脐带血清和成人自体血清体外培养人骨髓间充质干细胞的比较

背景:为避免骨髓间充质干细胞培养过程中应用异种血清的排斥以及提高骨髓间充质干细胞培养效率,制备脐带血清体外扩增培养骨髓间充质干细胞是目前的研究热点.目的:比较脐带血清和成人自体血清体外培养人骨髓间充质干细胞的生物学特性.设计、时间及地点:开放性实验,于2006-10/2008-06在中南大学湘雅二医院细胞生物实验室完成.材料:16份骨髓标本由中南大学湘雅二医院和湖南省湘潭市中心医院提供.方法:采用Ficoll Paque细胞分离液从16例新鲜成人骨髓穿刺液中分离骨髓间充质干细胞,分别用含体积分数为10%脐带血清、成人自体血清的DMEM/F12培养基培养,取第4代细胞进行实验.流式细胞仪检测细胞表面抗原,成骨诱导体系及脂肪诱导体系分别诱导各组骨髓间充质干细胞定向分化.通过细胞形态观察、免疫表型及免疫化学染色进行鉴定.主要观察指标:①细胞形态.②骨髓间充质干细胞计数和细胞周期.③表面标志物的鉴定.④免疫组织化学染色观察骨髓间充质干细胞诱导成骨、成脂情况.结果:①脐带血清组培养的骨髓间充质干细胞在增殖数量和速度上优于成人自体血清组(P＜0.05).两种血清培养条件下,只有少数细胞处于活跃的增殖期,脐带血清组S期细胞多于成人自体血清组(P＜0.05).②脐带血清组培养的骨髓间充质干细胞表达CD29、CD73、CD105的阳性率均在90%以上,高于成人自体血清组(P＜0.05);而CD31、CD34、CD45的阳性率均在2%以下,CD31阳性率低于成人自体血清组(P＜0.05).③脐带血清培养的骨髓间充质干细胞诱导为成骨细胞阳性率和脂肪细胞阳性率均高于自体血清组(P＜0.05).结论:脐带血清组培养的骨髓间充质干细胞的纯度和体外诱导分化能力比成人自体血清组高,更适合临床应用.

Bone marrow mesenchymal stem cells cultured by umbilical cord blood serum versus adult autoserum in vitro

BACKGROUND:Studies concerning bone marrow mesenchymal stem cells (BMSCs) cultured by umbilical cord serum in vitro were hot topic to avoid the heterogenous serum rejection during BMSC culture and improve culture efficiency of BMSCs.OBJECTIVE:To compare biological feature of BMSCs by the umbilical cord serum and adult autoserum in vitro.DESIGN,TIME AND SETTING:The opening experiment was performed at the Laboratory of Cell Biology,Xiangya Second Hospital,Central South University,China from October 2006 to June 2008.MATERIALS:Sixteen bone marrow samples were provided by Xiangya Second Hospital,Central South University and Xiangtan Central Hospital of Hunan Province.METHODS:BMSCs were obtained from 16 fresh adult bone marrow by gradient centrifugation with Ficoll Paque,cultured with DMEM/F12 with 10% umbilical cord blood serum and 10% adult autoserum.The fourth passage was used in this study.The surface antigens of BMSCs were detected by flow cytometry.BMSCs could differentiate into ostenblasts and adipocytes under culture in conditioned medium for osteogeneais and adipogenesis and the differentiated BMSCs were identified by morphological observation,immunophenotype and immunochemical staining.MAIN OUTCOME MEASURES:The following parameters were measured:morphological observation;cell cycle and cell count;identification of surface antigen;observation of osteoblasts and adipocytea induced from BMSCs by immunochemical staining.RESULTS:The quantity and velocity of BMSCs in umbilical curd blood serum was better than those in adult autoserum (P ＜ 0.05)Only few cells were proliferating in both medium,BMSCs in S phase in umbilical cord blood serum was more than those in adult autoserum (P ＜ 0.05).The positive rate of CD29,CD73 and CD105 on BMSCs in umbilical cord serum (above 90%) was higher than those in adult autoserum (P ＜ 0.05),and the positive rates of CD31,CD34 and CD45 were lower than 2%,and the positive rate of CD31 was lower than those in adult autoserum (P ＜ 0.05).The positive rate of BMSCs differentiated into osteoblasts and adipocytes in umbilical cord blood serum was also higher than these in adult autoserum (P ＜ 0.05).CONCLUSION:Purity and differentiated potency of BMSCs in umbilical cord blood serum are better than those in the adult autoserum in vitro.The umbilical cord serum is more adapt to clinical application.