度与照射频率呈正相关。     

D-半乳糖诱导大鼠脑损伤的糖基化机制

目的D-半乳糖（D-galactose）诱导大鼠体内不同糖基化水平,研究其脑损伤发生的机理。方法采用不同剂量D-半乳糖[150、75、37．5mg／（kg·d）]分别腹腔注射（ip）处理大鼠8周,诱导糖基化状态和脑损伤。采用硫代巴比妥酸（TBA）比色法测定糖化血红蛋白,硝基四氮唑蓝（NBT）比色法测定血清果糖胺;按文献方法分别测定血红细胞醛糖还原酶活性和晚期糖基化终末产物（AGEs）含量及脑组织中AGEs含量,羟胺法和比色法分别测定SOD和GSH-Px活性,硫代巴比妥酸法测定MDA含量;以Fura-2／AM作为钙荧光指示剂,双波长荧光分光光度法检测脑海马神经细胞胞质[Ca^2＋]i的变化;透射电镜观察脑海马神经细胞线粒体的变化。结果D-半乳糖处理8周后,大鼠血红细胞醛糖还原酶活性升高,糖化产物形成增多;脑组织中AGEs及脑细胞胞质[Ca^2＋]i含量明显升高,SOD及GSH-Px活性下降,MDA含量升高（P〈0．01,P〈0．05）,海马神经细胞线粒体出现病理性改变。结论D-半乳糖通过诱导体内蛋白糖基化和脑组织AGEs大量生成,降低抗氧化能力及胞质[Ca^2＋]i超负荷等,导致脑细胞损伤。     

谷氨酸、NMDA 和吗啡对培养大鼠星形胶质细胞内钙振荡的影响

目的:研究谷氨酸、NMDA、吗啡对原代培养的大鼠星形胶质细胞的胞内钙信号的影响及受体作用机制.方法:利用Leica AF6000活细胞工作站,检测谷氨酸、NMDA、吗啡分别灌流前后Fura-2/AM加载的星形胶质细胞内钙信号的动态变化,进一步观 察分别阻断代谢性谷氨酸受体5 (mGluR5)、NMDA受体(NMDA receptor,NMDAR)和阿片μ受体对诱导的胞内钙振荡的影响.结果:谷氨酸、NMDA、吗啡均可明显升高胞内游离钙的浓度([Ca2+]i),而将其相应受体拮抗后,星形胶质细胞[Ca2+]i升高的现象可以被显著抑制.结论:离体培养的星形胶质细胞膜上存在mGluR5、NMDAR和阿片μ受体,这些受体的激活可以升高星影胶质细胞的[Ca2+]i,且这些受体依赖的[Ca2+]i的调控机制可能是星形胶质细胞与神经元交互作用的重要途径之一.     

牛磺酸对大鼠脑神经细胞内钙稳态的影响

目的和方法 :利用激光扫描共聚焦显微镜和双波长荧光分光光度计 ,分别观察牛磺酸对无血清培养的单个海马神经细胞和分散的新生大鼠脑神经细胞内Ca2 浓度 ([Ca2 ]i)的影响 ,并探讨牛磺酸调节神经细胞内钙稳态的作用机理。结果 :牛磺酸主要通过刺激细胞内钙库释放 ,在一定剂量范围内 (0 .0 2～ 6 .4mmol/L)使神经细胞[Ca2 ]i 轻微升高 ,在 0 .4mmol/L时的升钙作用最大 (升钙百分率为 12 .2 0 %± 1.2 4% )。在测定介质中加入钙离子载体A2 3187(10 μmol/L) ,使神经细胞内的钙离子浓度升高 ,若此时加入牛磺酸 (1.6mmol/L) ,则神经细胞内钙离子的浓度下降。结论 :牛磺酸对细胞内钙离子有双向调节作用 ,牛磺酸可能是通过对钙稳态的调节作用来阻止细胞内钙超载 ,保护神经细胞、并发挥其增强动物学习记忆等方面功能的。     

视黄酸对海马神经元细胞内钙离子浓度的影响

探讨全反式视黄酸(all-trans retinoic acid,atRA)对原代培养的海马神经元胞内钙离子浓度的影响,以进一步了解atRA参与学习记忆可能机制。分离新生Wistar大鼠海马,采用添加B27的无血清培养液进行海马神经元原代培养,免疫荧光鉴定培养的神经细胞;以fura-2/AM温育海马神经元,采用钙离子测定系统动态观察视黄酸对海马细胞内钙离子浓度的影响。结果显示:(1)培养的神经元纯度达90%;(2)atRA作用于海马神经元,能引起海马神经元胞内钙离子浓度的升高;(3)这种升高与atRA浓度及神经元的发育时间相关;(4)钙离子升高的具体方式是通过细胞外钙离子内流;(5)视黄酸核受体alpha(RARα)的拮抗剂Ro41-5253(Ro)对atRA升高的神经元胞内钙离子浓度有抑制作用。atRA与RARα结合,促使海马神经元胞外钙离子的内流,这可能是atRA参与学习记忆的机制之一。     

17β-雌二醇快速诱导静止状态小鼠囊胚细胞胞内钙离子增加

用钙离子荧光探针fluo-3/AM标记囊胚细胞内钙离子,用激光共聚焦扫描显微镜连续检测17β-雌二醇(17β-E2)作用所致囊胚胞内钙离子浓度的动态变化,用荧光显微镜检测偶联牛血清白蛋白的17β-雌二醇(E2-BSA)所致囊胚胞内钙离子浓度的改变,以及在去钙镁M2液、传统雌激素受体阻断剂tamoxifen和磷脂酶C特异抑制剂U73122作用下17β-E2所致囊胚细胞内钙离子浓度的改变。结果显示:17β-E2和E2-BSA均可引起静止状态囊胚细胞内[Ca2 ]的快速升高;17β-E2诱导的囊胚细胞内[Ca2 ]的快速升高不依赖于胞外钙离子的内流,且不被传统雌激素受体阻断剂所阻断,而磷脂酶C特异性抑制剂可明显抑制该效应。     

适当浓度的过氧化氢诱导人支气管上皮细胞发生钙振荡

包括过氧化氢（Hzoz）在内的活性氧通过引起细胞内钙的变化而造成细胞损伤。然而,不同浓度的H202可以导致细胞内不同的钙变化,并激活不同的信号通路。细胞内钙振荡是其中的一种钙信号变化形式,钙振荡可以调控转录因子NF—KB的活性。该研究探讨可以诱导支气管上皮细胞内钙振苏发生的H2o2浓度。体外培养人支气管上皮细胞,采取钙离子荧光探针Fura_2标记细胞。并使用离子成像系统,观测不同浓度的H：0：（0～1000μmol／L）作用下细胞内钙浓度的变化。结果发现,低于50μmol／L的H202仅仅引起“钙火花”;50～500μmol／L的H202导致细胞内钙振荡的发生;而1000μmol／L的H202引起细胞内持续的高钙;同时也证实150μmol／L的H202诱发明显的钙振荡,而钙振荡随后引起了NF—KB活性的升高。该研究提示,适当浓度的H：0：可以诱发支气管上皮细胞内钙振荡的发生,推测可能是活性氧导致慢性气道炎症损伤的一个机制。     

VEGF165对血管内皮细胞内Mg^2＋浓度调节机制的研究

目的：探讨血管内皮生长因子165（VEGF165）对人脐带静脉内皮细胞（HLNECs）内游离镁离子浓度（[Mg^2＋]i）的调节机制.方法：采用荧光指示剂mag-fura-2及运用PTi阳离子测定系统动态检测HUVECs的[Mg^2＋].结果：经酪氨酸激酶阻断剂（tryrphostin A23和genistein）,磷脂酰3激酶阻断剂（wortmannin和LY294002）,磷脂酶Cγ阻断剂（U73122）预处理,均显著阻断VEGF165诱导的[Mg^2＋]i增加.但经磷脂酶C阻断剂无活性的类似物（U73343）和增殖激活蛋白激酶阻断剂（SB202190和PD9S059）预处理,不能阻断VEGF165诱导的[Mg^2＋]i增加：结论：VEGF165通过酪氨酸激酶／磷脂酰3激酶／磷脂酶口信号转导途径使细胞内的Mg^2＋库释和Mg^2＋,从而增加HUVECs的[Mg^2＋]i.     

蝙蝠葛碱拮抗缓激肽引起的钙稳态改变 及细胞骨架蛋白的异常磷酸

为研究蝙蝠葛碱 (dauricine , Dau) 拮抗缓激肽 (bradykinin , BK) 诱导的 Alzheimer 样钙稳态失衡及细胞骨架蛋白异常磷酸化的作用,采用双波长荧光分光光度计测定细胞内钙离子浓度 ([Ca2+] i) ,用 MTT 法检测细胞代谢水平,用免疫组织化学方法观察 tau 蛋白表达和磷酸化 . 结果表明,Dau (3 μmol/L , 6 μmol/L) 可抑制 BK 诱导的 [Ca2+]i 升高,保护 BK 引起的神经元代谢降低,拮抗 BK 引起的 tau 蛋白异常磷酸化和聚集 . 结果提示： Dau 可拮抗 BK 诱导的 Alzheimer 样钙稳态失衡及细胞骨架蛋白异常磷酸化的作用 .     

A myristoylated pseudosubstrate peptide of PKC-zeta induces degranulation in HMC-1 cells independently of PKC-zeta activity

Mast cells play a central role in allergic disease and host defense against several pathogens through the release of various bioactive compounds via degranulation. In this study, we found that a myristoylated pseudosubstrate of PKC-zeta (zeta-PS; myristoyl-SIYRRGARRWRKL, a PKC-zeta inhibitor) regulates mast cell degranulation. zeta-PS increased [Ca+2]i level at nanomolar concentrations in a PKC-zeta activity-independent manner in HMC-1 cells. Moreover, zeta-PS-induced [Ca+2]i generation was completely abrogated by phospholipase C (PLC), IP3 receptor or Galpha i/o inhibitor and zeta-PS potently induced degranulation in HMC-1 cells which was significantly inhibited by pretreating PLC inhibitors or a calcium chelator. Therefore, our results suggest that zeta-PS can induce degranulation in HMC-1 cells by triggering the calcium signal via a PKC-zeta-independent but Galpha i/o, PLC and IP3-dependent pathways.     

AT1 angiotensin II receptor mediates intracellular calcium mobilization in normal and cancerous breast cells in primary culture

Angiotensin II (Ang II) increases intracellular calcium concentration ([Ca2+]i) in both normal and cancerous human breast cells in primary culture. Maximal [Ca2+]i increase is obtained using 100nM Ang II in both cell types; in cancerous breast cells, [Ca2+]i increase (delta[Ca2+]i) is 135+/-10nM, while in normal breast cells it reaches 65+/-5 nM (P<0.0001). In both cell types, Ang II evokes a Ca2+ transient peak mediated by thapsigargin (TG) sensitive stores; neither Ca2+ entry through L-type membrane channels or capacitative Ca2+ entry are involved. Type I Ang II receptor subtype (AT1) mediates Ang II-dependent [Ca2+]i increase, since losartan, an AT1 inhibitor, blunted [Ca2+]i increase induced by Ang II in a dose-dependent manner, while CGP 4221A, an AT2 inhibitor, does not. Phospholipase C (PLC) is involved in this signaling mechanism, as U73122, a PLC inhibitor, decreases Ang II-dependent [Ca2+]i transient peak in a dose-dependent mode.Thus, the present study provides new information about Ca2+ signaling pathways mediated through AT1 in breast cells in which no data were yet available.     

硫酸酯化马尾松花粉多糖对小鼠脾脏B淋巴细胞免疫调节作用的研究

研究60％乙醇提取的马尾松花粉多糖组分D（PPM60-D）及其硫酸酯化物（SPPM60-D）对小鼠脾脏B淋巴细胞增殖、细胞内游离钙离子浓度（[Ca2＋]i）及抗体生成的影响。水煮醇沉法提取得到粗多糖,乙醇分级沉淀得到60％乙醇沉淀多糖PPM60,SephacrylS-400HR分离纯化得到多糖组分D,用氯磺酸-吡啶法对组分D进行硫酸酯化,尼龙毛法分离B淋巴细胞,MTT法测定其增殖,荧光分光光度计测定B淋巴细胞[Ca2＋]i,溶血空斑实验（PFC）和定量溶血分光光度（QHs）法测定B细胞抗体生成情况。结果显示,SPPM60-D相对于PPM60-D能更显著地提高B淋巴细胞的增殖以及[Ca2＋]i（P〈0．011：经TAK-242、LY294002、U73122、低分子肝素、维拉帕米和2-APB抑制剂作用后,均可抑制SPPM60-D和PPM60-D所致的[Ca2＋]i）升高（P〈0．05或P〈0．01）;PFC和QHS检测证实,SPPM60-D对于促进B淋巴细胞的分化及抗体的生成有显著作用,而PPM60-D的作用较弱。以上研究表明,PPM60-D经过硫酸酯化改性后,活性明显提高,推测SPPM60-D可与B淋巴细胞上TOLL样受体4（TLR4）结合,通过TLR4-P13K-PLC—IP3R信号通路使钙库释放激活的钙通道（CRAC）打开,从而使[Ca2＋]i）升高来激活B淋巴细胞,进而提高其体外增殖和抗体生成能力。     

Protease-activated receptor-1-induced calcium signaling in gingival fibroblasts is mediated by sarcoplasmic reticulum calcium release and extracellular calcium influx

Thrombin is a serine protease activated during injury and inflammation. Thrombin and other proteases generated by periodontal pathogens affect the behavior of periodontal cells via activation of protease-activated receptors (PARs). We noted that thrombin and PAR-1 agonist peptide stimulated intracellular calcium levels ([Ca2+]i) of gingival fibroblasts (GF). This increase of [Ca2+]i was inhibited by EGTA and verapamil. U73122 and neomycin inhibited thrombin- and PAR-1-induced [Ca2+]i. Furthermore, 2-APB (75-100 microM, inositol triphosphate [IP3] receptor antagonist), thapsigargin (1 microM), SKF-96365 (200 microM) and W7 (50 and 100 microM) also suppressed the PAR-1- and thrombin-induced [Ca2+]i. However, H7 (100, 200 microM) and ryanodine showed little effects. Blocking Ca2+ efflux from mitochondria by CGP37157 (50, 100 microM) inhibited both thrombin- and PAR-1-induced [Ca2+]i. Thrombin induced the IP3 production of GF within 30-seconds of exposure, which was inhibited by U73122. These results indicate that mitochondrial calcium efflux and calcium-calmodulin pathways are related to thrombin and PAR-1 induced [Ca2+]i in GF. Thrombin-induced [Ca2+]i of GF is mainly due to PAR-1 activation, extracellular calcium influx via L-type calcium channel, PLC activation, then IP3 binding to IP3 receptor in sarcoplasmic reticulum, which leads to intracellular calcium release and subsequently alters cell membrane capacitative calcium entry.     

Neural cell adhesion molecule induces intracellular signaling via multiple mechanisms of Ca2+ homeostasis

The neural cell adhesion molecule (NCAM) plays a pivotal role in the development of the nervous system, promoting neuronal differentiation via homophilic (NCAM-NCAM) as well as heterophilic (NCAM-fibroblast growth factor receptor [FGFR]) interactions. NCAM-induced intracellular signaling has been shown to affect and be dependent on the cytoplasmic Ca2+ concentration ([Ca2+]i). However, the molecular basis of this remains unclear. In this study, we determined [Ca2+]i regulating mechanisms involved in intracellular signaling induced by NCAM. To mimic the effect of homophilic NCAM interaction on [Ca2+]i in vitro, we used a peptide derived from a homophilic binding site of NCAM, termed P2, which triggers signaling cascades similar to those activated by NCAM-NCAM interaction. We found that P2 increased [Ca2+]i in primary hippocampal neurons. This effect depended on two signaling pathways. The first pathway was associated with activation of FGFR, phospholipase Cgamma, and production of diacylglycerol, and the second pathway involved Src-family kinases. Moreover, NCAM-mediated Ca2+ entry required activation of nonselective cation and T-type voltage-gated Ca2+ channels. These channels, together with the Src-family kinases, were also involved in neuritogenesis induced by physiological, homophilic NCAM interactions. Thus, unanticipated mechanisms of Ca2+ homeostasis are shown to be activated by NCAM and to contribute to neuronal differentiation.     

Sphingosine kinase-mediated calcium signaling by muscarinic acetylcholine receptors

Based on the finding that G protein-coupled receptors (GPCRs) can induce Ca2+ mobilization, apparently independent of the phospholipase C (PLC)/inositol-1,4,5-trisphosphate (IP3) pathway, we investigated whether sphingosine kinase, which generates sphingosine-1-phosphate (SPP), is involved in calcium signaling by mAChR and other GPCRs. Inhibition of sphingosine kinase by DL-threo-dihydrosphingosine and N,/N-dimethylsphingosine markedly inhibited [Ca2+]i increases elicited by M2 and M3 mAChRs in HEK-293 cells without affecting PLC activation. Activation of M2 and M3 mAChR rapidly and transiently stimulated production of SPP. Furthermore, microinjection of SPP into HEK-293 cells induced rapid and transient Ca2+ mobilization. Pretreatment of HEK-293 cells with the calcium chelator BAPTA/AM fully blocked mAChR-induced SPP production. On the other hand, incubation of HEK-293 cells with calcium ionophores activated SPP production. Similar findings were obtained for formyl peptide and P2Y2 purinergic receptors in HL-60 cells. On the basis of these studies we propose, that following initial IP3 production by receptor-mediated PLC activation, a local discrete increase in [Ca2+]i induces sphingosine kinase stimulation, which ultimately leads to full calcium mobilization. Thus, sphingosine kinase activation most likely represents an amplification system for calcium signaling by mAChRs and other GPCRs.     

氨对脑细胞胞浆游离钙含量的影响

目的与方法：采用Ｆｕｒａ－２／ＡＭ探针技术观察ＮＨ４Ｃｌ对离体急性分离之Ｗｉｓｔａｒ乳鼠大脑细胞胞头游离钙「Ｃａ＾２＋」ｉ含量的影响。结果：ＮＨ４＾＋浓度为２．５ｍｍｏｌ／Ｌ时脑细胞内「Ｃａ＾２＋」ｉ含量升高。在一定范围内,随着ＮＨ４＾＋浓度的加大,细胞内Ｃａ＾２＋持续升高。ＮＨ４＾＋的升钙作用主要被Ｎｉｃａｒｄｉｐｉｎｅ所阴断,其变化特征类以ＫＣｌ。结论：ＮＨ４＾＋主要通过影响电压依赖性钙离子通