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目的 :探讨硒在体外诱导小鼠胚胎干细胞向胰岛细胞分化中的促进作用。方法 :将ES D3小鼠胚胎干细胞悬浮培养 7d形成拟胚体 ,然后进行贴壁培养 ,加入分化培养液 1诱导 6d ,然后用分化培养液 2诱导 6d ,最后用含不同浓度的亚硒酸钠的培养基诱导 2 0d。对诱导后获得的胰岛素产生细胞团 (IPCC)细胞进行双硫腙染色 ,对染色结果进行体视学分析。进行胰岛素刺激释放试验 ,用ELISA法测定胰岛素释放量。RT PCR法检测胰腺细胞相关基因的表达。结果 :亚硒酸钠浓度在 0 .0 3～ 810 μmol/L之间时 ,经过系列诱导后获得的IPCC其DTZ阳性率随着亚硒酸钠浓度的提高而显著升高。当亚硒酸钠浓度为 2 70 μmol/L时DTZ阳性率上升至最高点 ,随后开始缓慢下降 ;胰岛素分泌释放试验也证实 2 70 μmol/L亚硒酸钠诱导的细胞释放的胰岛素最多。RT PCR结果进一步表明诱导获得的IPCC具有类胰岛组织的功能。结论 :硒对胚胎干细胞向胰岛细胞分化具有促进作用
Objective: To investigate the promotion of selenium on inducing mouse embryonic stem cells to differentiate into islet cells in vitro. METHODS: ES D3 mouse embryonic stem cells were cultured in suspension for 7 days to form embryoid bodies. The cells were then cultured in adherent culture. The cells were treated with differentiation medium 1 for 6 days, then induced with differentiation medium 2 for 6 days, and then cultured in ES medium containing different concentrations of sodium selenite Medium for 20 days. Insulin-producing cell mass (IPCC) cells obtained after the induction were dithizone-stained and the staining results were subjected to stereological analysis. Insulin release test was performed and the amount of insulin released was measured by ELISA. RT-PCR assay of pancreatic cell-related gene expression. Results: When the concentration of sodium selenite was between 0. 0 3 and 810 μmol / L, the positive rate of DTZ in IPCC obtained after series induction was significantly increased with the increase of sodium selenite concentration. When the concentration of sodium selenite was 2 70 μmol / L, the positive rate of DTZ increased to the highest point and then began to decline slowly. Insulin secretion release test also confirmed that selenite-induced cells released 2 70 μmol / L insulin most. RT PCR results further show that induced IPCC has the function of islet-like tissue. Conclusion: Selenium can promote the differentiation of embryonic stem cells into islet cells