高浓度葡萄糖对小鼠表皮干细胞增殖的影响

目的 探讨高浓度葡萄糖对小鼠表皮干细胞增殖的影响.方法 利用Ⅳ型胶原的快速黏附法分离培养小鼠表皮干细胞,观察其形态学;通过免疫荧光法检测表皮干细胞标志物β1整合素和K19来鉴定表皮干细胞;将小鼠表皮干细胞分别放在葡萄糖浓度为5.6、15和30mmol/L的培养基里培养;MTT法检测细胞增殖情况,免疫印迹法检测细胞凋亡蛋白Bax和抗凋亡蛋白Bcl-2的蛋白表达水平.结果 体外培养的小鼠表皮干细胞呈克隆样生长,增殖速度较快,细胞形态呈铺路石样,边缘细胞呈梭形,细胞核质比较大;免疫荧光法检测到培养的小鼠表皮干细胞β1整合素和K19呈阳性表达且阳性表达率均为100％;MTT法检测到与对照组（5.6mmol/L浓度葡萄糖）相比,15mmol/L浓度葡萄糖刺激72h后检测到细胞增殖速度无明显改变,而30mmol/L浓度葡萄糖刺激24、48和72h后检测到细胞增殖速度降低,差异有统计学意义（P＜0.05）.Western blot法检测到与对照组相比,30mmol/L浓度葡萄糖刺激48h后凋亡蛋白Bax的蛋白表达量明显增加而抗凋亡蛋白Bcl-2蛋白表达水平明显降低（P＜0.05）.结论 本研究结果表明较高浓度（30mmol/L）葡萄糖可以明显抑制小鼠表皮干细胞的增殖,从干细胞角度进一步为糖尿病创面难愈的原因提供了实验室依据.

Effects of High Concentration of Glucose on the Proliferation of Mouse Epidermal Stem Cells

Objective To investigate the effects of high concentration of glucose on the proliferation of mouse epidermal stem cells.Methods Mouse epidermal stem cells were isolated and cultured by rapid adhesion with collagen IV,and then its morphology was observed.The epidermal stem cells were identified through the detection of β1 integrin and K19 which are markers of epidermal stem cells by immunofluorescence method.The cells were cultured with different concentrations of glucose medium including 5.6mmol/L,15mmol/L,30mmoL/L.The proliferation of cells were detected by MTT assay.The expression levels of apoptotic proteins Bax and anti-apoptotic protein Bcl-2 protein were detected with immunoblotting.Results The mouse epidermal stem cells cultured exhibited clonally growth,fast proliferation.The cell morphology was cobblestone with fusiform edge,and the nucleus is relatively large.The expression of β1 integrin and K19 in the epidermal stem cells were positive and the positive rate was 100%.Compared with control group（5.6mmol/L）,cell proliferation did not significant change in the high glucose （15mmol/L） stimulation group at the time of 72h after stimulation,but cell proliferation was significantly decreased （P 〈 0.05）in the high glucose （30mmol/L） stimulation group at the time of 24,48 and 72h after stimulation.The expression levels of apoptotic proteins Bax were increased and the expression levels of anti-apoptotic protein Bcl-2 protein were decreased in the high glucose group （30mmol/L） compared with control group（P 〈 0.05）.Conclusion This study shows that a higher concentration （30mmol/L） of glucose can significantly inhibit the proliferation of epidermal stem cell,which provides laboratory evidence for the causes of diabetic wound healing from the perspective of stem cells.