不同还原态叶酸/胆碱/同型半胱氨酸对人类端粒长度和hTERT表观修饰的影响

Telomeres play a critical role in maintaining the integrity and stability of the genome. Genomic instability is associated with numerous degenerative diseases, including most of tumors, immune dysfunction, osteoporosis, cardiovascular and neurodegenerative diseases. Telomerase is one of the key mechanisms to maintain the telomere length. Expression of the core enzyme- - catalytic subunit of telomerase, human telomerase reverse transcriptase(hTERT) is the major limiting factor for telomerase activity. Folates are essential micronutrents in the transfer and utilization of one-carbon groups in the process of DNA-biosynthesis, methylation and DNA damage repairing. Choline and folate are interrelated methyl donors and they are interknited at the point where homocysteine is converted to methionine. Folate and choline are micronutrents which benefit to maintain human genomic stability and proper methylation pattern in DNA. However, homocysteine is shown to be an independent risk factor for neurodegenerative and cardiovascular disease. Homocysteine is readily oxidized and thought to exert its effects through mechanisms involving radical oxygen species and oxidative damage. The project is based on the theory and methodology of nutrigenomics and epigenetcs, and focuses on to explore ① The effects of serum folate, homocysteine and the SNP located on the upstream of hTERT promoter on the telomere length and genomic stability in human lymphocytes, the influence of serum folate on the methylation pattern and expression of hTERT gene; ②Comparison of the intervention effects and differences of reduced folate derivates and choline on the modification of hTERT promoter in different kind of immortal cells in vitro. The research will benefit to the construction of strategy for nutrition intervention aimed on the maintaining of normal telomere structure and function.

端粒结构功能异常导致基因组完整性与稳定性下降，基因组不稳定与多数肿瘤、发育异常、免疫缺陷、骨质疏松和神经系统退行性疾病危险度上升密切关联。端粒酶核心成分即端粒酶反转录酶(hTERT)表达水平是决定端粒酶活和端粒长度的关键因素。叶酸是一组与DNA合成、甲基化和损伤修复高度相关的微营养素，叶酸与胆碱代谢在同型半胱氨酸（Hcy）转化为甲硫氨酸节点相交汇。前期工作提示叶酸和胆碱利于基因组稳定和DNA甲基化模式维持,而Hcy代谢易造成氧化胁迫。项目拟从营养基因组学与表观遗传学的角度,①分析血清叶酸、Hcy浓度和hTERT启动子上游SNP对人淋巴细胞端粒长度与基因组稳定性的影响,探讨血清叶酸对hTERT启动子CpG位点甲基化修饰与表达的影响；②分析比较不同还原态叶酸和胆碱离体干预对人端粒酶阳性/阴性细胞hTERT启动子甲基化修饰、转录及端粒长度的影响与差异。研究利于构建维护正常端粒结构的营养干预策略。

端粒是维护基因组完整与稳定的关键内因,基因组稳定性下降与大多数肿瘤、发育异常、免疫缺陷、心脑血管疾病和骨质疏松等退行性疾病的风险上升紧密关联。端粒酶是维持正常端粒长度所必需,其核心酶即催化反转录酶(hTERT)水平是决定端粒酶活的关键因素。项目从一碳单位关键微营养素的层面, 分析了①血清叶酸/Hcy浓度及hTERT启动子上游SNP对人淋巴细胞端粒长度和基因组稳定性的影响；②不同浓度氧化型叶酸对人类端粒酶阳性/阴性细胞株端粒长度和基因组稳定性的影响；③不同还原态叶酸对端粒酶阳性/阴性细胞株端粒长度和hTERT表达的影响；④ Hcy对血管内皮细胞遗传稳定性的影响。.人外周淋巴细胞分析发现，hTERT -1327 TT和-245 CC个体的端粒长度显著高于其它等位基因型；红细胞叶酸浓度与端粒长度呈显著正相关,未发现端粒长度与血清叶酸、维生素B12、Hcy间存在显著关联。.Hcy水平与叶酸和维生素B12浓度呈显著负相关，MTHFR 677 TT基因型的血清Hcy显著高于CC和CT个体。.基因组不稳定性分析显示基因组损伤与年龄呈显著正相关，未发现端粒长度与基因组稳定性显著相关。.研究发现BJ的端粒长度与叶酸浓度呈正相关，叶酸缺乏在BJ细胞中诱发端粒长度不稳定改变和染色体稳定性下降；维持人离体淋巴细胞基因组稳定的最适FA浓度也能维护人端粒酶阴性成纤维细胞株BJ的端粒结构稳定.但在任何浓度和形式的叶酸干预下，BJ细胞的hTERT的表达无显著改变。.端粒酶阳性人皮肤黑色素瘤细胞A375在叶酸缺乏时基因组稳定性下降, 但端粒长度无显著变化。不同氧化态叶酸缺乏可能导致A375细胞内甲基供体出现不同程度的减少，致使hTERT表达下调;直接参与甲基化途径的5-MeTHF对hTERT表达的影响强于FA。无论叶酸状态如何，A375细胞端粒都相对恒定，且与hTERT表达改变无明显对应关系。.综上，一碳单位代谢的关键微营养素对于基因组和端粒的稳定性、对于Hcy水平的调节具有重要作用。