葡萄糖及O-GlcNAc糖基化对人骨髓间充质干细胞增殖、凋亡、衰老的影响

Effects of glucose on human bone marrow mesenchymal stem cell growth, apoptosis and senescence

中文关键词:  骨髓间充质干细胞  葡萄糖  糖基化  细胞增殖  细胞凋亡  细胞衰老

英文关键词:Mesenchymal stem cells  Glucose  Cell growth  Cells apoptosis  Cell senescence

中文摘要:

  【摘要】 目的：探讨微环境中不同浓度葡萄糖是否可通过蛋白质O位氮乙酰葡糖胺（O-GlcNAc）糖基化修饰影响人骨髓间充质干细胞增殖、周期、凋亡及衰老。方法：构建常规浓度葡萄糖（A组）（5.5mmol/L）、高浓度葡萄糖（B组）（25mmol/L）微环境模型及常糖糖基化（C组）（常糖+Thiamet-G 1.0mmol/L）、高糖糖基化（D组）（高糖+Thiamet-G 1.0mmol/L）细胞模型。人骨髓间充质干细胞在各组中培养，于第3、5、7天用WST-1细胞增殖及细胞毒性检测试剂盒测定各组细胞增殖情况；于第5天用流式细胞仪检测各组细胞周期和细胞凋亡情况；于第7天用细胞衰老β-半乳糖苷酶染色试剂盒检测各组细胞衰老情况；于第5天用Real-time PCR、Westen-bolt检测不同处理组Cyclin D1和caspase-3的表达水平。结果：第3、5、7天时细胞增殖结果显示，与A组相比，B组、C组和D组细胞增殖明显下降（P<0.05）。细胞周期结果显示，B组、C组和D组相较于A组细胞周期G1期阻滞（P<0.05）。细胞凋亡结果显示，与A组相比，B组、C组和D组凋亡明显增多（P<0.05）。细胞衰老染色显示，与A组相比，B组、C组和D组细胞衰老明显增多（P<0.05）。Real-time PCR结果显示，Cyclin D1的mRNA相对值A组、B组、C组和D组分别为1.01±0.31、0.31±0.07、0.42±0.1、0.18±0.04，其他三组显著低于A组（P<0.05）；caspase-3的mRNA相对值A组、B组、C组和D组分别为1.09±0.82、5.73±1.54、3.43±0.59、6.82±2.13，其他三组显著高于A组（P<0.05）。Westen-bolt结果显示，A组hBMSCs内蛋白质O-GlcNAc糖基化水平高于B组；Cyclin D1的蛋白水平表达B组、C组和D组低于A组（P<0.05），caspase-3的蛋白质水平表达B组、C组和D组高于A组（P<0.05）。结论：微环境中高浓度葡萄糖和细胞蛋白质高水平O-GlcNAc糖基化修饰抑制人骨髓间充质干细胞增殖，阻滞细胞周期促进细胞凋亡及衰老。微环境中高浓度葡萄糖可诱导胞内蛋白质O-GlcNAc糖基化水平升高。微环境中高浓度葡萄糖诱导的蛋白质O-GlcNAc糖基化水平升高可能是其抑制增值促进凋亡和衰老的机制之一。

英文摘要:

  【Abstract】 Objectives: To investigate the effect on cell growth, cycle, apoptosis and senescence of human bone marrow mesenchymal stem cells in high concentration of glucose microenvironment or with high level of O-GlcNAcylation cell models, and to explore the underlying molecular mechanism. Methods: The extent of growth of hMSCs previously cultured in normal concentration of glucose(group A)(1.0mg/ml), high concentration of glucose(group B)(4.5mg/ml), normal glucose with thiamet-G(group C)(1.0mmol/L) and high glucose with thiamet-G(group D)(1.0mmol/L) was evaluated by WST-1, flow cytometer, β-galactosidase staining to identify the role of glucose and O-GlcNAcylation in the regulation of proliferation, cell cycle, apoptosis and senescence in hMSCs. In addition, the expressions of Cyclin D1 and caspase-3 were detected by real-time RT-PCR and western blot. Results: In group B, group C and group D, hMSCs grew fewer than which in group A on the third, fifth and seventh day(P<0.05). Also in group B, group C and group D, the induced cell cycle was arrested at G1 phase in hMSCs on the fifth day(P<0.05). The rate of apoptosis of hMSCs in group B, group C and group D was higher than that in group A(P<0.05). After treatment, the dimension of hMSCs in group B, group C and group D was larger than that in group A culture(P<0.05), and the positive cells of SA-β-gal staining were also more than those in group A on the seventh day(P<0.05). The real-time PCR showed thawt the expression of Cyclin D1 in group A, B, C and D was 1.01±0.31, 0.31±0.07, 0.42±0.1, 0.18±0.04, and it decreased in group A on the fifth day(P<0.05). The expression of aspase-3 in group A, B, C and D was 1.09±0.82, 5.73±1.54, 3.43±0.59, 6.82±2.13, and it increased in group A on the fifth day(P<0.05). Compared with group A, the expression of Cyclin D1 decreased and the expression of aspase-3 increased when hMSCs were cultured in group B, group C and group D on the fifth day(P<0.05). Conclusions: Our findings demonstrate the effect of glucose concentration and O-GlcNAcylation on regulating the growth, cycle, apoptosis and senescence of hMSCs, which provide insight into the mechanism of how glucose concentration regulating hMSCs.