CAJ | 학술논문

目的探讨诱导人骨髓间充质干细胞(hBMSCs)分化为成骨细胞过程中表型变化.方法抽取骨髓分离单个核细胞,用DMEM条件培养基诱导hBMSCs向成骨细胞分化；相差显微镜观察成骨细胞形态,噻唑蓝(MTT)比色法检测细胞增殖,分别应用对硝基苯磷酸盐法和BM-Purple法测定成骨细胞碱性磷酸酶(ALP)分泌量和含量,逆转录-聚合酶链反应(RT-PCR)检测细胞ALP mRNA的表达.结果在诱导第5、10、15、20d,改良Gomori染色细胞阳性率分别为(10.35±3.79)％、(11.48±5.92)％、(35.28±7.05)％和(46.7±6.18)％,诱导至12d,p1、p3、p5、p7、p9成骨细胞ALP活性测定结果分别为:60.90±2.95、61.36±1.95、37.42±4.06、20.67±1.98、7.63±1.08.培养代次小于(等于)p5的细胞可见ALP mRNA表达,而p7、p9细胞未见表达.结论 hBMSCs经定向诱导后可以分化为成骨细胞,随着诱导细胞传代次数的增加成骨表型降低.
목적 탐토유도인골수간충질간세포(hBMSCs)분화위성골세포과정중표형변화.방법 추취골수분리단개핵세포,용DMEM조건배양기유도hBMSCs향성골세포분화；상차현미경관찰성골세포형태,새서람(MTT)비색법검측세포증식,분별응용대초기분린산염법화BM-Purple법측정성골세포감성린산매(ALP)분비량화함량,역전록-취합매련반응(RT-PCR)검측세포ALP mRNA적표체.결과 재유도제5、10、15、20d,개량Gomori염색세포양성솔분별위(10.35±3.79)％、(11.48±5.92)％、(35.28±7.05)％화(46.7±6.18)％,유도지12d,p1、p3、p5、p7、p9성골세포ALP활성측정결과분별위:60.90±2.95、61.36±1.95、37.42±4.06、20.67±1.98、7.63±1.08.배양대차소우(등우)p5적세포가견ALP mRNA표체,이p7、p9세포미견표체.결론 hBMSCs경정향유도후가이분화위성골세포,수착유도세포전대차수적증가성골표형강저.
Objective To investigate the phenotype change of human bone marrow mesenchymal stem cells (hBMSCs) during the differentiation into osteoblasts.Methods Bone marrow mononuclear cells were induced to differentiate into osteoblasts with DMEM conditioned medium.Osteoblasts were observed under a phase contrast microscope.Methyl thiazol tetrazolium (MTT) assay was used to detect cell proliferation,the nitrophenylphosphate and BM-Purple were used to determin the amount and content of osteoblast alkaline phosphatase (ALP) secretion,and reverse transcription-polymerase chain reaction (RT-PCR) was used to detect ALP mRNA expression.Results During the induction of the first 5,10,15,20 days,the positive rate of Gomori staining ceils was 10.35％,11.48％,35.28％ and 46.7％.At 12th day after induction,the ALP acitivity of p1,p3,p5,p7,p9 osteoblasts was 60.90 ±2.95,61.36 ±1.95,37.42 ± 4.06,20.67 ± 1.98,and 7.63 ± 1.08 respectively.In the cells with the generation of culture less than (equal to) p5,visible ALP mRNA expression was detectable,while p7 and p9 cells did not express ALP mRNA.Conclusion After oriented induction,the hBMSCs can differentiate into osteoblasts,and with the increase in the passages of the induced cells,osteoblast phenotype amy be reduced.