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目的:研究心肌缺血早期升高的O-GlcNAc糖基化是否通过促进转录因子CAMP反应元件结合蛋白(CREB)的活性而引起心肌细胞形态变化。方法:在HEK-293T细胞内过表达调控O-GlcNAc糖基化的酶O-连接N-乙酰氨基葡萄糖转移酶(OGT)和O-连接N-乙酰氨基葡萄糖苷酶(OGA),Western blot检测细胞内的O-GlcNAc糖基化水平和CREB的磷酸化。原代培养大鼠心肌细胞,药物DON和PUGNAc改变细胞内的O-GlcNAc糖基化水平,光镜下观察细胞形态,Western blot检测细胞内的O-GlcNAc糖基化水平和CREB的磷酸化。结果:在HEK-293T细胞和原代培养的大鼠心肌细胞中改变O-GlcNAc糖基化水平,CREB磷酸化也随之改变,两者呈正相关。升高原代大鼠心肌细胞内的O-GlcNAc糖基化水平,心肌细胞数目减少,形态增大。结论:O-GlcNAc糖基化调节CREB的活性,两者呈正相关。心肌缺血早期升高的O-GlcNAc糖基化很可能通过促进CREB的活性而引起心肌细胞体积增大密度下降,可能是导致心肌重构的重要原因。
AIM: To investigate whether glycosylation of O-GlcNAc in early stage of myocardial ischemia causes cardiomyocyte morphological changes by promoting the activity of transcription factor CAMP response element binding protein (CREB). METHODS: O-threonine-O-linked N-acetylglucosaminyltransferase (OGT) and O-linked N-acetylglucosaminidase (OGA) were overexpressed in HEK-293T cells and detected by Western blot Intracellular O-GlcNAc glycosylation levels and CREB phosphorylation. Primary cultured rat cardiomyocytes, drug DON and PUGNAc changed the level of intracellular O-GlcNAc glycosylation. The morphology of cells was observed under light microscope. The levels of O-GlcNAc and the phosphorylation of CREB were detected by Western blot. Results: The changes of O-GlcNAc glycosylation level in HEK-293T cells and primary cultured rat cardiomyocytes resulted in the change of CREB phosphorylation. Increasing the level of O-GlcNAc glycosylation in primary rat cardiomyocytes decreased the number of cardiomyocytes and increased their morphology. Conclusion: O-GlcNAc glycosylation regulates the activity of CREB, the two are positively correlated. Glycosylation of O-GlcNAc, which is elevated early in myocardial ischemia, probably leads to a decrease in the density of cardiomyocytes by promoting the activity of CREB, which may be one of the important causes of cardiac remodeling.