摘要
目的通过透射电子显微镜对体外培养过程中不同代次的人骨髓基质干细胞的超微结构的观察分析,进一步了解骨髓基质干细胞向成骨细胞分化的过程。方法将临床自愿捐献骨髓的无血液系统疾病患者的骨髓体外分离培养诱导,分别在原代,1代,2代,3代细胞达到融合后,2.5%戊二醛4℃原位固定1h后,送透射电子显微镜检测,观察细胞内细胞器、细胞间质及钙盐沉积状况,免疫组化检测Ⅰ型胶原、碱性磷酸酶染色及VonKossa染色,放免法测定骨钙素分泌情况。结果透射电镜显示,1代、2代、3代的骨髓基质干细胞超微结构有明显变化,第1代细胞内出现大量的溶酶体;第2代细胞内粗面内质网、高尔基体、线粒体增多;第3代细胞外基质中出现排列规律的胶原纤维以及初期和成熟的钙化基质。免疫组化I型胶原表达第3代细胞达到最强;碱性磷酸酶染色第2、3代ALP染色蓝紫色颗粒加深、增多;放免法测定骨钙素分泌第3代细胞达到最强。结论骨髓基质干细胞经过体外培养诱导从原代到第3代逐步向成骨细胞方向分化,具备成骨细胞的特征。由透射电镜反映出的细胞器及细胞外间质的变化为进一步探讨骨髓基质干细胞的分化机制提供了一定的实验依据。
Objective To investigate the ultrastructure of human bone marrow mesenchymal stem cells （BMSCs） during differentiation and proliferation procedure in vitro by transmitting electron microscope （TEM）.Methods The human BMSCs were aspirated from the iliac crests, which will be induced into the phenotype of osteoblasts in vitro. Along with the proliferation and differentiation, the BMSCs were studied the cytobiological properties by ultrastructure observation with different passages. Type Ⅰ collagen, alkaline phosphatase and ostocalcin were also measured. The Von Cossa stain was done with different passages.Results Human BMSCs had achieved the osteogenic phenotype after being induced in vitro. There were obvious differences in ultrasturcture between each passage of BMSCs. In passage Ⅰ there were lots of lososomes; in passage Ⅱ there were many rough endoplasmic reticulums and mitochanadrions; in passage Ⅲ there were collagen fibers in order and calcified matrix. The BMSCs of passage Ⅲ had the strongest proliferative ability and phenotype of osteoblast.Conclusion Human BMSCs can meet the demand of bone tissue engineering , through culturing and induced in vitro. This test can prove that after being induced BMSCs can have the strong osteogenic activity.
出处
《现代口腔医学杂志》
CAS
CSCD
北大核心
2007年第4期381-384,共4页
Journal of Modern Stomatology
基金
上海市重点学科(特色学科)建设项目资助(T0202)
国家自然基金(30271426)
关键词
骨髓基质干细胞
成骨细胞
分化
超微结构
Bone marrow mensenchymal stem cells Osteoblast Differentiation Ultrastructure
