CAJ | 학술논문

目的研究成人骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)体外培养定向诱导分化为成骨细胞,探讨其作为骨组织工程种子细胞的可行性和应用价值.方法抽取健康成年骨髓,Ficoll密度梯度离心结合贴壁培养法经连续传代后改用含10 nmol/L地塞米松、10 mmol/Lβ-甘油磷酸钠、50 μmol/L维生素C的条件培养基培养,在相差显微镜下观察细胞形态变化,流式细胞仪细胞表面分子标志鉴定,免疫组化和RT-PCR检测Ⅰ型胶原蛋门的表达,同时测定细胞内碱件磷酸酶的含量.结果 BMSCs原代和传代培养的细胞具有活跃的增殖能力,诱导培养后细胞形态向成骨细胞转化,经RT-PCR、流式细胞仪、细胞碱性磷酸酶活性、糖原染色鉴定为成骨细胞.结论 BMSCs经合理的体外诱导培养后符合成骨细胞的形态特征和生物学特性,有望成为理想的骨组织工程种子细胞来源.
목적 연구성인골수간충질간세포(bone marrow mesenchymal stem cells,BMSCs)체외배양정향유도분화위성골세포,탐토기작위골조직공정충자세포적가행성화응용개치.방법 추취건강성년골수,Ficoll밀도제도리심결합첩벽배양법경련속전대후개용함10 nmol/L지새미송、10 mmol/Lβ-감유린산납、50 μmol/L유생소C적조건배양기배양,재상차현미경하관찰세포형태변화,류식세포의세포표면분자표지감정,면역조화화RT-PCR검측Ⅰ형효원단문적표체,동시측정세포내감건린산매적함량.결과 BMSCs원대화전대배양적세포구유활약적증식능력,유도배양후세포형태향성골세포전화,경RT-PCR、류식세포의、세포감성린산매활성、당원염색감정위성골세포.결론 BMSCs경합리적체외유도배양후부합성골세포적형태특정화생물학특성,유망성위이상적골조직공정충자세포래원.
Objective To establish an ideal culture method on the differentiation of the adult human bone marrow mesenchymal stem cells(BMSCs)to osteoblasts in vitro. Methods The bone marrows were obtained bv aspiration from healthy volunteers.BMSCs were isolated and purified by modified ficoll density gradient centrifugation combined with adherence culture method. MSCs were induced to osteoblasts in vitro in a condition medium in subculture including dexamethasone,vitamin C,and β-glycerophosphate. The morphological changes and growth characteristics were examined by phase-contrast microscopy. Cell surface markers CD34 and CD45 were tested by flow cytometric analyses. Alkaline phosphatase activity and expression of type I collagen were also studied.Results Adult BMSCs presented active proliferation in primary and passage cultures in vitro.Cells were spindleshaped.The cell surface markers CD34 and CD45 were positive before differentiation and were negative after differentiation. Cells were successfully induced into osteoblasts. They were identified through morphological character,specific surface antigens and osteogenic differentiation. Immunochemical expression of type I collagens of the differentiated cells revealed the characteristics of osteogenic differentiation with strong staining.Alkaline phosphatase activity were increase. Conclusion The osteoblasts could be induced from human BMSCs,which have typically morphological and biological characteristics of osteoblasts.