人体脂肪干细胞培养及成骨诱导分化  

Cultivation and osteogenic differentiation of human adipose-derived stem cells

作　　者：郭艳萍[1] 张爱君[2] 马志兵[2] 沈才齐[2] 金培生[2] 

机构地区：[1]徐州医学院研究生学院,江苏徐州221002 [2]徐州医学院附属医院整形外科,江苏徐州221002

出　　处：《徐州医学院学报》2014年第1期22-26,共5页Acta Academiae Medicinae Xuzhou

基　　金：江苏省科教兴卫重点人才资助项目（RC2011115）

摘　　要：目的 通过成人脂肪体外分离培养脂肪间充质干细胞（adipose-derived stem cells,ADSCs）研究其生理特性及在特定培养条件下向成骨分化,探讨脂肪源性干细胞作为骨组织工程的种子细胞的前景.方法 从成人脂肪中利用胶原酶消化法分离并体外培养干细胞,流式细胞仪检测细胞表面标记物,CCK8检测细胞活性,P3代脂肪干细胞通过成骨诱导液诱导向骨细胞分化,BCIP/NBT比色法染色检测碱性磷酸酶（AKP）,茜素红染色检测钙结节形成,RT-PCR检测碱性磷酸酶（ALP）、骨桥蛋白（OP）表达变化.结果 成人脂肪间质来源的ADSCs,能稳定传代,增殖能力旺盛;流式细胞检测证实其特异表达相关干细胞表面标记物;成骨诱导后表现出呈典型的成骨细胞形态;ALP染色阳性,茜素红染色阳性,诱导培养0、3、7、14、21、28天后RT-PCR定量检测证实细胞中ALP、OP阳性表达.结论 成人脂肪中可分离得到ADSCs,其稳定表达特异性的表面抗原,并且经过相应诱导培养后可向骨细胞分化,阳性表达OP、ALP,可作为优良的骨组织工程的种子细胞来源.Objective To establish the method of isolation, cultivation and osteogenic differentiation of adipose - derived stem cells （ADSCs） in vitro, so as to explore the potential of ADSCs as seed cells for engineered bone. Methods Collagenase enzymatic digestion was used to isolate ADSCs from human adipose tissue. Flow cytometry was used to de- tect cell surface markers. CCK8 assay was performed to examine cell viability. Osteogenic induced reagent was used to induce differentiation of ADSCs towards bone cells. In addition, we also performed BCIP/NBT to detect alkaline pbospha- tase （ALP） activity. Alizarin red staining was used to detect the formation of calcium node. Reverse transcription poly- merase chain reaction （RT- PCR） was performed to examine ALP and osteopontin （OP） expression. Results ADSCs could passage stably and proliferate highly. Flow cytometry data showed the expression of stem cell surface markers. AD- SCs showed typical osteogenic cell morphology after osteogenic differentiation. ALP staining and Alizarin red staining were positive. Furthermore, ALP and OP mRNA was expressed after differentiation at 0, 3,7, 14, 21, and 28 days. Conclu- sion ADSCs can be obstained from adipose tissue through enzymatic digestion. ADSCs can express specific antigens stably and can differentiate towards bone cells, expressing OP and ALP. ADSCs can become potential seed cells for engi-neered bone.

关 键 词：脂肪间充质干细胞 组织工程骨 成骨分化 碱性磷酸酶 骨桥蛋白 

分 类 号：R622[医药卫生—整形外科]