CAJ | 학술논문

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.
배경:탈개골기질시일충상용적조직공정지가재료,지방간세포시근년래발현적일충성체간세포,이자균가자체취재,이자복합배양적연구환미견보도.목적:관찰지방간세포여탈개골기질복합후삼유배양적생장、증식정황.방법:무균절취토복고구피하지방점,채용I형효원매교반소화법분리배양원대지방간세포,계열전대전지제3대,도치현미경하관찰세포형태급생장정황,면역형광세포화학염색검측세포표면항원적표체.절취토가골,제비탈개골기질.취제3대지방간세포,여탈개골기질복합후공배양,1주후소묘전경관찰세포점부화생장정황,2주후석사포매절편진행소목정-이홍염색.결과여결론:자토피하지방분리배양획득적지방간세포증식신속,제3대지방간세포표면항원CD44정양성표체,CD34위음성;제비적탈개골기질위삼유입체다공결구,구유량호적가소성,병유일정적궤계강도.복합배양후전경소묘、소목정-이홍염색현시,세포재탈개골기질표면화공극내점부생장량호,병능계속증식화분비포외기질.결과제시지방간세포여탈개골기질복합공배양생장증식량호,병능분비세포외기질.
BACKGROUND: Adipose-derived stem cells (ADSCs) and demineralized bone matrix (DBM) can be obtained from the samedonator. 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 cultureand subculturing, ADSCs were expanded to the 3~(rd) passage. Cell morphology and growth were observed under an invertedmicroscope, and the expression of cell surface antigen was detected using immunofluorescence staining. DBM was preparedusing rabbit ilium. The 3~(rd) passage of ADSCs was seeded into the allogenic DBM scaffold sample for co-culture. Cell adherenceand growth were observed under an inverted microscope at 1 week after co-culture, and the haematoxylin-eosin staining wasperformed at 2 weeks after co-culture.RESULTS AND CONCLUSION: ADSCs isolated from rabbit subcutaneous adipose tissues expanded rapidly. After the thirdpassage, most of the passage cells were positive for CD44 but negative for CD34. The prepared DBM was three-dimensionalstereochemical structure with more hollows; there were good plasticity and tolerable mechanical strength. Scanning electronmicroscopy and haematoxylin-eosin staining revealed that ADSCs adhered and grew well within the DBM scaffolds, and couldsynthesize cartilage extracellular matrix in the scaffold. These results show that the DBM can provide an appropriatemicroenvironment for the proliferation and expressions of extracellular matrix of ADSCs.