胚胎干细胞分化为胰岛素分泌细胞的诱导条件

The conditions of embryonic stem cells that are induced into insulin-secreting cells

目的:探索胚胎干细胞ES-D3分化为胰岛素分泌细胞的诱导条件。方法:ES-D3细胞培养于鼠胚成纤维细胞滋养层上,对数生长期时转入无血清含bFGF的DMEM液中,隔天换液,2周后,用DTZ染色、ELISA、免疫细胞化学染色等方法鉴定诱导生成的胰岛素分泌细胞及其所分泌的胰岛素。结果:ES-D3细胞在不含白血病抑制因子和滋养细胞的无血清培养体系中呈悬浮生长,并于第4天形成拟胚体,加入促分化因子bFGF可使胚胎干细胞向胰岛素分泌细胞分化,诱导生成的胰岛素分泌细胞在培养基中自我组装形成三维立体细胞簇。诱导第17天,DTZ染色发现大量被染成暗红色的胰岛素分泌细胞,免疫细胞化学染色亦证实了胰岛素分泌细胞的存在;同时,用ELISA法检测到胰岛素分泌。随着诱导天数增加,DTZ染色阳性细胞数及胰岛素含量均增加。结论:胚胎干细胞ES-D3在无血清含bFGF培养基中能被诱导分化为胰岛素分泌细胞,且该细胞能够分泌胰岛素。

Objective: To study the induction of embryonic stem cells(ESCs) into insulin-secreting cells(ISCs). Methods: ESCs were allowed to grow on mouse fetal fibroblast feeder layer, then transferred into serum-free DMEM supplemented with bFGF for more than 2 weeks. DTZ staining and immunocytochemistry were used to identify the induced ISCs, and insulin secretion was examined by ELISA. Results: ESCs grew and formed embryoid bodies at day 4, and the addition of bFGF promoted the differentiation of ESCs into ISCs. The induced ISCs self-assembled to form three-dimentional clusters, and are stained into crimson red by DTZ at day 17 after induction, and at the same time, insulin secretion was positively detected by ELISA and immunocytochemistry. Conclusions: ESCs can be induced to differentiate into ISCs in serum-free DMEM supplemented with bFGF, and the induced ISCs can secret insulin.