悬滴培养对人脂肪间充质干细胞成骨分化的影响  

Hanging-drop culture promote osteogenic differentiation of human adiposetissue-derived mesenchymal stem cells

作　　者：杨静[1] 张琪[2] 

机构地区：[1]锦州辽宁医学院研究生院,辽宁121001 [2]北京武警总医院口腔颌面外科,北京100039

出　　处：《解放军医学杂志》2012年第6期602-605,共4页Medical Journal of Chinese People's Liberation Army

摘　　要：目的探讨悬滴培养对人脂肪来源间充质干细胞(hADSCs)成骨诱导分化的影响。方法利用酶消化法分离培养hADSCs,采用流式细胞术检测其表面标志的表达。对hADSCs进行悬滴培养,贴壁后添加诱导剂对其进行成骨诱导分化,以平板培养组为对照,诱导分化7d及21d时,利用RT-PCR检测成骨基因Runx2及Osteopontin的表达。结果分离培养的hADSCs表达干细胞表面标志CD44、CD90、CD105,不表达CD34、CD45;悬滴培养3d的hADSCs可形成大小均一的球状三维结构;RT-PCR结果表明,在成骨诱导分化的不同时间点,hADSCs悬滴培养组的成骨基因Runx2及Osteopontin表达均比平板培养组高,表明悬滴培养可促进hADSCs的成骨诱导分化。结论悬滴培养有利于hADSCs的成骨诱导分化,有助于提高其成骨诱导效率。Objective To investigate the effect of hanging-drop culture on osteogenic differentiation of human adiposederived mesenchymal stem cells（hADMSCs）.Methods hADMSCs were isolated by enzyme digestion and cultured.The primary culture and second passage of hADMSCs were observed with phase-contrast microscope.To confirm the immunophenotype of hADMSCs,surface markers of CD44,CD90,CD105,CD34 and CD45 were detected by flow cytometry.hADMSCs were then cultured in the hanging drops.The phenotypes of hADMSCs spheroid aggregate in hanging drops were then observed with phasecontrast microscope.After the adherence of cells,hADMSCs were cultured in osteogenic differentiation medium to achieve osteogenic differentiation.The expression of Runx2 and Osteopontin was detected by reverse transcription–polymerase chain reaction（RT-PCR） at 7th and 21st days.Results Most of hADMSCs grew as spindle-shaped cells with long cytoplasmic processes as observed under the phase-contrast microscope.hADMSCs shared the immunophenotypes of both multipotential stem cell and embryonic stem cell markers,as they were positive for CD44,CD90 and CD105,and negative for CD34 and CD45 expressions by flow cytometry analysis,hADMSCs cultured in the hanging drops could form cell aggregates after 24 hours of culture,and could form three-dimensional spheroid aggregates with uniform size after 72 hours of culture as observed under the phase-contrast microscope.RT-PCR showed that the expression levels of Runx2 and Osteopontin in hanging-drop cultures were much higher than that of control.Hanging-drop culture could promote osteogenic differentiation of hADMSCs.Conclusions Hanging-drop culture is beneficial in promoting the efficiency of osteogenic differentiation of human adipose-derived mesenchymal stem cells.

关 键 词：间质干细胞 细胞培养技术 细胞分化 骨生成 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]