不同代次人脐带间充质干细胞的生物学特性及分化  

Biological features and differentiation of human umbilical cord mesenchymal stem cells at different passages

作　　者：刘雨思 王凤昌[1] 夏海雄 许键炜[1] 杨燕[1] 崔冬冰[1] 王黎龙 何志旭[1,2,3] 舒莉萍[1,2,4] 

机构地区：[1]贵州医科大学,组织工程与干细胞实验中心,贵州省贵阳市550004 [2]贵州医科大学,基础医学院免疫学教研室,贵州省贵阳市550004 [3]贵州医科大学,儿童医学中心,贵州省贵阳市550004 [4]贵州医科大学,实验动物中心,贵州省贵阳市550004

出　　处：《中国组织工程研究》2017年第13期2015-2022,共8页Chinese Journal of Tissue Engineering Research

基　　金：贵州省“125计划”重大科技专项(黔教合重大专项字[2013]020)资助~~

摘　　要：背景:人脐带间充质干细胞作为组织工程及再生医学领域的新兴种子细胞,其应用价值有赖于能否在长期体外传代扩增后仍保持稳定生物学特性和多向分化能力,需要进一步的实验研究。目的:研究不同培养代次人脐带间充质干细胞重要生物学属性的差异。方法:采用组织块法分离培养人脐带间充质干细胞,分别采用细胞计数法与MTT法检测第3-6代细胞增殖活性;流式细胞仪检测第1-9代细胞表面标志物;细胞染色法分析第3-6代细胞核型。取第3代人脐带间充质干细胞,检测其分化为神经细胞、成骨细胞及肝样细胞的能力。结果与结论:(1)细胞计数法与MTT方法检测结果一致,第3-6代脐带间充质干细胞的增殖曲线均呈"S"型,在一二天时处于缓慢增长期,2-4 d时处于对数增长期,四五天时处于平台期,五六天时处于衰退期;(2)第1-9代脐带间充质干细胞表面高表达CD90、CD44、CD73,不或低表达CD11b、CD18、CD34、CD43及HLA-DR;(3)第3-6代脐带间充质干细胞核型均为正常人体核型;(4)脐带间充质干细胞可分化为神经细胞、成骨细胞及肝样细胞;(5)结果表明,人脐带间充质干细胞具有高度增殖活性和多向分化特性。BACKGROUND：As a kind of emerging seed cells in the field of tissue engineering and regenerative medicine,human umbilical cord mesenchymal stem cells are applied depending on whether it can remain stable biological characteristics and multi-directional differentiation capacity after long-term passage in vitro, which needs further experimental studies.OBJECTIVE：To investigate differences of vital biological features of human umbilical cord mesenchymal stem cells over different passages.METHODS：Human umbilical cord mesenchymal stem cells taken from Wharton＆#39;s jelly were isolated and cultured using tissue explant method. The proliferation abilities of cells cultured for 3-6 generations were detected using both cell counting method and MTT method. Expressions of surface markers of the cells after expanding over nine passages were comprehensively determined by flow cytometry. Karyotype analysis of cells cultured for 3-6 generations was assessed using cell staining method. The 3rd generation of cells were used to detect differentiation abilities into nerve cells,osteoblasts and hepatocytes.RESULTS AND CONCLUSION：Proliferation curve of cultured cells was ＆quot;S＆quot; shape, and it was in the slow growth phase within 1-2 days, in the increased logarithmic growth phase within 2-4 days, in the plateau growth phase within 4-5 days,and in the declining growth phase within 5-6 days. Furthermore, two methods of testing results were consistent.Expression levels of cell surface markers CD90, CD44, CD73 were all above 90% while the expression levels of CD11b,CD18, CD34, CD43 and HLA-DR were very low at passages 1-9. No abnormality in cell karyotype was found in cells at passages 3-6. Human umbilical cord mesenchymal stem cells could differentiate into nerve cells, osteoblasts and hepatocytes. All theses experimental findings show that the human umbilical cord mesenchymal stem cells have high proliferation activity and multi-directional differentiation properties.

关 键 词：干细胞 脐带 间质干细胞 组织工程 脐带脐血干细胞 间充质干细胞 生物学特性 组织块贴壁培养 增殖 表型 染色体制备 诱导分化 三胚层 

分 类 号：R394.2[医药卫生—医学遗传学]