摘要
本研究建立大鼠骨髓间充质干细胞(BMMSC)体外分离培养、表型鉴定和标记的方法,为临床进行细胞移植治疗提供合格的细胞。无菌条件下取大鼠股骨、胫骨,用冲洗法冲出骨髓,贴壁培养法分离纯化BMMSC,体外扩增;流式细胞术进行免疫表型鉴定,并进行成骨,成脂分化鉴定;利用携带绿色荧光蛋白(GFP)基因的慢病毒液转染大鼠BMMSC。结果表明,贴壁培养法能有效分离纯化大鼠BMMSC,细胞呈均一的成纤维细胞样;流式细胞术显示,第4代BMMSC中CD29、CD44表达阳性,CD34、CD45表达阴性。在相应的诱导培养条件下,BMMSC均能成骨及成脂分化。BMMSC用慢病毒液转染后表达GFP。结论:贴壁培养法简单、可行和稳定;分离培养的细胞具有BMMSC的生物学特性和多向分化潜能;BMMSC经慢病毒液转染后表达GFP,标记效率高,可作为标记细胞的一种有效手段,适合体内示踪。
This study was purposed to establish the methods for isolation,culture,identification and labeling of bone marrow mesenchymal stem cells（BMMSC）,so as to provide quantified seed cells for cell transplantation.Bone marrow was collected from SD rat by flushing femur and tibias under sterile condition and BMMSC were purified by adherent culture and amplified in vitro.The immunophenotypes of BMMSC were identified by flow cytometry,the ability of differentiation to osteogenic and adipogenic lineages was detected by alizarin red and oil red O respectively.The BMMSC were transfected by using lentivirus with green flurescence protein（GFP） gene so as to determine GFP expression in BMMSC.The results demonstrated that the method of adherent culture could effectively isolate and purify rat BMMSC which displayed homogenous fibro-like morphology.The flow cytometry showed that BMMSC expressed CD29,CD44,not expressed CD34,CD45.The BMMSC could differentiated into osteoblasts and adipocytes two mesenchymal lineages when grown in specific medium for each lineage.After being transfected by lentivirus,BMMSC could express GFP.It is concluded that the adherent culture is simple,effective,feasible method to separate MSC from the bone marrow of adult rats;the separated and cultured cells exhibit the biological characteristics of BMMSC and differentiating potential.BMMSC can express GFP efficiently and stably in vitro after being transfected by lentivirus,which can be used to label cells for tracing in vivo.
出处
《中国实验血液学杂志》
CAS
CSCD
2011年第6期1472-1476,共5页
Journal of Experimental Hematology
基金
广东省科技计划项目
编号2010B060900027
关键词
骨髓间充质干细胞
慢病毒转染
贴壁培养法
bone marrow mesenchymal stem cell
lentivirus transfection
adherent culture
