摘要
目的:探讨体外诱导培养人骨髓间质干细胞(BMSCs)向成骨细胞分化的方法和技术,为骨组织工程研究提供理想的种子细胞.方法:抽取无系统疾病自愿捐献骨髓患者的骨髓,体外分离诱导培养BMSCs,按照诱导条件加入时间的先后次序分为早期诱导组(A组)和延迟诱导组(B组),镜下观察2组细胞形态学变化,计算倍增时间,并测定2组骨钙素的含量和Ⅰ型胶原的表达,研究该细胞生物学特性及其体外成骨能力.结果:(1)镜下观察:A组细胞贴壁,呈菱形,5～7 d融合,成纤维细胞集落样生长;B组细胞贴壁,呈梭形,3～5 d融合,呈指纹状生长方式.(2)倍增时间:A组第1、2、3、4代倍增时间分别为18.86、17.45、18.71、20.99 h;B组第1、2、3代倍增时间分别为17.54、19.59、25.46 h,第4代停止生长,2组各代倍增时间差异均有统计学意义(P＜0.05).(3)骨钙素:A组各代均可检测到骨钙素,Ⅰ型胶原自原代就有阳性表达,第3、4代细胞达到最强;B组原代、第1代细胞中未检测到骨钙素,诱导后才分泌骨钙素,Ⅰ型胶原各代均有阳性表达,但不均匀,第4代细胞停止增殖,无阳性表达.结论:应用成骨细胞条件培养液在体外能够促进人BMSCs向成骨细胞分化,验证了BMSCs是一种比较理想的骨组织工程种子细胞.早期诱导是较为完善的体外诱导培养条件和方法.
Objective: To investigate the techniques of inducing human bone marrow mesenchmal stem cells (BMSCs) into osteoblasts, in order to provide ideal seed cells for tissue engineering. Methods: Bone marrow was aspirated from volunteer without systematic disease. Which was separated and induced into osteoblasts in vitro according to the sequence of adding the inducible media, two groups were divided: the early induced group and later induced group. To observe their changes of morphology cytokinetics and measuring type I collagen immunology chemistry Von Kcossa, etc. And the BMSCs were respectively term of their cytobiological properties and osteogeneic activity. Results: Observing the early induced group on the microscope, the BMSCs were composed of diamond-shaped cells and approached nearly confluence in 5~7 days formed characteristic colonies of fibroblast unit formation. With the same way observing the later induced group on the microscope, the BMSCs were composed of spindle-shaped cells and approached nearly confluence in 3~5 days formed characteristic colonies of finger print shape. The doubling time of group A from generation Ⅰ to Ⅳ were 18.86 h, 17.45 h, 18.71 h and 20.99h respectively. The doubling time of group B from generation Ⅰ to Ⅲ were 17.54 h, 19.59 h, 25.46 h respectively. The passage Ⅳ ceased to growing. There was different obviously in doubling time of the every passage of two groups, osteocacin could be detected in every passage. Type Ⅰ collagen had positive expression on the primary passage Ⅲ and passage Ⅳ access to the climax. Osteocacin could not be detected in the primary and passage Ⅰ, but could be detected after being induced. Type Ⅰ collagen expressed in each passage but uneven. The passage Ⅳ ceased to generation without positive expressed. Conclusion: The usage of inducible media can promote the BMSCs differentiation of osteoblasts and indicates that BMSCs is a kind of ideal seed cells for boning tissue engineering. Inducing earlier is a consummate condition and method o
出处
《新疆医科大学学报》
CAS
2006年第11期1021-1025,共5页
Journal of Xinjiang Medical University
基金
上海市重点学科(特色学科)建设项目资助(T0202)
关键词
组织工程
种子细胞
人骨髓间质干细胞
tissue engineering
seed cells
human bone marrow mesenchymal stem cells
