大鼠骨髓间充质干细胞的原代培养及鉴定

Primary Culture and Identification of Bone Marrow Mesenchymal Stem Cells from Rat

目的:研究大鼠BMSCs(骨髓间充质干细胞)原代培养与纯度鉴定的方法。方法:无菌环境中,从SD大鼠股骨与胫骨端采集骨髓,先行酶消化,利用全骨髓细胞悬液贴壁法对提取BMSCs实施传代培养,选取生长良好的第3代细胞进行鉴定;对BMSCs实施成脂与成骨诱导分化,同时经由油红O(ORO)与茜素红(ARS)染色法对诱导分化效果加以鉴定;借助流式细胞术(FCM)对CD34、CD44与CD90这3类BMSCs表面标志物的表达展开分析。结果:BMSCs是长梭状贴壁细胞,生长状态为纤维细胞样漩涡状;在第3代BMSCs传代期间,其第1-3 d发展至生长潜伏期,呈较慢速的生长;第3-5 d发展至对数生长期,呈高速生长;待至第7 d长速增殖最大,速度停止上升进入平缓期;BMSCs成骨、成脂诱导结束后,对其诱导分化鉴定发现:细胞出现明显形态学变化,通过ORO对脂肪染色,细胞显示橘红色;待成骨诱导培养结束,通过ARS对钙盐染色,显示红色,且出现矿化结节沉积,说明BMSCs具有良好的成骨、成脂分化能力;FCM测定发现:CD34表达呈阴性(1.09%),CD90(96.8%)与CD44(92.4%)皆呈阳性,与BMSCs表型相符。结论:经由全骨髓黏附培养技术有效分离BMSCs,且完成培养。

Objective:To study the methods of primary culture and purity identification of bone marrow mesenchymal stem cells.Methods:In a sterile environment,the bone marrow was collected from the femur and tibia of SD rats,digested with enzymes first,and subcultured the extracted BMSCs by using the whole bone marrow cell suspension adherence method,and the third generation cells with good growth were selected for identification;BMSCs were induced to differentiate into adipogenic and osteogenic,and the differentiation effect of BMSCs was identified by oil red O(ORO)and alizarin red(ARS)staining;CD34,CD44 and CD90 were detected by flow cytometry(FCM).The expression of three types of BMSCs surface markers was analyzed.Results:BMSCs are long spindle-shaped adherent cells,and the growth state is fibroblast-like swirl.During the passage of the third generation BMSCs,they developed to the growth latent period on the 1st-3d,showing a slower growth rate,and developed on the 3rd-5d.In the logarithmic growth phase,it grows at a high speed.After the 7th day,the growth speed reaches the maximum,and the speed stops increasing and enters the plateau phase.After the induction of osteogenesis and adipogenicity of BMSCs,the identification of the induced differentiation found that the cells showed obvious morphological changes,Staining fat by ORO,the cells showed orange red;after the end of osteogenic induction culture,staining calcium salt by ARS showed red,and mineralized nodules appeared,indicating that BMSCs had good osteogenic and adipogenic differentiation capabilities;FCM It was found that the expression of CD34 was negative(1.09%),and both CD90(96.8%)and CD44(92.4%)were positive,which was consistent with the phenotype of BMSCs.Conclusions:BMSCs were effectively isolated and cultured by the whole bone marrow adherent culture technique.