【摘要】：背景 表觀遺傳修飾的改變?cè)谀[瘤的發(fā)生中發(fā)揮重要的作用。5-羥甲基胞嘧啶(5hmC)與DNA的去甲基化有關(guān),是DNA分子表觀遺傳調(diào)控的重要方式之一,其在多種腫瘤,如腎癌、乳腺癌、膠質(zhì)瘤等中的總體水平和調(diào)控模式均發(fā)生了明顯變化。染色體10/11易位家族蛋白2(TET2)是一種雙氧酶,可催化5-甲基胞嘧啶(5mC)生成5hmC,繼續(xù)催化生成5-甲酰胞嘧啶(5fC)和5-羧基胞嘧啶(5caC)。TET2突變的發(fā)生在血液系統(tǒng)疾病中常見且影響5hmC水平,而TET2蛋白的表達(dá)量和定位情況也與5hmC的水平高低直接相關(guān)。研究證實(shí)表觀遺傳學(xué)改變也參與了垂體腺瘤的發(fā)生,但5hmC是否參與其中尚不清楚。無功能垂體腺瘤(NFPAs)中5hmC的水平及改變機(jī)制,尚無相關(guān)研究證實(shí)。NFPAs作為一種發(fā)病率高、危害嚴(yán)重、治療困難的腫瘤,有必要對(duì)其發(fā)病機(jī)制進(jìn)行進(jìn)一步的研究。目的 本研究旨在檢測NFPAs全基因組胞嘧啶修飾的變化,分析5hmC生成的催化酶TET2編碼基因的變異、蛋白表達(dá)及定位等對(duì)5hmC水平的影響,初步探索5hmC水平變化的機(jī)制。研究內(nèi)容包括3個(gè)部分。第一,收集樣本及臨床資料并分組,測定NFPAs和正常垂體全基因組5hmC總體水平；第二,對(duì)所有NFPAs樣本進(jìn)行TET2外顯子部分區(qū)域測序,檢測有無變異；第三,進(jìn)行NFPAs和正常垂體的TET2免疫組化染色。方法 選取NFPAs 57例(57例均進(jìn)行超高效液相色譜-電噴霧串聯(lián)質(zhì)譜(UPLC-ESI-MS/MS)分析和TET2外顯子部分區(qū)域測序,26例進(jìn)行免疫組化染色),正常垂體6例(5例進(jìn)行UPLC-ESI-MS/MS分析,1例進(jìn)行免疫組化染色),收集并整理臨床資料,NFPAs分為侵襲性和非侵襲性兩組,由北京協(xié)和醫(yī)院神經(jīng)外科兩位經(jīng)驗(yàn)豐富的醫(yī)生根據(jù)影像學(xué)資料和手術(shù)術(shù)野所見分別獨(dú)立判斷。通過質(zhì)譜檢測的方法比較NFPAs和正常垂體全基因組5hmC的總體水平,通過PCR擴(kuò)增TET2編碼區(qū)進(jìn)行測序?qū)ふ易儺?通過免疫組化染色分析TET2蛋白的表達(dá)和定位,表達(dá)水平以軟件分析的H-score值表示。結(jié)果NFPAs中5hmC的總體水平明顯低于正常垂體(0.38‰(0.13～1.23‰)vs2.47‰(0.82-2.86‰),P0.0001),5caC的總體水平則明顯升高(0.20%o(0.04～0.24%o) vs 0.16‰(0.14-0.19‰), P=0.005)。 5hmC水平低的病例,腫瘤直徑更大(34.6±12.7mm vs 27.4±8.5mm, P=0.023), Ki-67指數(shù)更高(≤3%/3%的例數(shù)分別為9/5 vs 16/0,P=0.014)。低5hmC組的年齡、性別、病程、侵襲性、P53及Knosp分級(jí)與高5hmC組無明顯差異。非侵襲組相比侵襲組,5hmC水平無明顯差異(0.44%o (0.15～1.04%o) vs 0.35%o (0.13～1.23%o), P=0.30)。測序檢測到TET2 c.86CG、c.5162TG和c.5284AG(正鏈)3個(gè)單核苷酸多態(tài)性(SNP)位點(diǎn),對(duì)應(yīng)有TET2 p.P29R、p.L1721W和p.I1762V 3個(gè)位點(diǎn)氨基酸改變。其中TET2 R29組相比P29組5hmC水平明顯降低(0.25‰(0.13～0.80‰)vs 0.46%o (0.16-1.23‰), P=0.013), TET2 p.L1721W、p.l1762V對(duì)5hmC水平無明顯影響。NFPAs中5hmC水平和TET2總表達(dá)量、細(xì)胞核表達(dá)量呈中度正相關(guān)(r=0.461,P=0.018; r=0.458, P=0.019)。免疫組化樣本按質(zhì)譜檢測的5hmC水平高低分成兩組,高5hmC組相比低5hmC組TET2總表達(dá)量和細(xì)胞核表達(dá)量明顯升高(192.78±79.87 vs 129.58±60.18,P=0.032；121.49±49.21 vs 80.07±36.68,P=0.023)。非侵襲組相比侵襲組,TET2表達(dá)量和定位無明顯差異。結(jié)論1、NFPAs存在全基因組5hmC的缺失,5hmC水平越低預(yù)示著腫瘤直徑越大、Ki-67指數(shù)越高,提示DNA羥甲基化修飾的總量降低可能參與NFPAs的腫瘤發(fā)生,并且和腫瘤的大小、Ki-67指數(shù)相關(guān)。2、存在TET2 p.P29R變異的樣本5hmC的水平明顯降低,TET2 p.P29R變異影響5hmC水平的機(jī)制尚不清楚。隨著TET2總表達(dá)量、細(xì)胞核定位的增多5hmC總體水平升高,提示5hmC水平的變化可能受到TET2表達(dá)量和亞細(xì)胞定位的影響。
[Abstract]:Background Epigenetic modification plays an important role in tumorigenesis. 5-hydroxymethyl cytosine (5hmC) is related to DNA demethylation and is one of the important ways of DNA molecular epigenetic regulation. It has been found that the overall level and regulation pattern of 5-hydroxymethyl cytosine (5hmC) in various tumors, such as renal cancer, breast cancer, glioma, etc. have changed significantly. Body 10/11 translocation family protein 2 (TET2) is a hydrogen peroxidase that catalyzes 5-methyl-cytosine (5mC) to form 5-hmC and continues to catalyze the formation of 5-formyl-cytosine (5fC) and 5-carboxy-cytosine (5caC). Mutations in TET2 are common in hematological diseases and affect the level of 5-hmC. The expression and localization of TET2 protein are also directly related to the level of 5-hmC. Studies have confirmed that epigenetic alterations are also involved in the development of pituitary adenomas, but it is not clear whether 5hmC is involved in the development of pituitary adenomas. Objective To detect the changes of cytosine modification in the whole genome of NFPAs, analyze the effect of the mutation of TET2 gene, protein expression and localization on the 5hmC level, and explore the mechanism of the changes in the 5hmC level. Group A, 5 hmC genome level of NFPAs and normal pituitary were measured; secondly, some regions of exon TET2 were sequenced in all samples of NFPAs, and the mutations were detected; thirdly, TET2 immunohistochemical staining was performed in NFPAs and normal pituitary. Methods 57 cases of NFPAs (57 cases) were selected for ultrahigh performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-M). S/MS analysis and TET2 exon partial region sequencing, 26 cases of immunohistochemical staining, 6 cases of normal pituitary gland (5 cases of UPLC-ESI-MS/MS analysis, 1 case of immunohistochemical staining), collection and collation of clinical data, NFPAs were divided into two groups: invasive and non-invasive, by Beijing Union Medical College Hospital neurosurgery two experienced doctors according to imaging. The total levels of NFPAs and 5 hmC of normal pituitary genome were compared by mass spectrometry. The TET2 coding region was amplified by PCR and sequenced. The expression and localization of TET2 protein were analyzed by immunohistochemical staining. The expression level was expressed by H-score value of software analysis. The overall level of 5 hmC in NFPAs was significantly lower than that in normal pituitary (0.38 8240 (0.13-1.23 82) vs 2.47 82 (0.82-2.86 82), P 0.0001, 0.82-2.86 82), 0.82-2.86 82), while the overall level of 5 caC was significantly higher (0.20% o (0.20% o (0.04-0.24% o) vs 0.16 82 (0.14-0.14-0.19 82), P = 0.005) in patients with lower level of normal pitpituitary (0.38 82 82 (0.13-1.13-1.23 82) vs 2.47 82 (0.47 023), higher Ki-67 index There was no significant difference in age, sex, course of disease, invasiveness, P53 and Knosp grade between the low 5hmC group and the high 5hmC group. There was no significant difference in 5hmC level between the non-invasive group and the invasive group (0.44% o (0.15-1.04% o) vs 0.35% o (0.13-1.23% o), P = 0.30). There were three single nucleotide polymorphisms (SNP) loci in the positive strand, corresponding to three amino acid changes at TET2 p.P29R, p.L1721W and p.I1762V. The levels of 5 hmC in TET2 R29 group were significantly lower than those in P29 group (0.25 (0.13-0.80) vs 0.46% o (0.16-1.23), P = 0.013, TET2 p.L1721W, p.l1762V) and TET2 had no significant effect on 5 hmC levels. Total expression of TET2 and nuclear expression were moderately positively correlated (r = 0.461, P = 0.018; r = 0.458, P = 0.019). Immunohistochemical samples were divided into two groups according to the level of 5 hmC detected by mass spectrometry. Total expression of TET2 and nuclear expression in 5 hmC group were significantly higher than those in 5 hmC group (192.78 [79.87] vs 129.58 [60.18], P = 0.032; 121.49 [49.21] vs 80.07 [36.68], P = 0.032, respectively. There was no significant difference in the expression and localization of TET2 between the non-invasive group and the invasive group. Conclusion 1. There was a deletion of 5hmC in the whole genome in NFPAs. The lower the 5hmC level indicates that the larger the tumor diameter, the higher the Ki-67 index, suggesting that the decrease of DNA hydroxymethylation may be involved in the tumorigenesis of NFPAs, and is related to the tumor size and Ki-67 index. With the total expression of TET2, the overall level of 5 hmC increased, suggesting that the change of 5 hmC level may be affected by the expression of TET2 and subcellular localization.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R736.4

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2 姜海洋;垂體腺瘤術(shù)后低鈉血癥與血漿B型腦鈉肽水平的相關(guān)性研究[D];蘭州大學(xué);2015年

3 陶曉e，

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