小鼠胚胎干细胞体外定向诱导分化成骨细胞  

Directed Differentiation of Murine Embryonic Stem Cells into Osteoblasts In vitro

作　　者：宋晓平[1] 窦忠英[1] 雷安民[1] 华进联[1] 冯秀亮[1] 屈雷[1] 

机构地区：[1]西北农林科技大学动物科技学院,杨凌712100

出　　处：《农业生物技术学报》2006年第1期7-10,i0001,共5页Journal of Agricultural Biotechnology

基　　金：国家重点基础研究发展规划(973)项目(No.G1999054301);国家高技术研究与发展计划(863)项目(No.2002AA216161);国家自然科学基金项目(No.39970363);陕西省重点攻关项目资助

摘　　要：将小鼠5d龄ES-D3细胞源的类胚体(EBs)单细胞,按5×104/mL接种6孔细胞培养板,在DMEM基础培养基中使EBs单细胞贴壁培养。于第14!21天时,分别添加60%成骨细胞条件培养液(A组)、50μg/mL维生素C"50mmol/Lβ-磷酸甘油(B组)和50μg/mL维生素C"50mmol/Lβ-磷酸甘油"1μmol/L地塞米松(C组),并设不添加诱导剂对照组(D组)。第22天时用1%茜素红染色显示阳性细胞,计算成骨细胞诱导形成率,数据用方差分析和多重比较检验。结果表明,A组细胞增殖呈团状聚集,茜素红染色阳性细胞结节多分布于聚集的细胞群内及其边缘,成骨细胞诱导形成率为10.04%,与对照组比较,差异极显著(P<0.01);B组诱导分化的细胞分泌物形成网状结构,茜素红染色阳性细胞分布在这些网状结构中,成骨诱导形成率为7.43%,与对照组比较差异显著(P<0.05);C组茜素红染色阳性细胞分散而均匀,但未出现网状结构,成骨细胞诱导形成率提高至27.57%,与对照组比较差异极显著(P<0.01)。The cells of 5-day embryoid bodies （EBs） derived from routine ES-D3 lineage were inoculated into each well of 6-well culture plate at 5×10^4/mL in DMEM. During the period from 14 to 21 days of the EBs-derived single cells attachment DMEM was supplemented with the concentration of 60% of conditioned-medicum（group A）, 50 μg/mL ascorbic acid＋50 mmol/L β-glycerophos- phate（group B）, 50 μg/mL ascorbic acid-1-50 mmol/L β- glycerophosphate ＋ 1 μmol/L dexamethasone（group C）, and without inducer （group D）, respectively, and at the 22th day the positive cells were characterized by the formation of discrete mineralized bone nodules that were stained with 1% alizarin red S, and the ratio of obstoblast differentiation were calculated and analyzed by variance analysis and multiple comparison. The results indicated that in group A the cells were dump-like growth and the positve cells stained with alizarin red mostly located inside or around, and the ratio ofobstoblast differentiation was 10.04%, which was extremely significent differences with group D（P 〈0.01）. In group B the secretion of differentiated cells formed net-like structures, and the stained-positive cells located in the centre of the structures and the ratio of obsteoblast differentiation was 7 A3%（P 〈0.05）. In group C the differentiated obsteoblats spreaded uniformly without net-like structures, and the ratio of obsteoblast differentiation was 27.57% （P〈0.01）.

关 键 词：ES细胞 成骨细胞 诱导分化 骨组织工程 

分 类 号：S188[农业科学—农业基础科学]