滑液间充质干细胞在无血清培养条件下生物学特性及体外三维软骨构建

摘要

目的 探讨滑液间充质干细胞(SF-MSCs)在无血清培养条件下的生物学特性,及其结合支架材料体外构建三维软骨的能力.方法 获取人的SF-MSCs,分别用有血清培养液和无血清培养液(MSCM-sf)体外培养,比较2种培养基培养的SF-MSCs增殖能力和细胞形态变化;取第3代MSCM-sf培养的SF-MSCs,检测细胞表面标志物的表达水平,并进行三向(成软骨、成骨、成脂)诱导分化实验以及结合聚羟基乙酸/聚乳酸(PGA/PLA)支架材料成软骨诱导实验.结果 MSCM-sf培养的SF-MSCs形态和增殖能力均优于有血清培养液培养细胞.第3代MSCM-sf培养的SF-MSCs表面阳性标志物CD73 (99.5％)、CD90(98.9％)和CD105 (96.5％)表达水平均大于95％,表面阴性标志物(CD34、HLA-DR和CD11b)总表达水平小于2％;三向诱导分化染色呈阳性;可与PGA/PLA复合物体外诱导成软骨.结论 SF-MSCs经MSCM-sf培养后能在维持细胞干性的前提下实现大量扩增,并可结合PGA/PLA生物支架体外构建三维软骨.%Objective To explore the biological characteristics of synovial fluid-derived mesenchymal stem cells (SF-MSCs) cultured in serum-free medium and the ability of in vitro reconstruction of three-dimensional cartilage combined with scaffold material.Methods Human SF-MSCs were cultured in serum medium and mesenchymal stem cells medium-serum free (MSCM-sf) respectively,then the proliferative ability and morphology of SF-MSCs were compared;The third passage SF-MSCs cultured in MSCM-sf were identified by flow cytometry,three-way(chondrogenic,osteogenic,adipogenic) differentiation assay and induced for chondrogenic differentiation when combined with polyglycolic acid/ polylactic acid (PGA/PLA).Results SF-MSCs cultured in MSCM-sf had better morphology and proliferative ability than that cultured in serum medium.The expression levels of positive markers of the third passage SF-MSCs cultured in MSCM-sf,such as CD73 (99.5％),CD90 (98.9％) and CD105 (96.5％),were more than 95％.However,the overall negative markers (CD34,HLA-DR and CD11b) expressed less than 2％.Three-way differentiation staining was positive.The combination of SF-MSCs and PGA / PLA can be induced into cartilage in vitro.Conclusions SF-MSCs cultured in MSCM-sf can be amplified under the condition of maintaining the stem cell characteristics,and can be combined with PGA/ PLA scaffold to construct three-dimensional cartilage in vitro.