密度梯度离心法结合贴壁培养法分离培养大鼠骨髓间充质干细胞的生物学特性  

Biological characters of rat bone marrow-derived mesenchymal stem cell isolated and cultured with density gradient centrifugation combined with attachment culture method

作　　者：李冬梅[1] 金连弘[1] 张宇[1] 李呼伦[2] 张宝东[1] 刘慧雯[1] 

机构地区：[1]哈尔滨医科大学组胚教研室,黑龙江省哈尔滨市150086 [2]哈尔滨医科大学神经生物教研室,黑龙江省哈尔滨市150086

出　　处：《中国临床康复》2006年第17期18-20,共3页Chinese Journal of Clinical Rehabilitation

摘　　要：目的:建立一种体外分离纯化、培养扩增大鼠骨髓间充质干细胞的方法,观察成年大鼠骨髓间充质干细胞的生物学特性。方法:实验于2003-03/2004-05于哈尔滨医科大学组胚教研室完成。①用密度梯度离心法结合贴壁培养法分离纯化成年大鼠骨髓间充质干细胞。②采用倒置显微镜、透射电镜观察,免疫细胞化学法鉴定骨髓间充质干细胞膜抗原和细胞基质蛋白属性。③体外加成骨、成软骨诱导剂,14d分别做碱性磷酸酶组织化学、Ⅱ型胶原免疫组织化学染色,观察骨髓间充质干细胞的成骨分化及成软骨分化结果。结果:①成年大鼠骨髓间充质干细胞呈均一的成纤维细胞样,具有一般干细胞的超微结构特点。②广泛表达Vimentin,CD29,CD44,不表达CD14,CD34。③加入成骨诱导剂14d后,骨髓间充质干细胞呈多层重叠生长,现碱性磷酸酶强阳性,阳性率为90.5%。④经成软骨诱导后,Ⅱ型胶原免疫组化染色,诱导14d的骨髓间充质干细胞呈现广泛的棕褐色染色,在细胞密集处细胞周围可见黄褐色染色。结论:密度梯度离心结合贴壁培养法能有效分离纯化成年大鼠骨髓间充质干细胞,用此种方法培养的细胞具有骨髓间充质干细胞的一般生物学特性。AIM： To establish a method of in vitro isolation, purification and amplification of bone marrow-derived mesenchymal stem cells （BMMSCs） of adult rat, and obseirve the biological characteristics of BMMSC of adult rats. METHODS： This experiment was conducted at the Department of Histology and Embryology of Harbin Medical University from March 2003 and May 2004. ①BMMSCs of adult rat were isolated and purified by density gradient centrifugation combined with attachment culture method. ②MMSCs membrane .antigen and excellular matrix protein property were detected wth immunocytochemical method under an inverted microscope , transmission electron microscope, ③Osteogeneic inducer and＇ chondrocyte inducer were added in vitro and alkaline phasphotase histochemical and type Ⅱ collagen immunohistological staining were performed on day 14 , respectively . Osteogenous differentiation and cartilage differentiation of BMMSCs were observed. RESUILTS：①The morphology of BMMSCs of adult rats was identical fibroblast-like and had the characteristics of uhrastructure of stem cells, ② Vimentin , CD29 and CD44 antigen could be detected, but not CD14 and CD34 antigen. ③14 days after osteogeneic inducer was added, BMMSCs grew with many layers overlapped, and alkaline phasphotase presented 90.5% strong positive. ④After induced by cartilage , type Ⅱ collagen immunohistological staining was performed . BMMSCs induced for 14 days presented brown and light brown and staining appeared around the cells in the cell-crowded area. CONCLUSION： Density gradient centrifugation combined with attachment method can be used to isolate and differentiate BMMSCs . Cells cultured with this method have general biological characteristics of BMMSCs.

关 键 词：骨髓细胞 干细胞 生物学 大鼠 生物学特性 

分 类 号：R551.3[医药卫生—血液循环系统疾病]