成骨诱导的兔骨髓基质干细胞成骨活性的表达及维持  

Expression and maintenance of osteogenic activity of induced rabbit bone marrow stromal cells

作　　者：陈光兴[1] 杨柳[1] 唐康来[1] 段小军[1] 李忠[1] 周红星[1] 顾祖超[1] 林炎水[1] 

机构地区：[1]第三军医大学西南医院关节外科中心,重庆市400038

出　　处：《中华创伤骨科杂志》2004年第7期735-739,共5页Chinese Journal of Orthopaedic Trauma

基　　金：国家自然科学基金资助项目(30270375);全军十五医药卫生科研重点基金资助项目(01Z072)

摘　　要：目的观察成骨诱导的兔骨髓基质干细胞(BMSCs)体内、外环境下成骨活性的表达及维持。方法观察BMSCs在体外成骨诱导培养条件下的成骨分化特性;构建兔BMSCs与活骨组织共培养模型模拟体内“成骨环境”,将成骨诱导的MSCs置于共培养及普通传代培养条件下进行传代培养,观察经成骨诱导的BMSCs在体外及模拟体内的培养条件下细胞的表型维持情况。结果药物成骨诱导培养的BMSCs,其ALP活性及骨钙素均显著高于普通培养组(P<0.05);经过诱导培养的BMSCs,其Ⅰ型胶原、骨钙素免疫组化阳性。RT-PCR法半定量测定Ⅰ型胶原mRNA,成骨诱导培养的Ⅰ型胶原mRNA表达量明显高于普通传代培养对照组。药物成骨诱导后的细胞在体外普通传代培养传5代后,细胞碱性磷酸酶(ALP)活性、骨钙素水平及Ⅰ型胶原表达稳定维持在较高水平,保持其成骨细胞的表型;在共培养条件下,ALP活性、骨钙素水平Ⅰ型胶原表达保持在高水平,且ALP活性、骨钙素水平在大部分时间点均高于普通传代培养。结论药物成骨诱导培养呈现促BMSCs向成骨方向转化的特点,能使ALP、骨钙素及Ⅰ型胶原表达短期内达到高水平;经成骨诱导的BMSCs在体外或模拟的体内传代培养条件下,均能维持成骨表型,保持成骨活力。Objective To investigate the expression and maintenance of osteoblast phenotype differentiation of rabbit bone marrow stromal cells(BMSCs) induced by conditional culture in vitro and vivo. Methods A coculture model was designed to simulate “osteogenic enviroment”and the osteogenic characteristics of induced BMSCs under different culture conditions( induction culture,co-culture and passage culture) were studied. Results Induced BMSCs maintained ALP (alkaline phosphatase) activities and expressed osteocalcin and typeⅠ collagen steadily in vitro and in co-culture (simulated osteogenic enviroment in vivo) . Osteoblast differentiation phenotypes of induction cultured and co-cultured BMSCs were verified by ALP activities, ALP staining and expression of typeⅠ collagen and osteocalcin, but BMSCs cultured in DMEM/F12 medium did not show significant osteogenic capacity. This indicated that the BMSCs cultured in induction medium and cocultured with fresh bone particles for 2 weeks had significant osteogenic capacity. But in induction condition, BMSCs expressed obvious osteogenic differentiation phenotypes in a shorter time . Conclusions Induced BMSCs maintain steadily osteogenic differentiation phenotype during passage culture. BMSCs cultured in induction medium and co-cultured with fresh bone particles for 2 weeks both have significant osteogenic capacity. But in induction condition, BMSCs express osteogenic differentiation phenotype in a short time.

关 键 词：骨髓基质干细胞 成骨分化 共培养 成骨诱导 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学]