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目的:目前小鼠胚胎干细胞(mouse embry-onic stem cells,mESC)常规应用高浓度葡萄糖(25 mmol/L)培养基培养,但是在应用干细胞向糖尿病胰岛beta细胞分化的研究中发现,慢性高糖培养可促进干细胞的凋亡、降低细胞分化的效率及分化后胰岛beta细胞对葡萄糖的反应性,因此本研究拟选择合适的较低浓度的葡萄糖以优化胚胎干细胞的培养基、提高胚胎干细胞的生长、分化效率。方法:mESC传代4或12 h后,将传统的25 mmol/L葡萄糖培养基分别换为5、10、15、25 mmol/L葡萄糖培养基培养,均用碱性磷酸酶(AP)染色计数细胞集落形成情况、台盼兰染色测定细胞数目,MTT法测定细胞活力及用4’,6-联脒-2-苯基吲哚(4’,6-diamidino-2-phenylindole,DAPI)染色检测细胞凋亡情况。结果:mESC传代4 h后即调换葡萄糖浓度:(1)各葡萄糖浓度组(5、10、15 mmol/L)与25 mmol/L组相比,集落形成明显减少。(2)各葡萄糖浓度组与25 mmol/L组相比,mESC增殖及细胞活力均受到不同程度明显影响。mESC传代12 h后调换葡萄糖浓度:(1)各葡萄糖浓度组(5、10、15 mmol/L)与25 mmol/L组相比,集落形成无明显变化,且AP染色呈强阳性。(2)15 mmol/L葡萄糖组对mESC增殖及细胞活力均无明显影响。(3)15 mmol/L葡萄糖组与25 mmol/L葡萄糖组细胞核形态正常,均未见明显凋亡。结论:ESC传代12 h后将培养基中传统的高糖(25 mmol/L)降低为15 mmol/L,不影响mESC细胞活力及多能分化潜能和未分化状态。
OBJECTIVE: Currently, mouse embryo-derived stem cells (mESCs) are cultured in high concentration of glucose (25 mmol / L), but in the study of stem cell differentiation to diabetic islet beta cells, chronic high glucose Culture can promote the apoptosis of stem cells, reduce the efficiency of cell differentiation and differentiation of pancreatic beta cells to glucose responsiveness, the study intended to select the appropriate lower concentrations of glucose to optimize the culture medium of embryonic stem cells to improve the growth of embryonic stem cells , Differentiation efficiency. Methods: After cultured for 4 or 12 h, the traditional 25 mmol / L glucose medium was changed to 5, 10, 15 and 25 mmol / L glucose medium respectively. All cells were counted with alkaline phosphatase (AP) The number of cells was determined by trypan blue staining, the cell viability was determined by MTT assay and the cells were detected by 4 ’, 6-diamidino-2-phenylindole (DAPI) staining Apoptosis. Results: After 4 h of passage, the concentration of glucose was changed after the passage of mESC. (1) The colony formation of each glucose concentration group (5, 10, 15 mmol / L) was significantly reduced compared with 25 mmol / L group. (2) Compared with 25 mmol / L group, the proliferation and cell viability of mESCs in each glucose concentration group were significantly affected by different degrees. The concentration of glucose was changed 12 h after passage of mESC. (1) There was no significant change of colony formation in each glucose concentration group (5, 10, 15 mmol / L) compared with 25 mmol / L group, and AP staining was strongly positive. (2) 15 mmol / L glucose group had no significant effect on the proliferation and cell viability of mESCs. (3) The nuclei of 15 mmol / L glucose group and 25 mmol / L glucose group were normal, and no obvious apoptosis was observed. CONCLUSION: After 12 h culture, the high glucose (25 mmol / L) in culture medium was reduced to 15 mmol / L without affecting the viability, pluripotentiality and undifferentiated status of mESC cells.