人脐带源间充质干细胞分离培养方法的改进

发布时间：2018-05-03 23:09

本文选题：干细胞 + 脐带脐血干细胞　；参考：《中国组织工程研究》2014年10期

【摘要】：背景:脐带来源的间充质干细胞因其具有高度的自我更新和多向分化潜能,以及取材方便等优点而日益受到关注。目的:建立一种改进的人脐带间充质干细胞分离、培养方法,并对其生物学特性进行分析。方法:无菌条件下获取足月妊娠分娩胎儿脐带,利用改良的组织块贴壁法分离培养脐带间充质干细胞,即将传统组织贴壁法中本应丢弃的组织转移到新的培养瓶中进行二次贴壁培养,取第3代脐带间充质干细胞进行生物学特性分析。结果与结论:组织贴壁后第5-7天可见有梭形细胞从组织块边缘爬出,第10天左右可形成明显的细胞克隆。将组织块转移到新培养瓶中继续培养,2 d后即可见有细胞爬出,细胞生长速度较快,5 d即可形成细胞克隆。传代后的细胞形态均一,呈成纤维细胞样的长梭形。流式细胞仪检测细胞高表达CD90、CD105,不表达CD34、CD45、HLA-DR。细胞增殖能力旺盛,平均倍增时间为50 h左右,41.24%的细胞处于G2/S期。体外可诱导分化为成骨细胞和脂肪细胞。上述实验结果证明二次贴壁培养出的细胞也具有间充质干细胞的生物学特性,而且通过这种培养方法获得的原代间充质干细胞数是传统方式培养的2倍。
[Abstract]:Background: umbilical cord derived mesenchymal stem cells (MSCs) have attracted more and more attention due to their high potential for self-renewal, multi-differentiation and easy access to materials. Aim: to establish an improved method for isolation and culture of human umbilical cord mesenchymal stem cells and analyze its biological characteristics. Methods: umbilical cord was obtained under aseptic condition. Umbilical cord mesenchymal stem cells were isolated and cultured by modified tissue mass adherence method. The tissue which should be discarded in the traditional tissue adherence method was transferred to a new culture bottle for secondary adherent culture, and the biological characteristics of the third generation umbilical cord mesenchymal stem cells were analyzed. Results & conclusion: shuttle cells could be seen crawling out from the edge of tissue mass on the 5th to 7th day after tissue adhesion, and obvious cell clones could be formed on the 10th day. After the tissue mass was transferred to the new culture flask for 2 days, it was observed that some cells were crawling out, and the cell clone could be formed at a rapid growth rate of 5 days. After passage, the cells were homogeneous in shape and appeared as long fusiform as fibroblasts. Flow cytometry was used to detect the high expression of CD90, CD105, and no expression of CD34, CD45, HLA-DR. The average doubling time was about 50 h. 41.24% of the cells were in G _ 2 / S phase. Osteoblasts and adipocytes can be induced to differentiate into adipocytes in vitro. The results show that the secondary adherent cells also possess the biological characteristics of mesenchymal stem cells, and the number of primary mesenchymal stem cells obtained by this method is twice as much as that of traditional culture.
【作者单位】：天津大学化工学院制药工程系系统生物工程教育部重点实验室;解放军沈阳军区总医院;北京工业大学生命科学与生物工程学院;军事医学科学院放射与辐射研究所;
【分类号】：R329

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