edaravone体外诱导rbmscs分化为神经元样细胞

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1、Edaravone体外诱导rBMSCs分化为神经元样细胞胡资兵曾荣魏劲松林颢吴少科孙欣郑鸿（广东医学院附属医院骨科，湛江524001）【摘要】目的探讨新型氧自由基清除剂依达拉奉诱导大鼠骨髓基质干细胞（ratbonemarrowstromalcells,rBMSCs）分化为神经元样细胞的可行性。方法采用密度梯度离心法和贴壁法分离纯化1月龄SDrBMSCs，体外培养扩增与传代，流式细胞术检测rBMSCs表面标记，克隆培养计算克隆形成率，分别用茜素红染色和油红O染色鉴定rBMSCs成骨成脂分化；实验分依达拉奉处理组和空白对照组，处理组用终浓度0.02g/L依达拉奉的无血清L-D

2、MEM诱导6h，对照组继续用10%胎牛血清、L-DMEM培养。观察细胞形态变化，免疫细胞化学鉴定神经元烯醇化酶、胶质纤维酸性蛋白、巢蛋白、微管相关蛋白2的表达。结果流式细胞术检测CD29、CD44、CD105阳性，CD34、CD45阴性；7d和14d克隆形成率为49%、92%；茜素红和油红O染色阳性；分化的rBMSCs胞体收缩，突起伸出；诱导后细胞神经元烯醇化酶阳性表达率（81.6±3.2）%，微管相关蛋白2阳性表达率(26±2.5)%，胶质纤维酸性蛋白阳性表达率(5.6±1.9)%，巢蛋白阳性表达率(9.0±2.8)%。对照组均阴性表达。两组NSE、GFAP、Nesti

3、n比较均有统计学意义（P<0.001）,处理组NSE阳性表达明显高于MAP2、GFAP（P<0.005）。结论依达拉奉可以诱导rBMSCs分化为神经元样细胞，从而为脊髓损伤的治疗提供种子细胞和理论基础。【关键词】骨髓基质干细胞；神经分化；神经元样细胞；脊髓损伤Differentiationofratbonemarrowstromalcellsintoneuron-likecellsinvitrobyedaravone【Abstract】ObjectiveToexplorethepossibilityofdifferentiationofratbonemarrow-deri

4、vedstromalcellstoneuron-likecellsbyedaravoneinvitro．MethodsBonemarrowstromalcellswereseparatedandpurifiedfromone-month-oldSDratbyusingdensitygradientcentrifugationcombiningattachmentmethodforculture,passageandproliferation.SurfacemarkersofrBMSCsweredetectedbyflowcytometryandcolonyforminge

5、fficiencycalculatedwithclonalculture.AlizarinredandoilredOstainingwasusedtoidentifyosteoblastsandadipogenicdifferentiation.Inthisexperimenttherearetwogroupscontainingedaravoneandcontrol.rBMSCswasinducedbyedaravoneatafinalconcentrationof0.02g/Landserum-freeL-DMEMfor6h.However,thecontrolgro

6、upcontinuedtoculturewith10%fetalcalfserumandL-DMEM.MorphologicobservationandimmunocytochemicalanalysiscontainingexpressionofNSE,GFAP,MAP2andnestinwereperformed．ResultsCD29,CD44andCD105werepositive,butCD34andCD45werenegative.Colonyformingefficiencywas49%and92%after7-dayand14-daycolonalcult

7、ure.AlizarinredandoilredOstainingwerepositive.Bodycontractionandlongprocessionwereshownindifferentiatedstromalcells.TheimmunocytochemicaldetectionshowedthatthepercentageofNSE,MAP2,GFAPandNestinpositivecellswere(81.6±3.2)%,(26±2.5)%,(5.6±1.9)%and(9.0±2.8)%,respective