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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 isolation and cultivation of human orbital adipose stromal cells (h OADSCs) in vitro and their differentiation into adipocytes. Methods Human orbital adipose stromal cells were isolated by tissue culture method and cultured in primary culture and passaged in vitro. The second generation of hADADSC cells were collected and the expression of cell surface antigens CD29, CD31, CD44 and CD45 were detected by flow cytometry. Adipogenic induction culture medium was used to induce hADADSC cells to differentiate into mature adipocytes and stained with Oil Red O. The morphology of h OADSC was observed under inverted phase contrast microscope. The cell adherent rate, cell doubling time and CCK-8 cell proliferation were measured and the cell growth curve was drawn. Results h OADSC was isolated by tissue culture method. The cells were spindle or spindle-shaped, and the cells proliferated rapidly after passage. The expression rates of cell surface markers CD29, CD31, CD44 and CD45 were 99.0%, 1.5%, 99.1% and 1.2% respectively. h OADSC differentiated into mature adipocytes under the action of adipogenic induction medium in vitro, and intracellular fat droplets were stained red with oil red O. Addition culture h OADSC cells adherent rate was 92.6%, cell doubling time was 1.98 d, the cells can be grown in vitro propagation of more than 10 generations of amplification. CONCLUSION: h OADSC was successfully isolated and cultured using tissue culture method. It can differentiate into mature adipocytes under the action of adipogenic induction medium in vitro.