体外诱导骨髓间充质干细胞成骨表型的变化．  

Experimental study on changes in osteogenic phenotype of induced proliferative human marrow-deo rived mesenchymal stem cells in vitro

作　　者：柳向东[1] 邓辰亮[2] 李卫[1] 黎明[1] 曹谊林[3] 

机构地区：[1]复旦大学附属金山医院烧伤整形科,上海200540 [2]上海交通大学附属第六人民医院整形外科 [3]中国医学科学院整形外科医院

出　　处：《中华实验外科杂志》2013年第1期132-134,共3页Chinese Journal of Experimental Surgery

基　　金：国家高技术研究发展863计划资助项目（2002AA205011）;湖北省自然科学基金资助项目（2007ABA168）

摘　　要：目的探讨诱导人骨髓问充质干细胞（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（MTr）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（AIJP）分泌量和含量，逆转录－聚合酶链反应（RT－PCR）检测细胞ALPmRNA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ALPmRNA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。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 cells was 10. 35% , 11. 48% , 35.28% and 46. 7%. At 12th day after induction, the ALP acitivity of pl, p3, pS, pT, 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 oassa^es of the induced cells, osteoblast ohenotvoe amy be reduced.

关 键 词：骨髓间充质干细胞 分化 成骨细胞 碱性磷酸酶 

分 类 号：R3[医药卫生—基础医学]