摘要
目的建立一种简便有效的体外分离纯化及培养扩增大鼠骨髓间充质干细胞(BMSCs)的方法,研究BMSCs的生物学特性,为后续细胞移植和基因治疗提供理想的种子细胞。方法全骨髓贴壁培养法分离大鼠BMSCs,体外培养和连续传代,在倒置相差显微镜下连续观察细胞的形态变化,利用MTT法测定其生长曲线,流式细胞仪鉴定其表面抗原,成骨、成脂肪诱导分化鉴定其多向分化潜能。结果大鼠BMSCs体外培养生长状况良好,可见BMSCs呈均一的梭形和多角形。流式细胞仪检测显示BMSCs表达CD29、CD90,不表达CD34、CD45。经体外诱导后具有多向分化潜能。结论全骨髓贴壁培养法适合体外分离、扩增和纯化大鼠BMSCs,培养的大鼠BMSCs具有间充质干细胞的一般生物学特性,为后续基因修饰BMSCs及其体内移植实验奠定良好的基础。
Objective To establish a simple and effective method of isolation,purification and culture of rat bone marrow-derived mesenchymal stem cells in vitro,and explore their biological characteristics in order to provide an ideal seed cell for stem cells transplantation and gene therapy.Methods BMSCs were isolated by the attachment culture method.They were cultured and passaged continuously,their morphological changes were observed under an inverted phase contrast microscope.Their growth curves were measured by MTT method,and their membrane antigens were detected by flow cytometry.Osteoblastic differentiation and adipocytic differentiation were induced in order to study their potential of multiple differentiation.Results The morphology of BMSCs was identical shuttle-like or polygon-like and the cells showed active proliferative ability.CD29 and CD90 antigen expression was positive in BMSCs,while CD34 and CD45 antigen expression was negative,assayed by flow cytometry.The cells showed their potential of multiple differentiations after induced.Conclusion The attachment culture method can be used to isolate,expand and purify BMSCs in vitro.Cultured BMSCs show stable and rapid growth and have general biological characteristics of mesenchymal stem cells,which lay solid foundation for gene-modified BMSCs and their transplantation in vivo.
出处
《海南医学》
CAS
2011年第8期25-29,共5页
Hainan Medical Journal