【作者】 王颖；

【导师】 高天明；

【作者基本信息】 中国人民解放军第一军医大学 ， 神经生物学， 2003， 硕士

【摘要】 目的：探讨离子通道在培养大鼠海马神经元凋亡性容积减少（AVD）中的作用。 方法：用星形孢菌素（Staurosporine,STS）诱导的培养海马神经元凋亡模型，检测STS作用后神经细胞容积12h内的动态改变和12h时的细胞存活率，分别加入不同的钾通道和氯通道阻断剂，观察它们对细胞容积减少和凋亡的保护作用，细胞存活率的检测用细胞计数和MTT两种方法。用全细胞膜片钳技术观察培养海马神经元STS作用前后氯离子通道活动的变化。 结果：STS（2μM）持续作用12h，活细胞数是加药前的39.04％，TUNEL染色证明死亡的细胞多为凋亡。经STS作用后2h，培养海马神经元即呈现容积明显减少，并且随时间延长，容积递减，12h后容积减少到原来的78.1％。细胞外高K⁺（25mM）能够明显抑制STS诱导的神经元容积减少，12h细胞存活率也升高到74.53％；广谱钾通道阻断剂TEA完全阻断了STS诱导的细胞容积减少，并且将12h细胞存活率提高到65.96％；BK通道阻断剂IBTX和paxilline分别使12h细胞存活率升高到63.92％和64.32％；而SK_Ca通道阻断剂apamin（1μM）和A型钾通道阻断剂4-AP（500μM）没有发现其保护作用。 使用氯通道阻断剂DIDS（0.5mM）也可以抑制海马神经元的AVD，并且12h后细胞存活率明显升高，达到68.4%;sITS（DIDS同类氯通道阻断剂，0.smM）对神经元凋亡的保护作用结果与DIDS的结果一致。用MTT法检测细胞存活率，单独使用钾通道阻断剂TEA（5 mM），细胞存活率为66.0%，单独使用氯通道阻断剂DIDS，细胞存活率为83 .7%，同时使用TEA和DIDS，细胞存活率为115%。 用全细胞膜片钳技术观察STS诱导培养海马神经元前后氯离子通道活动的变化。用斜坡电压模式记录的结果显示，10min内正常细胞在钳制电压下电流未发生明显变化，氯通道阻断剂DIDS可以抑制所记录到的电流，加药10mln就使电流峰值降低到原来的36%，向浴槽液中加凋亡诱导剂STS smin后，+8伽mV钳制电压下电流即增大，为加药前的131.3%，加药后10min电流进一步增大，是加药前的巧5.9%，而且电流随时间延长具有继续增大的趋势。用跃级电压模式记录的结果做I一V曲线，氯通道在所测试电压下呈现外向整流特性，在一6肠mV一+40mv钳制电压下加药前后电流未发生明显改变，在+60mV钳制电压下，加药前电流为61.2士3.3%，加药后smin为101.3士n.2%;在+80mV钳制电压下，加药前电流为100.0%，加药后sntin为巧4.1士14.6%。 结论:研究结果表明钾通道和氯通道参与了凋亡性细胞容积减少，这也可能是的钾通道和氯通道介导细胞凋亡的重要机制之一。更多还原

【Abstract】 Our previous studies have shown that activation of potassium channel is involved in the apoptosis of hippocampal neurons induced by in vivo ischemia and in vitro hypoxia. Recently, cell shrinkage is proposed as an early prerequisite to apoptotic events leading to cell death. To understand the mechanism underlying ionchannel-mediated neuronal apoptosis, the present study examined the temporal changes in neuronal cell body volume and the involvement of ion channels in the apoptotic volume decrease in a model of staurosporine (STS)-induced apoptosis of cultured hippocampal neurons. STS induced an early cell body volume decrease and this cell shrinkage was completely blocked by high extracellular K+, TEA or DIDS. Raising extracellular K+ concentration or tetraethylammonium (TEA, a nonselective potassium channel blocker) significantly prevented STS-induced neuronal cell death. Similar neuroprotections by treatment with the selective high-conductance calcium-activated potassium channel (BK) blockers iberiotoxin and paxilline was also observed whereas "A" current and SK channel blockers, 4-AP and apamin, showed no neuroprotective effect. Moreover, chloride channel blockers, DIDS and SITS, also significantly prevented STS-induced neuronal cell death. With whole-cell patch-clamp recordings, we found chloride channel currents increased significantly withtime when holding potential at 60mV and 80mV after 5min and 10 min of STS exposure. These results indicate that potassium channel, especially BK channels, and chloride channel contribute to STS-induced neuronal apoptosis and apoptotic volume decrease, which is probably one of the mechanisms underlying mediation of neuronal apoptosis by potassium channel更多还原