脐血间充质干细胞的分离培养鉴定和生物学特性.doc

脐血间充质干细胞的分离培养、鉴定及生物学特性研究 师玲玲1，2 刘赴平2 王立生1 王德文2 刘仁强2 黄志森2 陈少彬2 林栋2 崔四平2 许惠芯2 朱毅瑜2 温金花2 邓妙玲2 1：暨南大学 2：东莞市中心血站 摘要：目的：探讨体外分离、培养及纯化人类脐血间充质干细胞（hUCBMSC）的适宜体系，观察人类脐血间充质干细胞生物学特性。方法：收集人类脐血间充质干细胞，密度梯度离心法分离脐血中单个核细胞；贴壁培养和适宜培养基筛选人类脐血间充质干细胞，显微镜下观察细胞生长形态变化，细胞计数绘制细胞生长曲线，流式细胞仪检测细胞抗原表达，分析细胞周期。体外诱导脐血间充质干细胞成脂成骨分化。结果：采用密度为1.077g/ml的淋巴细胞分离液（Ficoll）以1500rm转速离心获得的单个核细胞，在Mesencult培养基中培养，约3-5h开始出现贴壁生长，24h后贴壁细胞增多，呈明显纺锤状，10d后开始出现细胞克隆，3w后呈漩涡状生长。原代培养时间为15d，P1代倍增时间为26h。经流式细胞仪鉴定，贴壁生长的细胞表达CD29、CD44、CD105,不表达CD34、CD45。分化潜能鉴定体外培养细胞可以成脂、成骨分化。结论：上述贴壁细胞的生长形态、流式表形、分化潜能具有hUCBMSC生物学特征，实验中采取的培养体系适宜hUCBMSC的分离培养。 关键词：MSC 分离培养 鉴定 生物学特性 Research of isolation，identification and biological characteristics of human umbilic cord blood mesenchymal stem cells Shi ling-ling1 Liu fu-ping2 Wang li-sheng1 Wang de-wen2 Liu ren-qiang2 Huang zhi-sen2 Chen shao-bin2 Lin dong2 Cui si-ping2 Xu hui-xin2 Zhu yi-yu2 Wen jin-hua2 Deng miao-lin2 1：Jinan University 2：Dong Guan Central Blood Station Abastract: OBJECT To study feasible isolation /cultivation system for human umbilical cord blood mesenchymal stem cell, observe its’ biological characteristic. METHORD Collect hUCBMSC, isolate monocyte by sensity-gradient centrifugation, filtrate hUCBMSC by attachment culture,observe growthform by microscope，portray growth curve，detect antigen express by flow cytometer, analyze the cell cycle. Test the potential for bone-formation lipocyte-formation of human umbilical cord blood mesenchymal stem cell. RESULT The primary cells can be procured by density gradient centrifugation. Adherence growth can be observed since cultured in Mesencult culture medium for 3-5h., mor and more adherent cells has appeared, cell clone has emerged in 10d. It takes 15d for primery culture.The cells that adherent grow express CD29/CD44/CD105, and its don’t express CD34/CD45. It can be induced in adipocyte or . Conclusion growth morphology、phenotype、potential for differentiation of the cells accord with the characteristic of MSCs. The cult