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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-Glc NAc)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m RNA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m RNA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h BMSCs内蛋白质O-Glc NAc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-Glc NAc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-Glc NAc糖基化水平升高。微环境中高浓度葡萄糖诱导的蛋白质O-Glc NAc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。
Aims: To investigate whether different concentrations of glucose in microenvironment can affect the proliferation, cycle, apoptosis and senescence of human bone marrow mesenchymal stem cells through the glycosylation modification of O-GlcNAc. Methods: The normal glucose (A group) (5.5 mmol / L), high concentration glucose (B group) (25 mmol / L) mmol / L), high glycosylation (D group) (high glucose + Thiamet-G 1.0mmol / L) cell model. Human bone marrow-derived mesenchymal stem cells were cultured in each group. On the 3rd, 5th, 7th day, the proliferation of each group was measured by WST-1 cell proliferation and cytotoxicity detection kit. On the 5th day, The cell cycle and apoptosis were observed. Cell senescence was detected by cell senescence β-galactosidase staining kit on the 7th day. The expression of Cyclin in each group was detected by Real-time PCR and Westen-bolt on the 5th day D1 and caspase-3 expression levels. Results: The results of cell proliferation on the 3rd, 5th, 7th day showed that compared with the A group, the proliferation of B, C and D groups was significantly decreased (P <0.05). The results of cell cycle showed that cells in group B, C and D were arrested in cell cycle G1 (P <0.05) compared with those in group A. The results of apoptosis showed that compared with group A, the apoptosis of group B, group C and group D were significantly increased (P <0.05). Cell senescence staining showed that compared with group A, cell senescence of groups B, C and D were significantly increased (P <0.05). Real-time PCR results showed that relative mRNA expression of Cyclin D1 in group A, group B, group C and group D was 1.01 ± 0.31,0.31 ± 0.07,0.42 ± 0.1,0.18 ± 0.04, respectively, while the other three groups were significantly lower than group A (P <0.05). The relative values of m RNA of caspase-3 in group A, B, C and D were 1.09 ± 0.82, 5.73 ± 1.54, 3.43 ± 0.59 and 6.82 ± 2.13, respectively, Group A (P <0.05). The results of Westen-bolt showed that the protein level of O-GlcNAc in group B hMSCs was higher than that of group B; the level of protein in group B was lower than that in group C (P <0.05); the level of caspase -3 protein expression in group B, C and D group than in group A (P <0.05). CONCLUSION: Glycosylation of O-GlcNAc with high levels of glucose and cellular proteins in microenvironment inhibits the proliferation of human mesenchymal stem cells and arrests the cell cycle to promote apoptosis and senescence. High concentrations of glucose in the microenvironment can induce elevated intracellular protein O-GlcNAc glycosylation levels. The high level of glucose-induced protein O-GlcNAc glycosylation in the microenvironment may be one of the mechanisms by which its inhibition of proliferation enhances apoptosis and senescence.