摘要
目的:采用地塞米松、β-磷酸甘油和维生素C3种物质作为诱导骨髓间充质干细胞(bonemarrow stromal stem cells,BMSCs)向成骨细胞分化的基本辅剂,定向诱导犬第2代BMSCs,探讨BMSCs向成骨细胞分化的潜能和成骨细胞的特征性鉴定方法。方法:无菌条件下行犬髂骨穿刺,采集骨髓15～20mL,全骨髓10%FBS DMEM培养液进行原代培养。将第2代纯化的BMSCs细胞悬液(调整细胞密度为1×105/mL)接种于含地塞米松、β-磷酸甘油和维生素C的DMEM/FI2培养液的培养瓶(皿)中,体外扩增培养、传代。对细胞形态特征进行观察;行碱性磷酸酶(ALP)染色,骨桥素以及Ⅰ型胶原相关蛋白免疫荧光化学检测鉴定细胞性质。结果:原代培养的BMSCs形态呈长梭形或不规则形,呈均匀分布生长。诱导分化后的细胞形态呈长梭形或不规则形,呈均匀分布生长,传代后细胞体积略变大;BMSCs成骨诱导增殖后电镜下细胞呈多边形,可见ALP染色阳性;经成骨细胞诱导培养14d后,细胞骨桥素以及Ⅰ型胶原相关蛋白表达阳性。结论:BMSCs易于体外分离培养及扩增,地塞米松、β-磷酸甘油和维生素C体外定向诱导能够在短时间内获得大量成骨细胞,且所培养的细胞具有典型的成骨细胞形态和功能。
Objective To investigate isolation and culture of canine bone marrow-stromal stem cells（BMSCs） and conditions of induced differentiation of BMSCs into osteoblasts in vitro.Methods The BMSCs were isolated from bone marrow of canine iliac bone and cultured in DMEM/F12 culture media for one week,then the second generation BMSCs were continued to be induced by osteogenic medium（1 × 10-8 mol/L dexamethasone,10 mmol/L β-sodium glycerophosphate and 50 μg/mL vitamine C）.After BMSCs were induced by osteogenic medium for 7 to 14 days,the cellular morphology was observed,OBs was identified by alkaline phosphatase（ALP） staining,the expression of osteocalcin（BGP）,type Ⅰ collagen（COLⅠ）were detected by immunofluorescence.Results The primary cultured BMSCs were long spindle shape or irregular shape,and grew in evenly distributing.The cells were usually subcultured in 5 or 7 days.After subculture,the cells would be larger than before.After cultured by osteogenic medium for 7 to 14 days,the expression of ALP,BGP and COLⅠof the BMSCs were detected.Conclusions The BMSCs were isolated,cultured and amplified easily in vitro.The BMSCs have potential to differentiate into osteoblast cells,which can be used as seed cells for bone tissue engineering.
出处
《实用医学杂志》
CAS
北大核心
2012年第9期1401-1404,共4页
The Journal of Practical Medicine
基金
国家自然科学基金资助项目(编号:30772194)
关键词
骨髓祖代细胞
成骨细胞
诱导分化
组织工程
Myeloid progenitor cells
Osteoblast cell
Induced differentiation
Tissue engineering
