高浓度葡萄糖和亮氨酸联合作用对人脐带血间质干细胞增殖和凋亡的影响  

Combination of glucose and leucine at high concentrations on proliferation and apoptosis of human cord blood-derived mesenchymal stem cells

作　　者：王晓慧[1] 邱伟[2] 徐祥[2] 黄宏[2] 刘晓萍[1] 

机构地区：[1]青岛大学医学院组织胚胎学教研室,山东青岛266021 [2]第三军医大学大坪医院野战外科研究所干细胞移植&细胞治疗中心,重庆400042

出　　处：《中国生物制品学杂志》2013年第11期1588-1592,共5页Chinese Journal of Biologicals

基　　金：青岛市科技局资助课题(07-2-1-4-nsh)

摘　　要：目的探讨高浓度葡萄糖和亮氨酸联合作用对人脐带血间质干细胞(human cord blood-derived mesenchymal stem cells,hUCB-MSCs)增殖和凋亡的影响。方法贴壁培养法分离、纯化并传代培养hUCB-MSCs,采用流式细胞术检测第3代hUCB-MSCs表面抗原CD29、CD34和CD44的表达;将hUCB-MSCs分为葡萄糖组、亮氨酸组和联合刺激组,葡萄糖液和亮氨酸液终浓度分别为28和1.35 mmol/L,并设PBS对照组。37℃培养3 d后,采用MTT法检测细胞的增殖活力;流式细胞术Annexin-V/PI染色法检测细胞的凋亡情况;Western blot法检测细胞凋亡相关蛋白Bcl-2和Bax的表达。结果 hUCB-MSCs呈CD29+CD44+CD34-。药物处理3 d后,与PBS对照组相比,葡萄糖组和亮氨酸组hUCB-MSCs的增殖活力、凋亡率、Bcl-2和Bax蛋白的表达水平均无明显变化(P>0.05);而联合刺激组hUCB-MSCs的增殖活力受到明显抑制,凋亡率显著升高,Bcl-2蛋白的表达水平明显降低,Bax蛋白的表达水平明显升高(P<0.01)。结论高浓度葡萄糖和亮氨酸联合作用可抑制hUCB-MSCs增殖,促进其凋亡。本研究在一定程度上为干细胞治疗糖尿病提供了实验依据。Objective To investigate the effect of combination of glucose and leucine at high concentrations on the pro-liferation and apoptosis of human cord blood-derived mesenchymal stem cells （hUCB-MSCs）. Methods The hUCB-MSCs were isolated and purified by adherent culture method then subeultured. The expression of antigens CD29, CD34 and CD44 antigen on surface of hUCB-MSCs of passage 3 were determined by flow cytometry. The hUCB-MSCs were divided into three groups, and incubated with glucose at a final concentration of 28 mmol/L, leucine at a final concentration of 1.35 mmol/L and glucose （28 mmol/L） ＋ leucine （1.35 mmol/L） at 37 ℃ for 3 d respectively, using PBS as control. The proliferation activity of hUCB-MSCs was determined by MTF method, while the apoptosis by flow cytometry with An-nexin-V/PI staining, and the expressions of apoptosis-associated proteins Bcl-2 and Bax by Western blot. Results The hUCB-MSCs were positive for CD29 and CD44 while negative for CD34 antigens. The proliferation activities, apoptosis rates as well as Bel-2 and Bax expression levels of hUCB-MSCs 3 d after incubation with glucose and leueine showed no significant difference with those in PBS control group （P 〉 0. 05）. However, the proliferation activity of hUCB-MSCs in glucose ＋ leucine group were inhibited, while the apoptosis rate increased significantly, the expression level of Bcl-2 pro-tein decreased significantly, and the expression level of Bax protein increased significantly （P 〈 0. 01）. Conclusion The combination of glucose and leucine at high concentrations inhibited the proliferation and promoted the apoptosis of hUCB-MSCs, which provided a certain experimental basis for treatment with diabetes with stem cells.

关 键 词：葡萄糖 亮氨酸 人脐带血间质干细胞 细胞增殖 细胞凋亡 

分 类 号：R329.26[医药卫生—人体解剖和组织胚胎学]