地塞米松浓度与脐带间充质干细胞的成骨分化  

Osteogenic differentiation of human umbilical cord mesenchymal stem cells induced with different concentrations of dexamethasone in vitro

作　　者：洪敬欣 刘剑 李琳芳[2] 韩俊领[1,3] 

机构地区：[1]协和干细胞基因工程有限公司,天津市300384 [2]解放军第二军医大学东方肝胆外科医院病毒基因治疗实验室,上海市200438 [3]中国医学科学院,北京协和医学院血液学研究所,血液病医院,天津市300020

出　　处：《中国组织工程研究》2013年第23期4204-4211,共8页Chinese Journal of Tissue Engineering Research

摘　　要：背景:在地塞米松、维生素C和β-甘油磷酸钠的共同作用下,人脐带间充质干细胞可以向成骨细胞分化。作为糖皮质类激素的地塞米松,其浓度对人脐带间充质干细胞体外成骨分化存在着影响。目的:探寻人脐带间充质干细胞向成骨细胞诱导分化过程中地塞米松的优选浓度。方法:采用植块法从正常足月新生儿脐带中分离培养间充质干细胞。取第3代细胞进行日常细胞培养和诱导分化实验。流式细胞术检测所获细胞的表面标记表达情况,成脂、成骨诱导分化鉴定人脐带间充质干细胞。倒置相差显微镜观察成骨分化过程中细胞形态的变化。以含1×10-8mol/L,5×10-8mol/L,1×10-7mol/L3种浓度地塞米松的成骨诱导液对人脐带间充质干细胞进行成骨诱导。盐酸四环素荧光及Von Kossa染色法对比观察钙结节形成。反转录-聚合酶链反应法对比检测细胞骨桥蛋白基因表达。结果与结论:植块法分离的人脐带间充质干细胞形态均一,多为梭形,平行排列生长或旋涡状生长。流式细胞术检测CD29,CD73和CD90呈阳性表达,CD31,CD34和HLA-DR呈阴性表达。成脂诱导后可观察到细胞内有脂滴形成,油红O染色呈阳性。盐酸四环素荧光结果和Von Kossa染色结果显示,所形成的钙结节大小及数量,均随着地塞米松浓度的增加而增强、增多。反转录-聚合酶链反应检测结果显示,3种浓度地塞米松组的骨桥蛋白基因均有表达,且表达强度随着地塞米松浓度的增加而增强。提示成骨诱导液中地塞米松浓度为1×10-7mol/L时,能更有效地诱导人脐带间充质干细胞向成骨细胞分化。BACKGROUND： Under the combination effect of dexamethasone, vitamin C and β-glycerophosphate, human umbilical cord mesenchymal stem cells can differentiate into osteoblasts. Dexamethasone is a glucocorticoid hormone, and its concentration can affect the osteogenic differentiation of human umbilicai cord mesenchymal stem cells. OBJECTIVE： To explore the optimal concentrations of dexamethasone in the osteogenic differentiation of human umbilical cord mesenchymal stem cells. METHODS： Human umbilical cord mesenchymal stern cells were isolated from the normal full-term newborn umbilical cord by explant method. The passage 3 human umbilical cord mesenchymal stem cells were used for routine cell culture and induced differentiation experiments. The expression of surface markers of stem cells was detected with flow cytomerey, and the human umbilical cord mesenchymal stem calls were identified by osteogenic differentiation and adipogenic differentiation. The changes of cell morphology were observed under the inverted phase contrast microscope in the process of osteogenic differentiation. The induced medium for osteogenic differentiation containing 1 ×10^-8 mol/L, 5×10^-8 mol/L and 1×10^-7 mol/L dexamethasone was used for the osteogenic induction of umbilical cord mesenchymal stem cells. Tetracycline hydrochlodde fluorescence and Von Kossa staining were applied to observe the formation of calcium nodules. Reverse transcriptase-PCR was used to detect the expression of osteopontin gene. RESULTS AND CONCLUSION： The form of human umbilical cord mesenchymal stem cells separated by explant method was uniform, spindle and growing in parallel and vortex-like shape. The flow cytometry results showed that CD29, CD73 and CDg0 were positive, and CD31, CD34 and HLA-DR were negative. Intrecellular lipid droplets could be observed after adipogenic differentiation, and the formation size and quantity of calcium nodules were enhanced as the concentration of dexamethasone increased. The expression level of osteopontin gene could b

关 键 词：干细胞 脐带脐血干细胞 地塞米松 优选浓度{成骨分化 成脂分化 钙结节 骨桥蛋白 细胞培养 细胞鉴定 细胞形态 干细胞图片文章 

分 类 号：R394.2[医药卫生—医学遗传学]