丙二醛破坏大鼠海马神经元胞质钙离子稳态的信号机制

目的研究丙二醛（MDA）对原代培养的海马神经元胞质中钙离子稳态的破坏作用及可能的信号机制。方法以Fur2／AM为荧光指示剂,采用荧光分光光度法定量测定原代培养海马神经元胞质游离钙浓度变化。结果随着MDA浓度的升高和作用时间的延长,导致胞质中游离钙水平显著升高,破坏其钙稳态。MDA所导致的海马神经元胞质游离钙水平升高包括两个过程：100μmol／L的MDA可使胞质[Ca2＋]i水平在0—10min内的早期渐进升高过程,经历中间大约5min的平台期后,接下来15—30min的晚期显著升高。以细胞膜电压依赖的Ca2＋通道抑制剂nimodipine抑制外钙内流后,可显著抑制晚期胞质[Ca2＋]i水平的升高,以PLC的抑制剂U73122作用后,则可抑制早期胞质[Ca2＋]i水平的升高。结论100μmol／L的MDA作用下,海马神经元胞质中早期钙离子水平的升高和晚期钙离子水平的升高可能分别由不同的信号机制所介导。     

整合素介导小鼠卵内钙离子增加

为了研究整合素是否作为跨膜信号传递受体介导小鼠卵[Ca^2 ]i的变化并探讨其机制。本实验采用甘-精-甘-天冬-丝-脯(GLY-ARG-GLY-ASP-SER-PRO,RGD肽)、纤连蛋A(fibronectin,Fn)及抗整合素α6、β1的单克隆抗体作用于负载了钙探针Fluo-3／AM的去透明带小鼠卵,用激光共聚焦显微镜检测小鼠卵的荧光强度以反映卵[Ca^2 ];用无钙液替代有钙液、或用酪氨酸激酶抑制剂或蛋白激酶C的抑制剂预先作用于卵,然后再观察RGD肽所致卵[Ca^2 ]i的变化。结果显示整合素配体RGD肽或Fn作用于去透明带小鼠卵可引起卵[Ca^2 ]i增加,增加的程度与精子作用相似;去除培养液中的Ca^2 后,再用RGD肽、Fn作用仍可引起卵[Ca^2 ]i增加：用功能性的抗小鼠整合素α6、β1的单克隆抗体也可引起不同程度的卵[Ca^2]i增加,尤其以抗小鼠整合素α6、β1单克隆抗体的作用明显;用酪氨酸激酶抑制剂预先作用于鼠卵,RGD肽或精子作用都不再引起卵[Ca^2 ]i增加;蛋白激酶C抑制剂预先作用鼠卵,RGD肽及Fn也不再引起卵[Ca^2 ]i增加。实验证明。小鼠卵膜整合素与其配体结合可使卵内贮存钙离子释放,引起卵[Ca^2 ]i增加这一卵激活的早期事件;整合素介导小鼠卵激活需要酪氨酸激酶信号转导途径的参与;蛋白激酶C也参与了整合素介导的卵激活。     

三疣梭子蟹精子顶体反应前后胞内Ca~(2+)的变化

应用激光扫描共聚焦显微镜(LSCM)和Fluo-3/AM荧染技术对三疣梭子蟹精子顶体反应前后的胞内Ca2 变化进行了观察和检测.结果显示,在精子顶体反应过程中,胞内Ca2 主要分布在细胞核、穿孔器和胞质膜残存处,胞内Ca2 浓度([Ca2 ]I)总体上呈现先上升后下降的趋势.顶体反应前精子的平均荧光强度为35.95±5.71;穿孔器前伸、顶体囊膜翻转阶段精子的平均荧光强度为66.80±7.35;顶体囊膜脱落、顶体丝形成阶段精子的平均荧光强度为3.87±2.82;上述各阶段间精子荧光强度有极显著差异(P<0.01).顶体反应穿孔器前伸、顶体囊膜翻转阶段的精子相比顶体反应前精子,[Ca2 ]I显著提高;而在顶体囊膜脱落、顶体丝形成阶段,[Ca2 ]I则急剧下降,只在顶体丝基部胞质膜残存处有微量Ca2 存在.初步探讨了三疣梭子蟹精子顶体反应前后胞内Ca2 变化的功能.     

精子与极核融合是受精吗

单倍体的精子 (雄配子 )和卵细胞 (雌配子 )融合成一个二倍体的受精卵的过程叫受精 (fertilization) ,为有性生殖的基本部分和一个重要特征 ,受精卵进一步发育成 1个新个体。被子植物的受精作用过程中 ,两个精子进入胚囊后 ,1个精子与卵细胞融合形成受精卵并发育成胚 ,另 1个精子与极核 (或中央细胞 )融合形成三倍体的初生胚乳核而形成胚乳 ,这种现象叫做双受精 (dou-ble fertilization) ,是被子植物特有的一种受精现象 ,为俄国生物学家纳瓦申 (Nawaschin)在 1898年发现的。双受精现象也会在一些裸子植物 (麻黄属 )中见到 ,此属植物的 1个…     

pH改变对心肌细胞内Ca~(2 )浓度和细胞长度的影响

目的 :探讨细胞内 pH(pHi)改变对心肌细胞内Ca2 浓度 ([Ca2 ]i)和细胞长度的影响。方法 :心肌细胞内分别灌注 2 0mmol/L丙酸钠和 15mmol/LNH4Cl,建立细胞内酸碱中毒模型。荧光指示剂indo 1和SNARF 1载入大鼠心肌细胞内 ,用荧光显微镜同时测定心肌 [Ca2 ]i、pHi 和细胞长度。结果 :细胞内酸中毒早期 ,收缩期和舒张期[Ca2 ]i 轻度增加 ,细胞缩短 (CS)降低 ,细胞长度增加 ,心肌纤维对Ca2 的敏感性和CS/ [Ca2 ]i 降低 (P <0 .0 1) ;碱中毒时 ,收缩期和舒张期 [Ca2 ]i 均较对照组降低 ,CS增加 ,细胞长度变短 ,心肌纤维对Ca2 的敏感性和CS/[Ca2 ]i 增加 (P <0 .0 1)。结论 :酸中毒早期 [Ca2 ]i 和细胞长度增加 ,碱中毒时 [Ca2 ]i和细胞长度降低。酸、碱中毒对Ca2 敏感性的影响并非线性关系 ,即单位 pHi变化时酸中毒对敏感性的影响较碱中毒小     

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。     

柴胡皂甙(I)对胰腺腺泡的拟膜受体激动剂作用

应用检测淀粉酶分泌和单细胞[Ca2 ]i 的技术 ,研究了Bt2-cGMP和GDP对柴胡皂甙(I)[SA(I)]和CCK -8促大鼠胰腺腺泡酶分泌和增加[Ca2 ]i 的抑制作用。Bt2-cGMP对SA(I)和CCK -8促酶分泌的抑制有相似的剂量依赖性。Bt2 -cGMP对SA(I)刺激的酶分泌动力学的抑制较对CCK -8滞后并持续。SA(I)诱发的胰腺腺泡单细胞[Ca2 ]i 的变化与CCK -8的作用有所不同 :[Ca2 ]i 峰值上升较慢且持续较长 ,并在峰后[Ca2 ]i 再次升高。GDP亦抑制SA(I)刺激的酶分泌和[Ca2 ]i 增加的峰植。结果表明 ,SA(I)可激活胰腺腺泡细胞膜受体从而升高[Ca2 ]i 和促酶分泌。     

钙信号在卵母细胞激活中的作用

钙信号是胞内主要的第二信使之一,发挥广泛的作用如细胞分裂、细胞凋亡等,对细胞的生命活动起着非常重要的作用。在精子和卵母细胞中,钙信号对精子获能、顶体反应、卵母细胞成熟、受精及卵裂等一系列复杂的过程有非常重要的影响。现就Ca2 在卵母细胞中的释放机制、信号转导途径、调控功能作一综述。     

白色霞水母生活史的实验室观察

本文首次描述了白色霞水母从受精卵至碟状体的生活史。(1)包括受精卵、卵裂、囊胚以及浮浪幼虫等在内的胚胎发育各期均在开放的水体中,在20·8 -21·4℃浮浪幼虫于受精后14 h出现; (2)浮浪幼虫在定置前形成一种凸面的圆形浮浪幼体囊,除了浮浪体囊外,螅状体还可产生足囊和通过产生匍匐茎形成囊胞进而发育成新的螅状体; (3)尽管偶而产生2个碟状体但仍为典型的单碟型横裂; (4)新释放的碟状幼体绝大多数为8个缘叶, 8个感觉棍和8对钝圆的缘瓣,但畸形个体最多12个,最少6个缘叶; (5)雌雄个体间的交互作用对产卵和受精是非常重要的因子[动物学报52 (2) : 389 -395 , 2006]。     

异源四倍体鲫鲤的受精细胞学

异源四倍体鲫鲤的成熟卵子处于第二次减数分裂中期,精子通过受精孔进入卵内.精子入卵以后,受精孔立即被受精塞堵住.受精后8 min,受精卵出现明显的精子星光,同时进入第二次减数分裂后期,即将排出第二极体;13 min时,精子头部开始膨胀,趋向核化;18 min时,雌雄原核均已形成,并向胚盘中央靠近;23 min时,雌、雄原核开始接触;33 min时,雌、雄原核完全融合成为一个合子核;38 min时,受精卵开始第一次卵裂,53 min后分裂形成两个子核.该研究证明异源四倍体鲫鲤和大多数二倍体鱼一样,具有正常的受精细胞学程序,受精方式为单精受精.     

Imaging [Ca2+]i dynamics during signal transduction

The elevation of free intracellular Ca2+ activity ([Ca2+]i) is widely recognised as a central event in many signal transduction processes in cellular physiology. Recent advances in optical techniques for measuring [Ca2+]i as well as developments in quantitative low light level fluorescence microscopy have led to the application of these methods to the study of subcellular [Ca2+]i in many biological systems. In the following paper we describe some techniques in our laboratory to provide quantitative high spatio-temporal resolution measurements of [Ca2+]i in individual living cells during the signal transduction of cell surface receptor ligand interactions. In particular, we are studying the changes in [Ca2+]i induced by the micro-aggregation of immunoglobulin E (IgE) receptor complexes on the surface of rat basophilic leukemia (RBL) cells (a tumor mast cell line) by multivalent antigen. We seek to understand the mechanisms which are involved in the detection of these cell surface events which lead to changes in [Ca2+]i as well as the interactions between the various subcellular components which impart the delicate control of [Ca2+]i during cellular stimulation. The limitations and properties of the technology used for these studies will be discussed, and some illustrative examples of the type of [Ca2+]i changes found in this biological system will be given.     

谷氨酸促进大鼠海马神经元的内钙升高

谷氨酸能影响大鼠海马神经元胞内钙信号的变化,进而影响海马神经元神经冲动的发放和学习记忆过程。运用荧光测钙技术实时监测了大鼠海马神经元内钙信号的动态变化,同时分析了谷氨酸对其胞内钙信号的影响。试验表明：谷氨酸能够显著提高胞内游离钙离子的浓度;细胞外钙离子的存在、谷氨酸刺激时间及刺激频率的增加都能引起胞内钙信号不同程度的升高;但谷氨酸的过度刺激会引起钙离子浓度的超负荷,从而导致神经元结构和功能的损坏。     

An insulin-sensitive cation channel controls [Na+]i via [Ca2+]o-regulated Na+ and Ca2+ entry.

The insulin-stimulated cation channel previously identified in patch-clamped muscle preparations is here shown to be responsible for bulk Na+ entry into the cell. The mainly Na+ current of the channel was shown to be accompanied by an inhibitory Ca2+ component responsible for oscillations. Here, using quantitative fluorescence imaging of Fura-2- and SBFI-loaded soleus muscle, we measure changes in [Na+]i and [Ca2+]i related to channel function. Insulin increased [Na+]i and [Ca+]i in a transient spike of < 1-min duration. There was a momentary dip in [Na+]i related to inhibition of the channel by the Ca2+ spike, and changes in external Ca2+ were shown to alter [Na+]i via the cation channel, all effects being blocked by the specific channel inhibitor mu-conotoxin, but not by tetrodotoxin. The [Ca2+]i spike could also be induced by 8-bromo cyclic-guanosine 5'-monophosphate, an analogue of the channel-activator cyclic-guanosine 5'-monophosphate (cGMP). In addition it was noted that insulin reduced the [Ca2+]i rise upon subsequent muscle depolarization by a factor of 3.5. Insulin could be substituted with phorbol ester for the same effect and HA1004, a protein kinase inhibitor, blocked the reduction.     

Depolarization decreases the [Ca2+]i sensitivity of myosin light-chain kinase in arterial smooth muscle: comparison of aequorin and fura 2 [Ca2+]i estimates

Histamine stimulation of swine arterial smooth muscle is associated with a high [Ca2+]i sensitivity for increases in myosin light-chain phosphorylation. In contrast, KCl depolarization produces a relatively lower [Ca2+]i sensitivity (i.e., similar increases in [Ca2+]i induce less myosin phosphorylation). We evaluated whether 1) artifacts in the methodology for measuring [Ca2+]i or 2) true alterations in the [Ca2+]i sensitivity of myosin light-chain kinase were responsible for these apparent changes in the [Ca2+]i sensitivity of phosphorylation. The [Ca2+]i sensitivity of phosphorylation was higher with histamine stimulation regardless of whether the [Ca2+]i indicator was aequorin (which was loaded intracellularly by reversible hyperpermeabilization) or Fura 2 (which was loaded intracellularly by incubation of the tissues in Fura 2 AM). Aequorin and Fura 2 appeared to detect qualitatively similar stimulus-induced changes in [Ca2+]i with the exception that the initial response to histamine stimulation was different (histamine initially induced a large aequorin light transient and a relatively smaller increase in Fura 2 fluorescence). The [Ca2+]i sensitivity of myosin light-chain kinase extracted from KCl depolarized tissues was lower than the [Ca2+]i sensitivity of myosin light-chain kinase extracted from unstimulated or histamine stimulated tissues. These results suggest that depolarization specifically modifies myosin light-chain kinase to decrease its [Ca2+]i sensitivity. Changes in the [Ca2+]i sensitivity of myosin light-chain phosphorylation are not an artifact of the [Ca2+]i measurement technique.