中文摘要：

目的观察Rho激酶（ROK）及下游底物肌球蛋白轻链（MLC）在体外培养的神经元缺血及再灌注过程中的变化，探讨ROK在神经元受损后轴突萎缩中的作用和机制。方法培养N2a细胞并分为对照组、缺氧组、再灌注组、Y-27632干预缺氧组和Y-27632干预再灌注组共5组，Westernblot检测ROCKI和ROCKII蛋白的表达以及ROK下游MLC的磷酸化水平；噻唑蓝（MTT）比色法检测细胞活力；FITC标记的鬼笔毒环肽（phalloidin）染色神经元纤维状肌动蛋白（F－aetin）骨架，免疫荧光观察轴突细胞骨架重组变化。结果N2a细胞缺氧和再灌注损伤过程中ROCKII的表达分别升高了29．7％和57．4％，而磷酸化MLC（p-MLC）的表达分别升高了21．5％和49．3％，免疫荧光镜下观察正常N2a细胞轴突、树突形态清晰，纤维状肌动蛋白丝主要分布于细胞周边，应力纤维少，而经历缺氧和再灌注损伤的N2a细胞胞浆内应力纤维增多，周边肌动蛋白丝带模糊，同时轴突回缩，MTT提示细胞增殖率低于阴性对照，分别下降了22．7％和45．6％；加入浓度为30mol／L的Rho激酶抑制剂Y-27632的N2a则无此反应，且在再灌注过程中干预可使轴突已回缩的N2a重新出现丝状伪足，MTT提示加入Y-27632能显著提高N2a细胞缺氧及再灌注损伤24h后的存活率（P〈0．05）。结论神经元在经历缺氧及再灌注过程中ROCKII激活并导致轴突内MLC过度磷酸化，这可能是致使轴突内肌动蛋白细胞骨架重组而最终导致生长锥的塌陷和神经突起的回缩的主要机制；对ROK进行抑制则可以预防或逆转这一过程。

英文摘要：

Objective To investigate the mechanism of ROK causing collapse of the axon by neuron injury through observing the activation of Rho-ROK during neuron isehemia and ischemia-reperfusion culture in vitro. Methods in vitro,N2a cells cultivation by ischemia and ischemia-reperfusion were treated with 30 mol/L Y-27632, activation of ROK and phosphorylation level of MLC was detected by Western blotting, cell damage was analyzed by MTT, the cytoskeleton of N2a cells were scaned through immunofluorescence techniques. Results The activation of ROCKII proteinum increased 29.7% and 57.4% during neuron ischemia and ischemia-reperfusion injury,The phosphorylation level of MLC increased 21.5% and 49.3%, they induced a striking reorganization of actin cytoskeleton with a weakening of fluorescent intensity of the peripheral filament actin bands and formation of the long and thick stress fibers,but Pretreatment of Y-27632 could reversed the changes of uhra-sturcture on the cellular surface,. MTT assay show that Y- 27632 could prolong the survival time of the N2a cells after ischemia-reperfusion for 24 h. Conchtsion The activation of Rho-ROK had a exceptional hoist after N2a ischemia and ischemia-reperfusion injury, it is likely to induce the collapse of the growth cone. Suppression of Rho kinase activity could promote axonal growth.