Rho-ROK通路阻滞剂对小鼠缺氧神经元轴突再生和细胞活力的影响  

Effects of Rho-ROK Inhibitors on Axon Regeneration and Viability of Mouse Neuron Induced by Hypoxia in vitro

作　　者：王欢[1] 陈安民[1] 郭风劲[1] 张栋栋[2] 易成腊[2] 

机构地区：[1]华中科技大学同济医学院附属同济医院骨科,武汉430030 [2]华中科技大学同济医学院附属同济医院创伤外科,武汉430030

出　　处：《神经损伤与功能重建》2009年第2期98-101,共4页Neural Injury and Functional Reconstruction

基　　金：国家自然科技基金资助项目(30400449)

摘　　要：目的:探讨Rho-ROK通路阻滞剂对小鼠缺氧神经元轴突再生和细胞活力的影响。方法:取小鼠大脑皮质神经元培养并鉴定,进行体外缺氧干预,于缺氧前后加入Rho-ROK通道阻滞剂(Y-27632,法舒地尔和胞外酶C3a),用FITC标记的鬼笔毒环肽染色观察神经元纤维状肌动蛋白(F-actin)骨架的重组变化,用四甲基偶氮唑蓝法(MTT法)检测细胞活力。结果:正常神经元生长良好,轴突、树突形态清晰并交织成网,F-actin主要分布于细胞外周,形成周边肌动蛋白丝带,胞浆内应力纤维少;缺氧后,周边肌动蛋白丝带模糊,神经突起回缩,应力纤维增多,胞体膨胀,细胞趋于凋亡或破裂;缺氧前加入Y-27632、法舒地尔孵育则无此变化,缺氧后加入Y-27632、法舒地尔亦可明显逆转这一过程,而胞外酶C3a干预则无明显改善。MTT法显示Y-27632、法舒地尔能显著提高缺氧神经元再灌注24 h后的存活率(P<0.05),而胞外酶C3a无明显作用。结论:神经元缺氧可引起细胞骨架重组、生长锥塌陷、神经突起回缩,细胞趋于凋亡、破裂。于缺氧前后阻滞Rho-ROK通路均可抑制这一过程,但不同种类Rho-ROK通路阻滞剂的保护作用不同,其中Y-27632、法舒地尔作用显著,而胞外酶C3a作用不明显。Objective：To investigate the effect of Rho ROK inhibitors on axon regeneration of mouse cortical neurons after hypoxia Methods： Mouse cortical neurons were cultured and treated with hypoxia. The inhibitors of Rho kinase, Y-27632, Fasudil and Exoenzyme C3a, were applied to the cultures before and after hypoxia. The axon regeneration was determined by immunostaining of F-actin. The neuronal viability was measured by MTT method. Results： In control normal neuronal cultures, axons and dendrites formed the network, and the actin cytoskeleton was distributed in the periphery of neurons and formed the actin bands. Stress fibers were rarely observed in the endochylema. However, after exposure to hypoxia, actin bands became blurred and the neuronal processes were re- tracted. The cells were swollen and became apoptotic. The cellular changes induced by hypoxia could be reversed by Y 27632 and fasudil whereas Exoenzyme C3a showed little effects. Y-27632 and fasudil also could improve the viability of neurons after hypoxia. Conclusion： Hypoxia induces the recombination of cytoskeleton and collapse of growth cones in parallel with apoptosis of cortical neurons. Suppression of Rho kinase activity with different Rho-ROK inhibitors could inhibit this process, Y 27632 and fasudil show significant effects but exoenzyme C3a displays only a marginal effect.

关 键 词：缺氧 神经元 轴突 Rho/ROK阻滞剂 肌动蛋白 

分 类 号：R741[医药卫生—神经病学与精神病学] R743[医药卫生—临床医学]