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1、98NeuralInjuryandFunctionalReconstruction,march2009,Vol.4,No.2【DOI】10.3870/sjsscj.2009.02.007·论著·Rho2ROK通路阻滞剂对小鼠缺氧神经元3轴突再生和细胞活力的影响aaabb#王欢,陈安民,郭风劲,张栋栋,易成腊华中科技大学同济医学院附属同济医院a.骨科,b.创伤外科,武汉430030【摘要】目的:探讨Rho2ROK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Rho2ROK通道阻滞剂(Y227632,法舒地尔和
2、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(F2actin)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,F2actin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Y227632、法舒地尔孵育则无此变化,缺氧后加入Y227632、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Y227632、法舒地尔能显著提高缺氧神经元再灌注24h后的存活率(P<
3、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Rho2ROK通路均可抑制这一过程,但不同种类Rho2ROK通路阻滞剂的保护作用不同,其中Y227632、法舒地尔作用显著,而胞外酶C3a作用不明显。【关键词】缺氧;神经元;轴突;Rho/ROK阻滞剂;肌动蛋白【中图分类号】R741;R743【文献标识码】A【文章编号】10012117X(2009)0220098204EffectsofRho2ROKInhibitorsonAxonRegenerationandViabilityofMouseNeur
4、onInducedbyHypoxiainvitro▲▲WANGHuan,CHENAn2min,GUOFeng2jing,ZHANGDong2dong,YICheng2la.DepartmentofOrthopae2dics,TongjiHospital,TongjiMedicalCollege,HuazhongUniversityofScienceandTechnology,Wuhan430030,China【Abstract】Objective:ToinvestigatetheeffectofRho2ROKinhibitorsonaxonregenerationofmouseco
5、rticalneuronsafterhypoxiaMethods:Mousecorticalneuronswereculturedandtreatedwithhypoxia.TheinhibitorsofRhokinase,Y227632,FasudilandExoenzymeC3a,wereappliedtotheculturesbeforeandafterhypoxia.TheaxonregenerationwasdeterminedbyimmunostainingofF2actin.TheneuronalviabilitywasmeasuredbyMTTmethod.Resu
6、lts:Incontrolnormalneuronalcultures,axonsanddendritesformedthenetwork,andtheactincytoskeletonwasdistributedintheperipheryofneuronsandformedtheactinbands.Stressfiberswererarelyobservedintheen2dochylema.However,afterexposuretohypoxia,actinbandsbecameblurredandtheneuronalprocesseswerere2tracted.T
7、hecellswereswollenandbecameapoptotic.ThecellularchangesinducedbyhypoxiacouldbereversedbyY227632andfasudilwhereasExoenzymeC3ashowedlittleeffects.Y227632andfasudilalsocouldimprovethevia2bilityofneuronsafterhypoxia.Conclusion:Hypoxiainduce
