人大网膜间充质干细胞的体外培养、鉴定及诱导化分方法

Cultivation,Differentiation and Identification of Human Omental Adipose Derived Mesenchymal Stem Cells in Vitro

目的建立人大网膜脂肪间充质干细胞(adipose derived mesechymal stem cells,ADSCs)的原代培养及诱导分化为脂肪细胞的方法.方法术中取人大网膜脂肪组织,采用胶原酶消化法原代培养,经流式细胞仪鉴定细胞CD44、CD34、CD90和CD45分子、MTT法测定细胞生长曲线,取P3细胞以3-异丁基-1-甲基黄嘌呤、胰岛素、地塞米松及吲哚美辛诱导该细胞向脂肪细胞分化,油红O脂肪染色法进行脂肪细胞鉴定.结果培养出的细胞形态均一呈梭形或漩涡状生长,经流式细胞仪鉴定为ADSCs,细胞生长曲线表明P3细胞增值旺盛,用分化培养基诱导后,细胞形态由梭形变为圆形,细胞体积增大,胞浆内聚集脂肪颗粒,油红O染色鉴定为成熟的脂肪细胞.结论该方法能培养出形态均一的人大网膜ADSCs,经诱导后可向成熟脂肪细胞分化.

Objective To establish the primary culture and induced differentiation to fat cells of human omental adipose derived mesechymal stem cells （ADSCs）. Methods The omental adipose tissues were obtained h^v opration and fibroblast-like cells was primarily cultured by collagenase digestion. The ADSCs were identificated by detecting the expression of CD44, CD34, CD90 and CD45 with flow cytometry, cell growth curve was measured by MTF method, the P3 cells were induced to fat cells with 3 - isobutyl -1- methylxanthine, insulin, dexamethasone and indomethacin, and were identificated by oil red O. Results The cultured ceils were homogeneous spindle-shaped or swirling and were identifieated as ADSCs by flow eytometry. Cell growth curve showed the cells proliferated well. After induction with differentiation medium, cell became round, cell volume increased, fat particles aggregated in the cytoplasm, and the cells were identificated as mature fat cells by oil red O staning. Conclusion This method can culture homogeneous ADSCs, and ADSCs can differentiate into mature adipoeytes after induction.