天然可降解脱钙骨基质材料复合脂肪干细胞的三维培养  

Three-dimensional culture of adipose-derived stem cells combined with demineralized bone matrix scaffolds

作　　者：唐少锋[1] 李朝翠[2] 刘承杏[3] 

机构地区：[1]解放军成都军区昆明总医院脊髓损伤治疗科,云南省昆明市650032 [2]中国科学院昆明动物研究所遗传资源与进化国家重点实验室,云南省昆明市650223 [3]昆明医学院解剖教研室,云南省昆明市650031

出　　处：《中国组织工程研究与临床康复》2010年第12期2101-2105,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：解放军成都军区昆明总医院院管课题资助项目(YG2009016)~~

摘　　要：背景:脱钙骨基质是一种常用的组织工程支架材料,脂肪干细胞是近年来发现的一种成体干细胞,二者均可自体取材,二者复合培养的研究还未见报道。目的:观察脂肪干细胞与脱钙骨基质复合后三维培养的生长、增殖情况。方法:无菌切取兔腹股沟皮下脂肪垫,采用Ⅰ型胶原酶搅拌消化法分离培养原代脂肪干细胞,系列传代传至第3代,倒置显微镜下观察细胞形态及生长情况,免疫荧光细胞化学染色检测细胞表面抗原的表达。切取兔髂骨,制备脱钙骨基质。取第3代脂肪干细胞,与脱钙骨基质复合后共培养,1周后扫描电镜观察细胞黏附和生长情况,2周后石蜡包埋切片进行苏木精-伊红染色。结果与结论:自兔皮下脂肪分离培养获得的脂肪干细胞增殖迅速,第3代脂肪干细胞表面抗原CD44呈阳性表达,CD34为阴性;制备的脱钙骨基质为三维立体多孔结构,具有良好的可塑性,并有一定的机械强度。复合培养后电镜扫描、苏木精-伊红染色显示,细胞在脱钙骨基质表面和孔隙内黏附生长良好,并能继续增殖和分泌胞外基质。结果提示脂肪干细胞与脱钙骨基质复合共培养生长增殖良好,并能分泌细胞外基质。BACKGROUND：Adipose-derived stem cells（ADSCs）and demineralized bone matrix（DBM）can be obtained from the same donator.However,there are few studies on the co-culture of ADSCs as seed cells while DBM as scaffold.OBJECTIVE：To observe the growth and proliferation of ADSCs cultured combining with DBM.METHODS：The subcutaneous adipose tissue was cut and collected from the inguinal fat pads of rabbits under aseptic condition.Primary ADSCs were isolated subsequently by the methods of stirring digestion within collagenase type Ⅰ.After primary culture and subculturing,ADSCs were expanded to the 3rd passage.Cell morphology and growth were observed under an inverted microscope,and the expression of cell surface antigen was detected using immunofluorescence staining.DBM was prepared using rabbit ilium.The 3rd passage of ADSCs was seeded into the allogenic DBM scaffold sample for co-culture.Cell adherence and growth were observed under an inverted microscope at 1 week after co-culture,and the haematoxylin-eosin staining was performed at 2 weeks after co-culture.RESULTS AND CONCLUSION：ADSCs isolated from rabbit subcutaneous adipose tissues expanded rapidly.After the third passage,most of the passage cells were positive for CD44 but negative for CD34.The prepared DBM was three-dimensional stereochemical structure with more hollows;there were good plasticity and tolerable mechanical strength.Scanning electron microscopy and haematoxylin-eosin staining revealed that ADSCs adhered and grew well within the DBM scaffolds,and could synthesize cartilage extracellular matrix in the scaffold.These results show that the DBM can provide an appropriate microenvironment for the proliferation and expressions of extracellular matrix of ADSCs.

关 键 词：脱钙骨基质 脂肪源性干细胞 组织工程 培养 生物材料 

分 类 号：R318[医药卫生—生物医学工程]