大鼠神经元缺氧缺糖损伤后calpain1及αⅱ-spectrin表达变化规律

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1、录幽IIrlIIIIIIrlllllIIIIilIllIIFY1892380一、摘要中文论著摘要················································-·l英文论著摘要············································一····4二、英文缩略语···················································7三、论文靖{『言··················································

2、········8月U百。·。。。。。。。‘。。。。。。‘。。。。。‘。’。。。。。。。。。。。。。。。。。’。。’。。。。。。。’。。。。苎材料与方法·····································一·············9结果··························································11讨论·······················?··································18结论··················

3、········································23四、本研究创新性的自我评价·······································25五、参考文献······················································26六、附录综j苤··························································29在学期间科研成绩·······································

4、·······39致谢··························································40个人简介······················································41·中文论著摘要·大鼠神经元缺氧缺糖损伤后calpainl及all—spectrin表达变化规律目的颅脑损伤是法医实践工作中常见而严重的损伤类型，不同类型的颅脑损伤常伴有不同类型及不同程度的神经元损伤，颅脑损伤发生后的继发性神经元损伤的分子机制一直为国内外学者所关注。本研究通过进行大

5、鼠皮质神经元的培养，在纯化神经元的理想状态下，化学性作用建立OGD损伤模型，动态观察神经元损伤后不同时间形态学特征，并采用免疫荧光细胞化学染色技术、Westernblot等方法，在不同的时间点检测calpainl及其底物蛋白0【II．spectrin(240kDa)，以及a11．spectrin的裂解片段SBDPs(150kDa和120kDa)在蛋白水平表达的时间规律性，进一步分析calpainl和a11．spectrin在脑损伤后的继发性神经元损伤中所起作用，为法医学中应用生物学标记蛋白calpainl和a11．spectrin推断脑损

6、伤形成时间提供一定的实验性理论依据。实验材料与方法选择出生后1～2天的Wistar大鼠，取大脑皮质组织，剪碎，0．25％的胰酶消化，离心弃去上清，用含10％马血清、10％胎牛血清的DMEM／F12培养基悬浮沉淀组织，过滤使之成为单细胞悬液，按2x106个／ml的密度接种于预先包被多聚赖氨酸的六孔培养板中，置于细胞培养箱中培养；2天后加入为终浓度10l，tmol／L阿糖胞苷抑制胶质细胞、纤维细胞等杂细胞的生长；神经元培养至第8天时，随机分为对照组及若干损伤组，损伤组神经元用含O．5mmol／L连二亚硫酸钠的低糖DMEM培养基进行缺氧缺糖损伤

7、，作用30分钟后更换为正常培养基分别继续培养oh、1h、3h、6h、12h、24h、48h、96h；对照组细胞只常规换液一次。对照组及损伤组神经元分别进行活细胞倒置显微镜观察、免疫荧光细胞化学染色、Westernblot检测。结果当日禾本实验应用原代培养大鼠大脑皮质神经元，进行缺氧缺糖损伤，通过活细胞倒置显微镜观察发现神经元缺氧缺糖损伤后形态学发生了一系列改变。对照组神经元形态较好，细胞形态规整，边缘折光性强，周围光晕明显，胞体丰满，呈类圆形、近三角形及多极形，树突、轴突较长并相互交织成网，核仁明显，呈圆形。OGD损伤即刻，细胞形态基本没

8、有明显改变，细胞胞浆内出现少量空泡状结构。伤后1h，大部分神经元仍保持原有形态，少数细胞胞体周围光晕变暗，突起缩短。伤后6h．12h，神经元胞体明显皱缩，多数细胞突起缩短，甚至消失，光晕减弱甚