母体甲基供体缺乏对子代小鼠实验性结肠炎的影响及可能的表观遗传学机制研究

发布时间：2018-01-09 13:01

本文关键词：母体甲基供体缺乏对子代小鼠实验性结肠炎的影响及可能的表观遗传学机制研究　出处：《天津医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的溃疡性结肠炎(ulcerative colitis,UC)是一种慢性、反复发作性的结直肠连续性黏膜炎症性疾病,多以年轻患者多发。但其发病机制仍不明确。目前,认为UC是一种多因素、异质性、多基因遗传的疾病,多种因素包括环境、遗传、感染、免疫、肠道微生物等相互作用的结果。近年来发现表观遗传学是环境与遗传因素之间的桥梁,其中DNA甲基化是重要的表观遗传修饰之一,它能够调节哺乳动物细胞的正常发育、分化,基因表达和染色质结构。DNA甲基化需要甲基供体,包括叶酸、胆碱、B族维生素和甲硫氨酸。DNA高甲基化与基因沉默相关,而低甲基化则基因表达活跃。研究发现在UC患者肠道T淋巴细胞中,IFN-γ表达增加与甲基化水平降低相关,而且IFN-γ基因(IFNG)甲基化水平在调节黏膜细胞因子分泌中有重要作用。众多证据表明在哺乳动物发育过程中,妊娠期和新生儿期的环境因素通过影响表观遗传修饰,导致表型的永久性改变。因此,改变母体饮食因素可能通过影响DNA甲基化参与UC发生、发展。本研究以BALC/c鼠为研究对象,通过给予母鼠甲基供体缺乏饮食,构建子代小鼠实验性结肠炎模型,并检测血清叶酸、维生素B12和同型半胱氨酸水平,结肠黏膜IFN-γ表达水平,以及IFNG启动子区DNA甲基化水平,探讨母体甲基供体缺乏是否通过影响DNA甲基化从而参与UC的发病过程。方法1.动物模型建立:7周龄BALB/c雌鼠在孕前1个月开始分别喂养标准饮食(C,n=3)和甲基供体缺乏饮食(D,即无叶酸、胆碱、维生素B12和甲硫氨酸,n=4),持续到子代断乳(即出生后21天);子代小鼠与母鼠相同饮食直到被处死。子鼠出生后第23天,造模组以2.5%葡聚糖硫酸钠溶液(DSS)作为饮用水5天,饮食水量不限。子代小鼠分组如下:C/DSS-组、D/DSS-组、C/DSS+组、D/DSS+组,每组6只。2.结肠炎疾病活动指数(DAI):每日观察子代小鼠身体状况、体质量、粪便性状、肛周红肿糜烂和便血程度。根据体重减轻程度、粪便性状和便血程度评分,分数范围0-4分,根据DAI=总分数/3(0-4),评估结肠炎症程度。3.采用酶联免疫吸附法(ELISA)检测子代小鼠外周静脉血清叶酸、维生素B12和同型半胱氨酸(Hcy)水平。4.采用免疫组织化学SP法检测子代小鼠结肠黏膜组织中IFN-γ的表达水平。5.提取结肠组织样本基因组DNA,经过亚硫酸盐转化,采用Sequenom Mass Array甲基化检测方法检测IFNG启动子区Cp G岛甲基化水平。结果1.甲基供体缺乏的证据:与标准饮食组(C/DSS-和C/DSS+组)相比,甲基供体缺乏饮食组(D/DSS-和D/DSS+组)小鼠的血清叶酸(8.87±1.11比11.34±0.31nmol/L,P0.01)、维生素B12(409.2±56.27比676.1±51.66 ng/L,P0.01)水平均显著降低;同型半胱氨酸(8.45±0.35比6.77±0.36μmol/L,P0.01)水平显著增高。2.甲基供体缺乏对DAI和组织学评分的影响:在DSS处理后第5天,结肠炎症程度最高。C/DSS+组较C/DSS-组的DAI高,差异有统计学意义(P0.05);D/DSS+组较C/DSS+组的DAI增高,差异有统计学意义(P0.01),表明甲基供体缺乏饮食更加加重了小鼠结肠炎;C/DSS-组和D/DSS-组的DAI无统计学差异。D/DSS-组组织学评分高于C/DSS-组,D/DSS+组明显高于C/DSS+组,也表明甲基供体缺乏饮食加重了小鼠结肠炎。3.小鼠结肠黏膜组织IFN-γ的表达水平:C/DSS组和D/DSS组IFN-γ表达水平较C/DSS-组和D/DSS-组显著增高(c2=14.875,P0.01),而且D/DSS+组较C/DSS+组增高更明显(P0.01)。4.小鼠结肠黏膜组织IFN-γ表达水平与DAI的相关性:C/DSS+组和D/DSS+组的IFN-γ表达水平与DAI呈正相关(r=0.853、0.840,P均0.05)。5.IFNG启动子区CpG岛甲基化水平:对同一CpG位点四组样本之间甲基化水平分析未发现明显差异,对四组样本之间所有Cp G位点总甲基化水平分析也未发现明显差异。结论1.母体甲基供体缺乏能够加剧子代小鼠实验性结肠炎发病。2.甲基供体缺乏饮食可以引起子代结肠黏膜IFN-γ表达增高,参与实验性结肠炎发病。3.甲基供体缺乏并非通过IFNG启动子区Cp G岛低甲基化,从而导致IFN-γ表达增高,进而加重实验性结肠炎。
[Abstract]:The purpose of ulcerative colitis (ulcerative colitis UC) is a chronic, recurrent colorectal mucosa continuity of inflammatory disease in young patients with multiple. But its pathogenesis is still unclear. At present, UC is a multi factor, heterogeneity, multiple genetic disease, a variety of factors including the environment, heredity, infection, immunity, intestinal microbial interactions in recent years. Results show that epigenetics is a bridge between environmental and genetic factors, in which DNA methylation is one of the important epigenetic modifications, normal development, it can regulate mammalian animal cell differentiation, gene expression and staining of methyl donor the quality structure of.DNA methyl, including folic acid, choline, vitamin B and methionine.DNA hypermethylation is associated with gene silencing, and low methylation gene expression is active. The study found in the patients with intestinal UC T lymphocytes in IFN-. Gamma expression increases with the decrease of methylation level, and IFN- gamma (IFNG) gene methylation in regulating mucosal cytokine secretion plays an important role. Many evidence in mammalian development process, environmental factors during pregnancy and the newborn period on genetic modification by affecting the table, resulting in permanent phenotypic changes so. Change, maternal dietary factors may influence the methylation of DNA involved in UC occurrence, development. This research is based on the BALC/c rats as the research object, by giving the mice a diet lacking methyl donor, construction of offspring in mice with experimental colitis model, and serum folic acid, vitamin B12 and homocysteine levels in colonic mucosa of IFN- gamma expression. IFNG and promoter DNA methylation levels, to explore the pathogenesis of the lack of maternal methyl donor could influence DNA methylation to participate in UC. Methods 1. animal model: 7 weeks Old BALB/c female rats were fed a standard diet in the beginning 1 months before pregnancy (C, n=3) and methyl donor deficient diet (D, no choline, folic acid, vitamin B12 and methionine, n=4), continued to offspring weaning (21 days after birth); maternal and offspring were the same diet to be straight were killed. Twenty-third days after birth, the model with 2.5% dextran sulfate sodium (DSS) as drinking water for 5 days, the diet content is not limited. The offspring of mice following groups: C/DSS- group, D/DSS- group, C/DSS+ group, D/DSS+ group, 6 rats in each group.2. colitis disease activity index (DAI): daily observation of offspring the mouse body condition, body weight, stool, stool and perianal swelling and erosion degree. According to the degree of weight loss, stool and stool score, score 0-4 points, according to the total score of /3 DAI= (0-4), to assess the extent of inflammation of.3. by enzyme linked immunosorbent assay (ELISA) detection of transgenic mice peripheral static Vein serum folic acid, vitamin B12 and homocysteine (Hcy) levels of.4. were detected by SP immunohistochemical method in mice offspring in colon mucosa of IFN- gamma.5. expression level in colon tissue samples to extract genomic DNA by using Sequenom Mass Array bisulfite conversion methylation detection method to detect the IFNG level of Cp promoter G island methylation results of the 1. sub regions. The lack of evidence: methyl donor and standard diet (C/DSS- group and C/DSS+ group) compared to methyl donor deficient diet (D/DSS- group and D/DSS+ group) serum folic acid in mice (8.87 + 1.11 and 11.34 + 0.31nmol /L, P0.01), vitamin B12 (409.2 + 56.27 to 676.1 + 51.66 ng/L, P0.01) level were significantly decreased; homocysteine (8.45 + 0.35 6.77 + 0.36 mol/L, P0.01) were significantly higher in.2. methyl donor lack of scores of DAI and organization in the DSS after fifth days, the highest degree of.C/ colitis DSS+ group than in C/DSS- group DAI, the difference was statistically significant (P0.05); D/DSS+ group compared with C/DSS+ group, the DAI increased, the difference was statistically significant (P0.01), showed that the methyl donor deficient diet more aggravated colitis in mice; C/DSS- group and D/DSS- group DAI score higher than that of C/DSS- group.D/ group DSS- group significant difference, D/DSS+ group was significantly higher than that in group C/DSS+, also showed that the methyl donor deficient diet increased the expression of.3. in colonic mucosa of mice with colitis mice IFN- Gamma: expression of C/DSS group and D/DSS group IFN- levels compared with C/DSS- group and D/DSS- group increased significantly (c2=14.875, P0.01), and D/DSS+ group than in C/DSS+ group increased more significantly (P0.01).4. mice colonic mucosa IFN- expression level and DAI correlation: IFN- gamma C/DSS+ group and D/DSS+ group, the expression level was positively correlated with DAI (r=0.853,0.840 P 0.05).5.IFNG promoter CpG island methylation level in the same sub district Between the four groups of sample CpG sites methylation analysis found no significant differences between the four sample groups of all Cp G sites methylation level analysis also found no significant differences. Conclusion 1. maternal methyl donor deficiency increased the offspring of mice with experimental colitis.2. methyl donor deficient diet can cause the offspring of colonic mucosa IFN- expression increased participation in experimental colitis is not lack of donor.3. methyl Cp in the promoter region G Island hypomethylation by IFNG, resulting in increased expression of IFN- gamma, thereby increasing the experimental colitis.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R574.62

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