人眼眶脂肪基质细胞的分离培养、鉴定及成脂诱导分化的研究  

Isolation,culture,identification and adipogenic differentiation of orbital adipose-derived stromal cells in human

作　　者：赵平千[1] 张瑜[1] 杨文蕾[1] 

机构地区：[1]上海交通大学医学院附属仁济医院眼科,上海200127

出　　处：《山东大学耳鼻喉眼学报》2015年第4期65-68,73,共5页Journal of Otolaryngology and Ophthalmology of Shandong University

基　　金：上海交通大学医学院科技基金项目资助(13XJ10047)

摘　　要：目的探讨人眼眶脂肪基质细胞(h OADSC)体外分离培养、鉴定方法及其向脂肪细胞诱导分化的能力。方法采用组织块培养法分离人眼眶脂肪基质细胞并在体外进行原代培养及传代扩增。取第二代h OADSC细胞,用流式细胞仪检测细胞表面抗原CD29、CD31、CD44、CD45的表达情况;采用成脂诱导培养液体外诱导h OADSC细胞向成熟脂肪细胞分化并用油红O染色。倒置相差显微镜下观察h OADSC形态、测定细胞贴壁率、细胞倍增时间、CCK-8检测细胞增殖情况并绘制细胞生长曲线。结果组织块培养法分离得到h OADSC,细胞呈梭形或纺锤形生长,传代后细胞增殖迅速。细胞表面标志物CD29、CD31、CD44、CD45的表达率分别为99.0%、1.5%、99.1%、1.2%。h OADSC在体外成脂诱导培养液的作用下可分化为成熟的脂肪细胞,细胞内的脂肪滴被油红O染为红色。传代培养h OADSC细胞贴壁率为92.6%,细胞倍增时间为1.98 d,细胞可在体外生长传代扩增10余代。结论采用组织块培养法成功地分离培养了h OADSC,其在体外成脂诱导培养液的作用下可分化为成熟的脂肪细胞。Objective To investigate the method of isolation,culture and identification of human orbital adiposederived stromal cells（ h OADSCs） and it＇s ability of adipogenic differentiation in vitro. Methods The h OADSCs were isolated by tissue explant culture technique,then were primary cultured and subcultured in vitro. The second generation of h OADSCs was detached,and the expressions of cell surface antigen CD29,CD31,CD44 and CD45 were analyzed with flowcytometry to identify the cells. The h OADSCs were induced to be adipogenesis with the adipogenic induction medium in vitro and stained with oil red O. The morphology of the h OADSC was observed by phase contrast microscopy. The plating efficiency and doubling time of the h OADSCs were tested. The h OADSCs proliferation was detected by CCK-8,and the cell growth curve was made. Results The h OADSCs were isolated by tissue explant culture technique and exhibited a fibroblast- or spindle-like morphology. The h OADSCs grewrapidly after subculturing. The expression rate of cell surface antigen CD29,CD31,CD44 and CD45 was 99. 0%,1. 5%,99. 1% and 1. 2%,respectively. The h OADSCs were differentiated into adipocytes with the adipogenic induction medium in vitro and the intracellular lipid droplets were stained in red by oil red O. The plating efficiency and doubling time of the subcultured h OADSC were92. 6% and 1. 98 days,respectively. The h OADSCs could be subcultured for more than ten generations in vitro.Conclusion In this experiment,the h OADSCs were isolated,cultured by tissue explant culture technique and could be differentiated into mature adipocytes with the adipogenic induction medium in vitro.

关 键 词：眼眶 脂肪基质细胞 细胞培养 

分 类 号：Q813.1[生物学—生物工程]