摘要
目的比较全骨髓贴壁法和骨组织消化法分离获取骨髓间充质干细胞(bone marrow mesenchymal stemcells,BMMSCs)的效果。方法分离获取小鼠股骨,冲洗骨髓腔并收集骨髓悬液培养BMMSCs为全骨髓贴壁法;去骨髓的股骨剪碎、消化后培养BMMSCs为骨组织消化法。比较两组BMMSCs的原代细胞数量、细胞生长曲线、细胞传代增殖能力、免疫表型及成骨分化潜能。结果骨组织消化法的BMMSCs原代细胞数量明显多于全骨髓组,并且在第7天细胞就可以融合达到90%;骨组织组的BMMSCs生长快,并且细胞传代增殖能力比全骨髓组强,传代计数实验显示第5次传代后,骨组织组的细胞数量达全骨髓组的4倍(P<0.05);流式表型鉴定结果显示,两组细胞均高表达干细胞表面标志物Sca-1及表面黏附分子CD29,其中骨组织组Sca-1及CD29阳性细胞的比例均高于全骨髓组;骨组织组BMMSCs几乎不表达CD45和CD11b,而全骨髓组CD45阳性细胞的比例为3.18%,CD11b阳性细胞的比例为9.11%。两组BMMSCs均能分化为成骨细胞。结论骨组织消化法操作简单、成本低,且获得的BMMSCs纯度高,可短时间内纯化得到大量BMMSC细胞,是基础研究中值得推广的实验方法。
Objectives To compare the effects of culturing flashing-out bone marrow(FOB)and culturing digestedcompact bone(DCB)fragments in obtaining bone marrow mesenchymal stem cells(BMMSCs).MethodsThe mousefemur was obtained for cell culture.BMMSCs obtained in these two groups were conducted by collecting the bone mar-row suspension flushed out from femurs and digesting bone marrow cavity,respectively.Counts of primary cells,growth condition,cell surface markers and differentiation capacity were compared.ResultsThe number of primarycells in DCB group was significantly higher than that in FOB group.Cells in DCB group growed rapidly and reached90%confluenceinat at the 7 th day.Additionally,the potency of cell proliferation in DCB group was four times ofFOB group at the 5 th passage(P<0.05).Stem cell surface markers,such as Sca-1 and CD29 were highly expressed inboth groups,especially in DCB group.CD45 and CD11 b were merely expressed in DCB group.However,3.18%and 9.11%of cells in FOB group expressed CD45 and CD11 b,respectively.The ability of differentiating into osteoblastsin both groups were confirmed.ConclusionsCulturing DCB fragments is an effective method for the isolation of mouseBMMSCs with high purity,which is an experimental method worthy of promotion in basic research.
作者
杨雷
朱烁基
刘健
黄焕雷
郭惠明
陈寄梅
庄建
赵明一
吴秀山
朱平
YANG Lei;ZHU Shuo-ji;LIU Jian;HUANG Huan-lei;GUO Hui-ming;CHEN Ji-mei;ZHUANG Jian;ZHAO Ming-yi;WU Xiu-shan;ZHU Ping(Department of Cardiovascular Surgery,Guangdong Cardiovascular Institute,Guangdong Provincial People’s Hospital,Guangdong Academy of Medical Sciences,Guangzhou 510080,China;The Center for Heart Devel opment,State Key Laboratory of Developmental Biology of Fresh Water,College of Life Sciences,Hunan Nor mal University,Changsha 410081,China;Department of Pediatrics,The Third Xiangya Hospital,Central South University,Changsha 410013,China)
出处
《岭南心血管病杂志》
2020年第2期225-229,共5页
South China Journal of Cardiovascular Diseases
基金
国家重点研发计划项目(项目编号:2018YFA0108700,2017YFA0105602)
国家自然科学基金重点国际(地区)合作与交流项目(项目编号:81720108004)
国家自然科学基金面上项目(项目编号:81570279)
广东省自然科学基金研究团队项目(项目编号:2017A030312007)
广州市科学研究计划重点项目(805212639211)
广东省人民医院登峰计划专项。
关键词
骨髓间充质干细胞
小鼠
骨组织消化法
骨髓贴壁
mesenchymal stem cells
mice
digestion of compact bone fragments
flashing-out bone marrow
