外周血内皮祖细胞纯化与细胞鉴定.pdf

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1、中国科技论文在线http://www.paper.edu.cn#外周血内皮祖细胞纯化与细胞鉴定111231**赵娴，魏韩笑，周箐竹，李杉，王松梅，刘流（1.昆明医科大学第一附属医院；52.云南大学生命科学学院；3.昆明医科大学公共卫生学院）摘要：目的：提取纯化来源于机体本身具有优异新生血管及管腔形成能力的自体外周血内皮祖细胞（EndothelialProgenitorCell），为组织工程血管化的研究提供优质的、取材方10便的、血管化能力强的种子细胞。方法：抽取新西兰大耳白兔外周血，采用密度梯度离心法加贴壁

2、法，提取纯化自体外周血内皮祖细胞，取第三代细胞进行免疫荧光染色检测贴壁细胞CD34、CD133、vWF表面标志，及免疫双标染色鉴定内皮组细胞。结果：抽取的外周血用密度梯度离心法、贴壁培养法提取纯化并进行扩增，获得高纯度的EPCs，第三代外周血EPCs抗原CD34、CD133、vWF免疫荧光鉴定均为阳性；EPCs的免疫双标染色鉴定也证实，经过同15时摄取乙酰化低密度脂蛋白以及荆豆凝集素的免疫荧光复合染色，分离培养的细胞为血管内皮祖细胞。结论：兔外周血经密度梯度离心分离及贴壁法纯化，可获得纯度较高的内皮组细胞。

3、关键词：基础医学；外周血；内皮祖细胞；细胞纯化；免疫双标染色中图分类号：请查阅《中国图书馆分类法》20ThePurificationandIdentificationofEPCsDerivedfromPeripheralBlood111231ZhaoXian,WeiHanxiao,ZhouQingzhu,LiShan,WangSongmei,LiuLiu(1.TheFirstAffiliatedHospitalofKummingMedicalUniverstiy;252.TheLifeScienceofYu

4、nnanUniversity;3.ThePublicandHealthCollegeofKummingMedicalUniversity)Abstract:Aim：TheEndothelialProgenitorCellsderivedfromperipheralbloodwithexcellentablityofvascularizationwereisolatedandpurified,toapplyhighqualityandeasilybeobtainedseedcellsfortissueengi

5、neeringvascularization.Method:Endothelialprogenitorcells(EPCs)wereisolatedfrom30rabbitperipheralbloodandculturedinEndothelialcellmedium(EGM).TheexpressionofCD34,CD133,vWFonthethirdgenerationadherentcellsweredetectedwithimmunofluorescentstaining;(2)Bonemarr

6、owstemcells(BMSCs)wereisolatedfromrabbitbonemarrowbloodandculturedinmediumadded1%fetalbovineserum.TheexpressionofCD29,CD34,CD90onthethirdgenerationadherentcellsweredetectedwithimmunofluorescentstaining,andCellmorphologicalchangesandthe35EPCstubecavitystruc

7、turewereobservedundermicroscope.Result:HigherpurityEPCscouldbeisolatedwithFicoll.TheCD34、CD133andvWFofperipheralbloodderivedendothelialprogenitorcellswerepositivewithimmunofluorescentstainingonweek3weeks,andDiIlabeledacetyllowdensitylipoproteincholesterola

8、ndfluoresceinlabeledlectinimmunofluorescencedoublestainingwerepositive,whichwereconsistentwiththeEPCscharacteristic.Conclusion:EPCsisolatedfromperipheral40bloodwithFicolldensitygradientcentrifugationwerehighl