人脂肪来源的成体干细胞体外向软骨细胞诱导分化的研究  

The experimental study on the chondrogenic differentiation of human adult adipose-derived adult stem cells in vitro

作　　者：靳小兵[1] 孙永生[1] 娄思权[1] 张克[1] 

机构地区：[1]北京大学第三医院骨科,100083

出　　处：《中华实验外科杂志》2007年第6期649-651,共3页Chinese Journal of Experimental Surgery

摘　　要：目的研究人脂肪来源的成体干细胞(ADSC)体外能否向软骨细胞成功分化,探讨其作为组织工程软骨种子细胞的可行性。方法自成人皮下取少量脂肪组织,经机械剪切及Ⅰ型胶原酶消化后获取成体干细胞,体外培养扩增;流式细胞仪鉴定细胞表型;第5代细胞采取离心管中微块法培养,培养液中加入转化生长因子(hTGF-B2)等诱导剂诱导其向软骨细胞分化;诱导14 d的细胞团经消化后获得单细胞悬液,接种于玻片上行形态学观察,甲苯胺兰、免疫组织化学染色及逆转录-聚合酶链式反应(RT-PCR)用以鉴定软骨细胞表型。结果从人体皮下脂肪组织中可成功分离到ADSC,细胞可于体外大量扩增;第5代细胞大多数表达CD29、CD90,而CD14、CD45抗原表达阴性;RT-PCR结果提示诱导后的细胞可表达黏多糖(GAG)及Ⅱ型胶原,诱导后的细胞块分离成单个细胞后可见其形态较诱导前发生明显变化,甲苯胺兰染色和Ⅱ型胶原免疫组织化学染色为阳性。对照组未加诱导因子的细胞则未能观察到上述变化。结论成人皮下脂肪组织中可分离到大量ADSC;ADSC可在体外成功诱导向软骨细胞分化;ADSC有望作为组织工程软骨构建中的种子细胞。Objective To investigate the chondrogenic potential of human adipose-derived adult stem cells （hADSC） in vitro,discuss the possibility of them as the seeding cells in the construction of tissue engineered cartilage. Methods Some subcutaneous adipose tissues were obtained during the operation. After being dissected and digested with 0.1% collagenase type Ⅰ,hADSCs were isolated from the tissue. After proliferating in monolayer culture, CD14, CD4S, CD29 and CD90 were detected by flow cytometry;For chondrogenic induction, hADSCs were cultured in pellet in centrifuge tube with addition of human transforming growth factor beta2 （hTGFbeta2）. After induction for 14 days ,single cells released from the induced cell pellets were reseeded onto coverslips to observe the morphology change. Chondrogenic phenotype was examined by toluidine blue staining,immunohistochemistry for collagen type Ⅱ and reverse transcriptase polymerase chain reaction （RT-PCR）. Results hADSCs were successfully isolated from the adult adipose tissue, and the cells could proliferate in monolayer culture flourishly; Results of flow cytometry revealed the majority of hADSCs were positive for CD 29 and CD 90, only a small fraction of the cells expressed CD 14 and CD 45 ; RT-PCR analysis revealed that hADSCs produced aggrecan and collagen type Ⅱ 7 days after induction and continued throughout the culture period. Single cells, released from the hTGF-beta2 treated pellets reseeded in monolayer, were polygonal and triangle, and biosynthesis of glycosaminoglycan and collagen type Ⅱ was evidenced by positive staining of Toluidine blue and immunohistochemistry. All these findings were not detected in the control group not treated with chondrogenic media. Conclusion hADSCs are capable of undergoing chondrogenesis in vitro. The cells can be a better choice as the seeding cells in constructing tissue engineered cartilage.

关 键 词：干细胞 脂肪组织 分化 软骨 组织工程 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]