大耳白兔骨髓间充质干细胞的分离培养与生物学鉴定

Isolational Culture and Biological Identification of Japanese Large Ear White Rabbit Bone Marrow Mesenchymal Stem Cells

骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)来源于发育早期的中胚层和外胚层,作为干细胞中的种子细胞,具有强大的自我复制和更新能力,并对治疗、修复、补充受损或缺损的肝、肌肉等组织有修复和治疗作用。为了研究BMSCs的生物学特性和多化潜能,对日本大耳白兔BMSCs进行体外分离培养及生物学鉴定研究。取日本大耳白胎兔骨髓组织,采用全骨髓贴壁法分离纯化、体外培养,鉴定日本大耳白兔BMSCs生物学特性,并进行细胞形态学观察、生长动力学检测及其成骨、成软骨、成脂肪能力鉴定。结果表明,采用全骨髓贴壁法成功从日本大耳白胎兔骨髓中分离得到生长状态良好的大耳白兔BMSCs,细胞形态为长梭形,生长曲线呈典型“S”形;免疫荧光和RT-PCR结果显示,大耳白兔BMSCs表达CD29、CD44、CD73、CD105间充质干细胞表面标记物基因,但不表达原始造血祖细胞标志物CD45;经特异性染色和RT-PCR分析表明,分离的细胞是间充质干细胞;体外诱导大耳白兔BMSCs能够分化为骨骼细胞、软骨细胞、脂肪细胞。建立了大耳白兔BMSCs体外分离培养体系,为利用骨髓间充质干细胞进行组织与工程学研究和动物遗传资源保存研究奠定了一定的理论基础。

Bone marrow mesenchymal stem cells(BMSCs)derived from the early development of the mesoderm and ectoderm,as seed cells in stem cells,with strong self-replication and renewal ability,and have repair and therapeutic effects for the damaged or defective liver,muscle and other tissues.In order to study BMSCs,this article conducted in vitro isolation and culture and biological identification of Japanese large ear white rabbit bone marrow mesenchymal stem cells.The bone marrow tissue of Japanese large ear white rabbit was isolated,purified and cultured in vitro by whole bone marrow adhesion method to identify the biological characteristics of Japanese large ear white rabbit BMSCs,observe cell morphology,detect growth kinetics,and identify osteogenesis,cartilage and adipogenic ability.The results showed that BMSCs with good growth status were successfully isolated from the bone marrow of Japanese large ear white rabbits by whole bone marrow adhesion,with long spindle shape and a typical“S”shape in the growth curve.The results of immunofluorescence and RT-PCR showed that BMSCs of Japanese large ear white rabbits expressed CD29,CD44,CD73,CD105 mesenchymal stem cell surface marker genes,but didn’t express the original hematopoietic progenitor cell marker CD45.Specific staining and RT-PCR showed that the isolated cells were mesenchymal stem cells;in vitro induced BMSCs of Japanese large ear white rabbits to differentiate into bone,cartilage and fat cells.In this study,an in vitro isolation and culture system for BMSCs in Japanese large ear white rabbits was established,which laid a theoretical foundation for the use of bone marrow mesenchymal stem cells as tissue and engineering research and the conservation of animal genetic resources.animal genetic resources.