机构地区:[1]陆军军医大学大坪医院全军战创伤中心创伤实验室,重庆400042 [2]陆军军医大学大坪医院创伤,烧伤,复合伤国家重点实验室,重庆400042 [3]陆军军医大学大坪医院骨代谢与修复中心,重庆400042
出 处:《国际骨科学杂志》2018年第5期326-332,共7页International Journal of Orthopaedics
基 金:国家重点基础研究发展计划(2014CB942904);国家自然科学基金(81530071);国家自然科学基金(81472074)
摘 要:目的分离培养小鼠关节软骨表层细胞(ACSC)并鉴定其干细胞特性。方法运用纤连蛋白黏附法从3~5 d新生小鼠膝关节软骨表层分离细胞及培养,对分离细胞以流式细胞仪检测干细胞表面阳性标志物(CD44与CD90)和阴性标志物(CD45、CD31与CD34)的表达;实时荧光定量逆转录-聚合酶链反应(qRT-PCR)检测相关基因表达;单克隆形成实验检测克隆形成能力。三系诱导分化(成软骨、成骨与成脂分化)检测分离培养细胞的多向分化潜能。取第6代分离培养细胞做裸鼠皮下移植实验,检测其体内成软骨能力。结果纤连蛋白可以特异性地黏附ACSC,ACSC形态偏长,类似成纤维细胞。ACSC高表达CD44和CD90,但几乎不表达CD31、CD34和CD45。qRT-PCR检测结果显示,以ACSC表达的mRNA水平为1,则软骨细胞显著性低水平表达间充质干细胞标志物CD73(0.08±0.07,P<0.001)、CD90(0.07±0.01,P<0.001)、CD105(0.37±0.02,P<0.001)和软骨表层细胞标志物Prg4(0.42±0.01,P<0.001)、Erg(0.61±0.02,P<0.001)、Ten C(0.64±0.07,P<0.001),但显著性高水平表达软骨细胞标志物Col2(6.89±0.06,P<0.001)、Acan(6.51±0.04,P<0.001)、MATN1(14.57±4.21,P<0.01)。经过14 d培养,单个ACSC可以形成大于50个细胞的单克隆细胞团。体外诱导ACSC具备很强的成软骨、成骨与成脂分化能力,裸鼠体内ACSC具有成软骨能力。结论小鼠ACSC具有典型的干细胞特征。Objective To isolate and culture mouse articular cartilage superficial zone cell (ACSC), and identify their characteristics.Methods Cells were isolated from mouse articular cartilage superficial zone by fibronectin-conglutination assay. The expression of positive markers (CD44 and CD90) and negative markers (CD31, CD34 and CD45) at the surface of ACSC were detected by flow cytometry. Real-time fluorescent quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was used to detect related gene expression.The colony forming ability of ACSC was determined by single-cell colony forming unit assay.The multilineage differentiation potential of ACSC was identified through chondrogenesis, osteogenesis and adipogenesis induction experiment. The chondrogenesis potential of ACSC were identified by subcutaneously transplantation into athymic mice.Results Fibronectin could specifically adhere to ACSC , which displayed an elongated, fibroblastic cytoarchitecture. ACSC showed high expression of CD44 and CD90, while hardly express CD31, CD34 and CD45. Defining the amount of mRNA expressed by ACSC as 1, we detected the low expression of stem cell marker [ CD73 (0.08±0.07, P 〈0.001), CD90 ( 0.07± 0.01, P 〈0.001), CD105 (0.37±0.02, P 〈0.001) ]and cartilage superficial zone marker [ Prg4 (0.42± 0.01 , P 〈0.001), Erg ( 0.61± 0.02, P 〈0.001), Ten C (0.64±0.07, P 〈0.001)], but high expression of Col2 (6.89±0.06, P 〈0.001), Acan (6.51±0.04, P 〈 0.001) and MATN1 (14.57±4.21, P 〈0.01) . After culturing for 14 days, single ACSC formed colony containing more than 50 cells. ACSC were capable of chondrogenesis, osteogenesis and adipogenesis differentiation in vitro, and have high chondrogenesis potential in vivo.Conclusion Fibronectin-conglutination assay may be applied to obtain ACSC with characteristics of stem cells.
分 类 号:R329.2[医药卫生—人体解剖和组织胚胎学]
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