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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 high concentration of glucose and leucine on the proliferation and apoptosis of human cord blood-derived mesenchymal stem cells (hUCB-MSCs). Methods hUCB-MSCs were isolated, purified, subcultured and subcultured. The expression of CD29, CD34 and CD44 on the third generation of hUCB-MSCs was detected by flow cytometry. The hUCB-MSCs were divided into glucose group, leucine group And the combined stimulation group, the final concentration of glucose solution and leucine solution were 28 and 1.35 mmol / L, and PBS control group. After cultured at 37 ℃ for 3 days, the cell viability was detected by MTT assay. Cell apoptosis was analyzed by flow cytometry with Annexin-V / PI staining. Western blot was used to detect the expression of Bcl-2 and Bax . Results hUCB-MSCs showed CD29 + CD44 + CD34-. After 3 days of drug treatment, the proliferation activity, apoptosis rate, Bcl-2 and Bax protein expression of hUCB-MSCs in glucose group and leucine group had no significant change (P> 0.05) compared with PBS control group; However, the proliferation activity of hUCB-MSCs in combination group was significantly inhibited, the apoptosis rate was significantly increased, the expression of Bcl-2 protein was significantly decreased, and the expression of Bax protein was significantly increased (P <0.01). Conclusion The combination of high concentration of glucose and leucine can inhibit hUCB-MSCs proliferation and promote their apoptosis. This study provides an experimental basis for the treatment of diabetes stem cells to some extent.