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1、GFP年轻成年大鼠嗅鞘细胞的分离培养与形态学特征张洁元,段朝霞,陈莉发,李兵仓作者简介:张洁元(1982—),女,重庆,本科,助理实验师,主要从事脊髓损伤修复方面研究。电话:023-68757460基金项目:创伤、烧伤与复合伤国家重点实验室自主研究课题(SKLZZ200803)通讯作者:李兵仓,Tel:023-68757461,E-mail:bcli1118@yahoo.com.第三军医大学大坪医院野战外科研究所六室创伤、烧伤与复合伤国家重点实验室,重庆,400042【摘要】 目的 观察急性体外分离、培养的GFP大鼠OECs的形态学特征与生物学活性,为在体移
2、植示踪研究奠定基础。方法 于解剖显微镜下、无菌环境取2.5月龄的GFP大鼠嗅球最外两层,经酶消化法分散后将细胞接种于含20%胎牛血清的DMEM/F12培养基中。采用差速贴壁和阿糖胞苷抑制相结合的方法纯化,定期进行形态学观察并摄片。在培养第10d进行S-100和NGFRp75的免疫细胞化学染色鉴定,并计算OECs的纯度。结果 培养的GFP大鼠OECs在荧光倒置显微镜下呈绿色强荧光,形态以双极、多突起形为主,呈S-100、NGFRp75阳性,纯度在95%以上。结论 利用差速贴壁和阿糖胞苷化学抑制相结合的方法可分离出高纯度的GFP-OEC,其形态学特征与生物学活性
3、与普通OECs相同。【关键词】 GFP大鼠;嗅鞘细胞;细胞培养;免疫细胞化学【中图分类号】R322.81【文献标识码】AIsolated、CultureandMorphologiccharacteristicsofOlfactoryEnsheathingCellsfromGFPYoungAdultRatZHANGJie-yuan,DUANZhao-xia,CHENLi-fa,LIBing-cangDepartment6ofResearchInstituteofSurgery,DapingHospital.ThirdMilitaryMedicalUnivers
4、ity,StateKeyLaboratoryofTrauma,BurnsandCombinedInjury,ChongQing,400042,China.【Abstract】 Objective ToobservethemorphologiccharacteristicsandbiologicalactivityoftheOECsfromGFPratbyinvitroacuteisolation,incubation,andtolaythefoundationfortheresearchontrackingcelltransplantation.Method
5、s TheOECsweredissociatedfromthefirsttwoouterlayersoftheolfactorybulbsoftheyoungadultGFPrat(2.5monthsold)underanatomicalmicroscopebythemethodofenzymaticdigestion,culturedinDMEM/F12mediumwith20%foetalcalfserum,andpurifiedbythemethodofcombiningthedifferentratesofcellatadhesionwiththeA
6、ra-Cinhibitionofcell.ThemorphologicalchangesofculturedOECswereregularlyobservedandphotographs.On10thcultureday,theOECsinculturewereidentifiedbytheimmunocytochemistrytechniqueofS-100andNGFRp75,andthepurityofthepositivecellswascalculated.Results TheculturedOECsweregreenhyperfluoresce
7、nceobservedinfluorescentinvertedmicroscope,presentedmainmorphologicaltypes:bipolar,process-bearingmultipolar.Morethan95%culturedcellswereidentifiedtobeOECs,whichexpressedS-100andNGFRp75.Conclusion ThehighpurityGFP-OECswhichcanbeculturedsuccessfullyinvitrobythecombinedpurificationme
8、thod,havethesamemorphologi