本文關(guān)鍵詞：母體甲基供體缺乏對子代小鼠實驗性結(jié)腸炎的影響及可能的表觀遺傳學(xué)機制研究　出處：《天津醫(yī)科大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 潰瘍性結(jié)腸炎 甲基供體 DNA甲基化 干擾素-γ 干擾素-γ基因 CpG島

【摘要】：目的潰瘍性結(jié)腸炎(ulcerative colitis,UC)是一種慢性、反復(fù)發(fā)作性的結(jié)直腸連續(xù)性黏膜炎癥性疾病,多以年輕患者多發(fā)。但其發(fā)病機制仍不明確。目前,認(rèn)為UC是一種多因素、異質(zhì)性、多基因遺傳的疾病,多種因素包括環(huán)境、遺傳、感染、免疫、腸道微生物等相互作用的結(jié)果。近年來發(fā)現(xiàn)表觀遺傳學(xué)是環(huán)境與遺傳因素之間的橋梁,其中DNA甲基化是重要的表觀遺傳修飾之一,它能夠調(diào)節(jié)哺乳動物細(xì)胞的正常發(fā)育、分化,基因表達(dá)和染色質(zhì)結(jié)構(gòu)。DNA甲基化需要甲基供體,包括葉酸、膽堿、B族維生素和甲硫氨酸。DNA高甲基化與基因沉默相關(guān),而低甲基化則基因表達(dá)活躍。研究發(fā)現(xiàn)在UC患者腸道T淋巴細(xì)胞中,IFN-γ表達(dá)增加與甲基化水平降低相關(guān),而且IFN-γ基因(IFNG)甲基化水平在調(diào)節(jié)黏膜細(xì)胞因子分泌中有重要作用。眾多證據(jù)表明在哺乳動物發(fā)育過程中,妊娠期和新生兒期的環(huán)境因素通過影響表觀遺傳修飾,導(dǎo)致表型的永久性改變。因此,改變母體飲食因素可能通過影響DNA甲基化參與UC發(fā)生、發(fā)展。本研究以BALC/c鼠為研究對象,通過給予母鼠甲基供體缺乏飲食,構(gòu)建子代小鼠實驗性結(jié)腸炎模型,并檢測血清葉酸、維生素B12和同型半胱氨酸水平,結(jié)腸黏膜IFN-γ表達(dá)水平,以及IFNG啟動子區(qū)DNA甲基化水平,探討母體甲基供體缺乏是否通過影響DNA甲基化從而參與UC的發(fā)病過程。方法1.動物模型建立:7周齡BALB/c雌鼠在孕前1個月開始分別喂養(yǎng)標(biāo)準(zhǔn)飲食(C,n=3)和甲基供體缺乏飲食(D,即無葉酸、膽堿、維生素B12和甲硫氨酸,n=4),持續(xù)到子代斷乳(即出生后21天);子代小鼠與母鼠相同飲食直到被處死。子鼠出生后第23天,造模組以2.5%葡聚糖硫酸鈉溶液(DSS)作為飲用水5天,飲食水量不限。子代小鼠分組如下:C/DSS-組、D/DSS-組、C/DSS+組、D/DSS+組,每組6只。2.結(jié)腸炎疾病活動指數(shù)(DAI):每日觀察子代小鼠身體狀況、體質(zhì)量、糞便性狀、肛周紅腫糜爛和便血程度。根據(jù)體重減輕程度、糞便性狀和便血程度評分,分?jǐn)?shù)范圍0-4分,根據(jù)DAI=總分?jǐn)?shù)/3(0-4),評估結(jié)腸炎癥程度。3.采用酶聯(lián)免疫吸附法(ELISA)檢測子代小鼠外周靜脈血清葉酸、維生素B12和同型半胱氨酸(Hcy)水平。4.采用免疫組織化學(xué)SP法檢測子代小鼠結(jié)腸黏膜組織中IFN-γ的表達(dá)水平。5.提取結(jié)腸組織樣本基因組DNA,經(jīng)過亞硫酸鹽轉(zhuǎn)化,采用Sequenom Mass Array甲基化檢測方法檢測IFNG啟動子區(qū)Cp G島甲基化水平。結(jié)果1.甲基供體缺乏的證據(jù):與標(biāo)準(zhǔn)飲食組(C/DSS-和C/DSS+組)相比,甲基供體缺乏飲食組(D/DSS-和D/DSS+組)小鼠的血清葉酸(8.87±1.11比11.34±0.31nmol/L,P0.01)、維生素B12(409.2±56.27比676.1±51.66 ng/L,P0.01)水平均顯著降低;同型半胱氨酸(8.45±0.35比6.77±0.36μmol/L,P0.01)水平顯著增高。2.甲基供體缺乏對DAI和組織學(xué)評分的影響:在DSS處理后第5天,結(jié)腸炎癥程度最高。C/DSS+組較C/DSS-組的DAI高,差異有統(tǒng)計學(xué)意義(P0.05);D/DSS+組較C/DSS+組的DAI增高,差異有統(tǒng)計學(xué)意義(P0.01),表明甲基供體缺乏飲食更加加重了小鼠結(jié)腸炎;C/DSS-組和D/DSS-組的DAI無統(tǒng)計學(xué)差異。D/DSS-組組織學(xué)評分高于C/DSS-組,D/DSS+組明顯高于C/DSS+組,也表明甲基供體缺乏飲食加重了小鼠結(jié)腸炎。3.小鼠結(jié)腸黏膜組織IFN-γ的表達(dá)水平:C/DSS組和D/DSS組IFN-γ表達(dá)水平較C/DSS-組和D/DSS-組顯著增高(c2=14.875,P0.01),而且D/DSS+組較C/DSS+組增高更明顯(P0.01)。4.小鼠結(jié)腸黏膜組織IFN-γ表達(dá)水平與DAI的相關(guān)性:C/DSS+組和D/DSS+組的IFN-γ表達(dá)水平與DAI呈正相關(guān)(r=0.853、0.840,P均0.05)。5.IFNG啟動子區(qū)CpG島甲基化水平:對同一CpG位點四組樣本之間甲基化水平分析未發(fā)現(xiàn)明顯差異,對四組樣本之間所有Cp G位點總甲基化水平分析也未發(fā)現(xiàn)明顯差異。結(jié)論1.母體甲基供體缺乏能夠加劇子代小鼠實驗性結(jié)腸炎發(fā)病。2.甲基供體缺乏飲食可以引起子代結(jié)腸黏膜IFN-γ表達(dá)增高,參與實驗性結(jié)腸炎發(fā)病。3.甲基供體缺乏并非通過IFNG啟動子區(qū)Cp G島低甲基化,從而導(dǎo)致IFN-γ表達(dá)增高,進(jìn)而加重實驗性結(jié)腸炎。
[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.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R574.62

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