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目的:探讨叶酸(Folic acid,FA)缺乏在培养的人胚肾细胞(HEK-293)中对细胞组蛋白修饰水平的影响。方法:人胚肾细胞分两组培养,一组正常培养,一组无叶酸培养。细胞提取组蛋白后通过高效液相色谱一线性离子阱/静电场轨道阱高分辨质谱(HPLC-LTQ/Orbitrap Ms)检测组蛋白的修饰以比较叶酸缺乏对人胚肾细胞组蛋白修饰的影响。结果:用高分辨质谱方法成功检测到人胚肾细胞的五个组蛋白变体H1,H3,H4,H2a和H2b上的33个组蛋白修饰位点,其中23个修饰位点为uniprot数据库上已经报道的组蛋白修饰位点,而其余10个为未报道修饰位点。通过质谱比较正常和叶酸缺乏组人胚肾细胞修饰谱发现H3K79me1和H3K79me2在叶酸缺乏培养组中检出率较低。进一步用蛋白免疫印迹的方法也证明了在叶酸缺乏的人胚肾细胞中H3K79me1水平低于正常培养组。结论:细胞中叶酸缺乏影响组蛋白甲基化包括H3K79me2和H3K79me1修饰水平,提示细胞外营养因素叶酸水平可影响组蛋白修饰水平从而参与疾病如神经管畸形(Neural tube defect,NTD)的发生。
Objective: To investigate the effect of folic acid (FA) deficiency on cellular histone modification in cultured human embryonic kidney cells (HEK-293). Methods: Human embryonic kidney cells were divided into two groups, one group of normal culture, one group without folic acid culture. Histones were extracted from the cells and the histone modifications were detected by HPLC-LTQ / Orbitrap MS to compare the effects of folic acid deficiency on the histone modifications of human embryonic kidney cells. RESULTS: Totally 33 histone modifications on H1, H3, H4, H2a and H2b were detected in human embryo kidney cells by high-resolution mass spectrometry, of which 23 were uniprot databases Histone modification sites have been reported, while the remaining 10 are unreported modification sites. Comparison of normal and folic acid deficiency human embryonic kidney cell line by mass spectrometry showed that the detection rate of H3K79me1 and H3K79me2 in folic acid deficiency culture group was lower. Further Western blotting also demonstrated that H3K79me1 levels were lower in folic acid-deficient human embryonic kidney cells than in normal cultures. CONCLUSION: Folic acid deficiency in cells affects histone methylation, including H3K79me2 and H3K79me1 modification, suggesting that extracellular nutrient folic acid levels may influence histone modification levels and thereby contribute to diseases such as neural tube defect (NTD).