CAJ | 학술논문

目的探讨Nucleostemin(NS)在大鼠骨髓基质干细胞向成骨细胞诱导分化中的机制及作用.方法取4～6周龄雄性SD大鼠两侧股骨、胫骨骨髓基质细胞,原代及传代培养.取第3代骨髓间充质干细胞分别用普通和矿化培养基培养,通过相差倒置显微镜观察细胞生长、MTT法检测细胞增殖、碱性磷酸酶和茜素红钙染色了解成骨活性.免疫组化SABC法及免疫荧光法检测NS在细胞中的表达情况,比较NS分别在普通培养基和成骨细胞诱导培养基作用下的表达情况.结果大鼠骨髓间充质干细胞在矿化培养基诱导下其NS的表达较普通培养基培养下的表达明显减弱,且呈时间依赖性衰减.成骨细胞的NS表达则为阴性.在矿化液作用下,骨髓间充质干细胞可诱导为成骨细胞.MTT显示矿化液培养细胞生长潜伏期长,对数生长期较对照组延长.结论 NS作为核干细胞因子,在干细胞中和某些肿瘤细胞中表达丰富.细胞分化后,其表达明显减少.因此,NS很可能作为大鼠基质干细胞是否具有潜在分化能力的标志性因子.
목적 탐토Nucleostemin(NS)재대서골수기질간세포향성골세포유도분화중적궤제급작용.방법 취4～6주령웅성SD대서량측고골、경골골수기질세포,원대급전대배양.취제3대골수간충질간세포분별용보통화광화배양기배양,통과상차도치현미경관찰세포생장、MTT법검측세포증식、감성린산매화천소홍개염색료해성골활성.면역조화SABC법급면역형광법검측NS재세포중적표체정황,비교NS분별재보통배양기화성골세포유도배양기작용하적표체정황.결과 대서골수간충질간세포재광화배양기유도하기NS적표체교보통배양기배양하적표체명현감약,차정시간의뢰성쇠감.성골세포적NS표체칙위음성.재광화액작용하,골수간충질간세포가유도위성골세포.MTT현시광화액배양세포생장잠복기장,대수생장기교대조조연장.결론 NS작위핵간세포인자,재간세포중화모사종류세포중표체봉부.세포분화후,기표체명현감소.인차,NS흔가능작위대서기질간세포시부구유잠재분화능력적표지성인자.
Objective to discuss the mechanism and the function of NS by differentiating rat bone marrow stem cells into osteoblasts in vitro.Methods The BMSCs isolated from the femur and tibia of 4～6-week-old male SD rat were cultured in vitro.The BMSCs of passage 3 were divided into two parts and cultured in different medium.Part A was sub cultured by L-DMEM medium and Part B added mineralization condition medium as the inducers.The morphological characters of BMSCs and cell proliferation were observed with phase contrast microscope and MTT test.Osteoblasts activities of the sub culturing cells were detected by alkaline phosphatase staining and alizarin red S staining.The expression of NS in BMSCs and induced cells were showed and compared by Immunohistochemical and immunofluorescence staining.Results NS in the induced BMSCs is hightly expressed than the cells which is cultured by L-DMED medium.The Immunohistochemical staining shows that the longer BMSCs induced the lower expression of NS.The NS is negative expressed in osteoblasts.The BMSCs can be induced into osteoblasts in mineralization condition medium.Alkaline phosphatase staining and alizarin red S staining were both positive.Conclusion NS is a nucleolar protein predominantly associated with proliferating in BMSCs.It was mainly localized in the nucleoli.When bone marrow stem cells were differentiated into osteocytes,NS expression hightly decreased compared to the undifferentiated cells.NS is a marker of undifferentiated BMSCs in SD rat and that it is involved in the regulation of proliferation of these cells.