【作者】 朱静；

【导师】 汪旭； 梁子卿；

【作者基本信息】 云南师范大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 叶酸是人体正常发育的重要微营养物质，其涉及dUMP到dTMP的合成，同时通过同型半胱氨酸(HC)合成甲硫氨酸(Met)和S-腺苷甲硫氨酸(SAM)的生化过程影响DNA的甲基化。在叶酸代谢中，亚甲基四氢叶酸还原酶(MTHFR)催化5，10-亚甲基四氢叶酸(5，10-methylene THF)向5-甲基四氢叶酸(5-methylTHF)转化，MTHFR基因位点突变可导致MTHFR活性降低，同型半胱氨酸水平升高。黄素腺嘌呤二核苷酸(FAD)是MTHFR的辅酶，而FAD的前体为核黄素。因此，叶酸、核黄素缺乏及MTHFR基因多态性可能对DNA结构和DNA甲基化过程、基因表达、染色体分离等形成一定的胁迫作用。 本研究采用多重聚合酶链式反应-限制性片段长度多态(Multiplex PCR-RFLP)技术筛查MTHFR基因677位点、1298位点基因型在正常个体与乳腺癌患者中的多态分布特点；以胞质阻滞微核分析(CBMN)研究各种MTHFR基因型个体淋巴细胞在叶酸、核黄素缺乏以及不同浓度组合条件下产生的遗传与细胞毒性效应，评价不同MTHFR基因型、叶酸与核黄素不同组合对人类基因组稳定性的影响。 研究表明： 1、677位点突变纯合体(TT)在乳腺癌患者(n=122)和对照组(n=99)中的分布频率分别为18.03％和9.09％，无显著性差异；1298位点突变纯合体(CC)在乳腺癌患者(n=119)和对照组(n=98)中的分布频率分别为9.25％和7.15％，也未表现显著性差异；677、1298位点组合基因型在乳腺癌患者和对照组中的分布频率无显著性差异。 2、在本实验浓度范围内，乳腺癌患者及对照个体遗传损伤和细胞损伤指标均具有以下趋势：高叶酸低核黄素组合浓度(HFLR)＜高叶酸高核黄素组(HFHR)＜低叶酸高核黄素组(LFHR)＜低叶酸低核黄素组(LFLR)。提示在叶酸含量较低时，过量的核黄素会对细胞造成损伤。分析各种因素对损伤的贡献发现，叶酸对遗传物质稳定性的影响最大，乳腺癌背景及核黄素对遗传物质稳定性的影响不显著。 3、本实验还讨论了基因型、叶酸、核黄素对淋巴细胞遗传损伤的影响及三者对造成遗传损伤的交互作用。结果发现叶酸、677位点基因型对细胞遗传损伤有显著影响，而核黄素仅对核质桥(NPB)的产生有显著影响，三者对造成遗传损伤更多还原

【Abstract】 Folic acid (FA) is an important micronutrient in human body. It is required for the synthesis of dTMP from dUMP. At the same time, FA is required for the synthesis of S-adenosyl methionine via a series of metablism, the common methyl donor required for the maintenance of methylation patterns in DNA that determine gene expression and DNA conformation and that assure some high methylation on some chromosomes that is greatly important to normally separate during mitotic division. In FA metablism, Methylenetetrahydrofolate reductase (MTHFR) acts as a critical juncture in folate metabolism by catalyzing the irreversible conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate (5-methyl THF), thereby committing folate metabolites to homocysteine remethylation and synthesis of the primary methyl donor for cellular methylation reactions, S-adenosylmethionine (SAM).The two common polymorphisms in the MTHFR gene, 677 C→T and 1298 A→C, have been implicated as risk factors for several cancers. Homozygosity for the 677 C→T polymorphism and compound heterozygosity for 677 C→T and 1298 A→C polymorphisms both reduce enzyme activity. Riboflavin is precursor of flavin adenine dinucleotide (FAD) and FAD is cofactor of MTHFR. So folic acid and riboflavin deficiency and methylenetetrahydrofolate reductase gene polymorphisms not only lead to point mutation but may also result in the generation of single- and double-stranded DNA breaks, chromosome breakage and of the activation of proto-oncogene.We employ Multiplex polymerase chain reaction-Restriction fragmentl ength polymorphism to identify MTHFR 677 C→T and 1298 A→C polymor phisms in breast cancer cases and healthy subjects. We also study the effect of the cytotoxicity and genotoxicity in Human lymphocyte in folic acid and ribofla vin deficiency by the cytokinesis-block micronucleus assay from which we obtai n a comprehensive information of the cytotoxicity and genotoxicity.The experime nt aimed to evaluate the effect of FA, riboflavin and MTHFR polymorphisms o更多还原