摘要
目的观察脊髓损伤（SCI）后RhoA活性变化和体外模拟脊髓缺血再灌注诱导的神经元损伤中Rho激酶（ROCK）下游底物肌动蛋白（F-actin）细胞骨架的重组，探讨ROCK通路对于SCI后轴突萎陷的作用机制。方法成年sD大鼠Allen’s制模，Westernblot检测RhoA蛋白表达，GSTPulldownassay RhoA活性变化；培养N2a细胞，置于含90％N2的37℃培养箱中模拟脊髓继发损伤中缺血缺氧和再灌注过程，MTT检测细胞活力，并用FITC标记的鬼笔毒环肽染色神经元F-actin，免疫荧光观察其重组。结果SCI后RhoA总蛋白表达无明显变化（P〉0．05），但活性逐步增高（P〈0．05）；正常N2a细胞F-actin主要分布于细胞周边，应力纤维少，缺氧后胞质周边肌动蛋白丝带模糊，轴突回缩，胞质内应力纤维增多；而加入Y-27632的N2a缺氧后无此过程，且缺氧后再加入Y-27632亦可明显逆转此过程；MTT显示Y-27632能显著提高N2a缺氧和再灌注24h后的存活率（P〈0．05），且保护作用在一定范围内和浓度成正比。结论SCI后神经元轴突中RhoA的活性增高并过度激活ROCK，影响轴突内F—actin细胞骨架的重组，从而导致生长锥萎陷和轴突回缩；对ROCK进行抑制可以预防轴突塌陷和神经元死亡，促进轴突再生。
Objective To investigate the mechanism of ROCK causing collapse of the growth cone after SCI through observing the activation of RhoA after SCI and the reorganization of F-actin of neuron during mimic ischemia injury in vitro. Method The model of SCI was made by Allen＇s method. The activation expressive variety of RhoA was detected through Western Blot and Pull down assay; In vitro, N2a cells induced by ischemia and ischemia-reperfusion were treated with different dilute Y-27632, cell damage was analyzed by MTF; On the other hand, the cytoskeleton of N2a cells were seaned through immunofluorescence techniques by confocal laser microscopy which stained with Fitc-phalloidin for F-actin visualization. Results The activation of RhoA proteins was increased significantly in the damaged local during the following phase of SCI. Ischemia 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 ultra-strnctttre on the cellular surface, and the protection of Y-27632 was related with its concentration in determinate bound. MT＇F assay showed that Y-27632 could prolong the survival time of N2a cells after mimic ischemia-reperfusion for 24 h. Conclusions The activation of Rho had exceptional hoist after SCI, it is likely to induce the collapse of the growth cone through ROCK. Suppression of Rho kinase activity could promote axonal growth on inhibitory spinal cord.
出处
《中华神经外科杂志》
CSCD
北大核心
2009年第3期283-287,共5页
Chinese Journal of Neurosurgery
基金
国家自然科技基金资助项目（30570554）
