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。
BACKGROUND：Human umbilical cord mesenchymal stem cells with capabilities for self-renewal and multi-differentiation have attracted widespread attention. OBJECTIVE：To develop an efficient method for isolation and culture of human umbilical cord mesenchymal stem cells, and to analyze the cellbiological features. METHODS：Mesenchymal stem cells were isolated and cultured from human umbilical cord by improved tissue cultivation. Immunophenotype and cellcycle were analyzed by flow cytometry. Growth curve was determined by MTT assay, and differentiation ability was evaluated by in vitro osteogenic and adipogenic induction as wel . RESULTS AND CONCLUSION：Some fusiform cells crawled out from human umbilical cord tissues after cultivation for 5 days and formed colonies about 10 days later. When the removed tissues were further cultured, more cells appeared again within 2 days and formed colonies after 5 days. The isolated cells exhibited similar morphology of fibroblast-like shape after passage. Furthermore, the cells expressed CD90, CD105, but were negative for the markers of CD34, CD45, HLA-DR. Population doubling time of the cells calculated from the result of MTT was about 50 hours and cellcycle analysis showed that 41.24%cells were in the G 2/S phrase. Therefore, the isolated cells had a high prolification ability. In addition, the isolated cells could be induced into osteoblasts＆amp;nbsp;and adipocytes in vitro. In a word, the results of this study demonstrated that the cells from the second tissues culture possessed the biological characteristics of mesenchymal stem cells and more primary umbilical cord mesenchymal stem cells were acquired through the improved method.
出处
《中国组织工程研究》
CAS
CSCD
2014年第10期1609-1614,共6页
Chinese Journal of Tissue Engineering Research
