本文選題：偏頭痛 + DNA甲基化��； 參考：《中國人民解放軍醫(yī)學院》2016年博士論文

【摘要】：背景：偏頭痛是一種常見神經(jīng)系統(tǒng)疾病,臨床主要表現(xiàn)為反復發(fā)作的單側中重度搏動性頭痛,伴惡心、嘔吐、畏光、畏聲,活動加重頭痛,嚴重影響患者生活質量、工作能力,被WHO列為第6位失能性疾病。由于復雜的發(fā)病機制仍不十分清楚,偏頭痛迄今依然缺乏可用于臨床的標志物,診斷高度依賴臨床癥狀,嚴重困擾患病風險評估、臨床鑒別診斷,以及預后判斷。目前推測表觀調節(jié)機制可能參與偏頭痛的發(fā)病過程,如DNA甲基化和miRNA的異常表達,但臨床研究很少。大量研究顯示降鈣素基因相關肽(CGRP)及其受體活性修飾蛋白1 (RAMP1)與偏頭痛發(fā)病密切相關,然而基因多態(tài)性研究并未發(fā)現(xiàn)與偏頭痛相關。本研究擬從臨床角度,探索外周血RAMP1啟動子區(qū)DNA甲基化水平、循環(huán)miRNA (miR-188-5p與miR-382-5p)及CGRP差異性表達是否與偏頭痛有關,能否作為偏頭痛新的臨床標志物。方法：以偏頭痛患者及年齡、性別匹配的健康對照作為研究對象,抽取靜脈血,分離血清,提取外周血細胞DNA和RNA,以及血清miRNA;亞硫酸鹽處理DNA,采用Sequenom公司的Mass ARRAY系統(tǒng)對RAMP1基因啟動子區(qū)CpG島進行甲基化定量分析;血清miRNA逆轉錄后,實時定量PCR檢測miR-188-5P和miR-382-5p相對表達;采用實時定量PCR檢測外周血CGRP mRNA的表達。分析外周血RAMP1基因啟動子區(qū)CpG島甲基化、CGRP mRNA,以及血清miR-188-5P和miR-382-5p和偏頭痛相關性。結果：①偏頭痛組RAMP1啟動子區(qū)平均甲基化水平較健康對照組有降低趨勢,8.41%±1.92% vs.9.90% ±3.88%,p=0.197：②有偏頭痛家族史的患者在(+25,+27,+31,相對于轉錄起始點)CpG單位的甲基化水平顯著高于無偏頭痛家族史的患者(13.92%±5.97%vs.8.77% ±6.61%,p=0.034)：③女性患者在(+89,+94,+96)CpG單位的甲基化水平顯著低于健康女性(2.18%±1.91% vs.5.85%±5.41%,p=0.02),而在男性患者和男性對照中并沒有類似差異;該位點甲基化水平低于3.50%的女性,其罹患偏頭痛的風險高于甲基化水平大于3.50%的女性(OR:7.313; 95%CI:1.439-37.164);④有偏頭痛家族史患者血清miR-188-5p相對表達量下降4.2倍(p=0.106),發(fā)作期miR-188-5p較非發(fā)作期相對表達量下降5.54倍(p=0.132),但未達到顯著統(tǒng)計學差異;⑤miR-382-5p的相對表達量在偏頭痛和對照組之間未見明顯差異;⑥外周血CGRP mRNA未見差異性表達。結論：①本研究提示RAMP1基因啟動子區(qū)DNA甲基化可能參與偏頭痛發(fā)病,在偏頭痛患者中平均甲基化水平有降低趨勢,2個CpG單位甲基化水平分別與陽性偏頭痛家族史和女性偏頭痛相關,其中(+89,+94,+96)CpG單位甲基化可能與女性罹患偏頭痛的風險有關;②血清miR-188-5p可能與偏頭痛陽性家族史和偏頭痛發(fā)作有關;③血清miR-382-5p水平和外周血CGRP mRNA表達與偏頭痛關聯(lián)性不密切。上述發(fā)現(xiàn)仍需進一步擴大樣本量驗證,其潛在的分子機制仍需闡明。
[Abstract]:Background: migraine is a common nervous system disease. The main clinical manifestations of migraine are recurrent unilateral moderate and severe pulsatile headache with nausea, vomiting, photophobia, fear of noise, aggravation of headache by activity, which seriously affects the patients' quality of life and ability to work. It was ranked the 6th incapacitated disease by WHO. As the complex pathogenesis is still unclear, migraine is still lack of clinical markers, diagnosis is highly dependent on clinical symptoms, serious trouble disease risk assessment, clinical differential diagnosis, and prognosis judgment. It is speculated that epiregulatory mechanism may be involved in the pathogenesis of migraine, such as DNA methylation and abnormal expression of miRNA, but there are few clinical studies. A large number of studies have shown that calcitonin gene-related peptide (CGRP) and its receptor activity modified protein 1 (RAMP1) are closely related to migraine, but gene polymorphism has not been found to be associated with migraine. The aim of this study was to explore whether the differential expression of circulating miRNA (miR-188-5p and miR-382-5p) and CGRP are related to migraine and whether they can be used as a new clinical marker for migraine. Methods: the patients with migraine and the healthy controls matched by age and sex were used as the study objects. The venous blood was collected and the serum was isolated from the patients with migraine. DNA, RNA and serum miRNAs were extracted from peripheral blood cells, DNA was treated with sulfite, methylation quantitative analysis of CpG island of RAMP1 gene promoter region was performed by mass ARRAY system of Sequenom Company, and miR-188-5P and miR-382-5p relative expressions were detected by real-time quantitative PCR after reverse transcription of miRNA in serum. The expression of CGRP mRNA in peripheral blood was detected by real time quantitative PCR. The relationship between CGRP mRNAs and miR-188-5P, miR-382-5p and migraine in peripheral blood was analyzed. Results the average methylation level of RAMP1 promoter in the migraine group was 8.41% 鹵1.92% vs.9.90% 鹵3.88% 鹵0.197: 2 in patients with family history of migraine. The methylation level of CpG units was significantly higher in patients with a family history of migraine (25,27,31, relative to the starting point of transcription). The methylation level of (89,94,96) CpG units in female patients was significantly lower than that in healthy women (13.92% 鹵5.97vs.8.77% 鹵6.61p0.034) compared with healthy women (2.18% 鹵1.91% vs.5.85% 鹵5.41 p0.02), but there was no similar difference between male patients and male controls. The methylation level of the locus was less than 3.50% of women. The risk of migraine was higher than that of women with more than 3.50% methylation level (OR: 7.313; 95 CI: 1.439-37.164). The relative expression of miR-188-5p in patients with family history of migraine decreased 4.2 times (p0.106), and the relative expression of miR-188-5p decreased 5.54 times (p < 0.132) in patients with family history of migraine. Significant statistical difference; There was no significant difference in the relative expression of 5miR-382-5p between migraine and control group. Conclusion this study suggests that DNA methylation in the promoter region of RAMP1 gene may be involved in the pathogenesis of migraine. The average methylation level in migraine patients tended to decrease, and two CpG unit methylation levels were associated with the family history of positive migraine and the female migraine respectively. (89, 94, 96) CpG unit methylation may be associated with the risk of migraine in women. 2Serum miR-188-5p may be related to migraine positive family history and migraine attack. The level of miR-382-5p in serum and CGRP mRNA expression in peripheral blood are not correlated with migraine. These findings need to be further expanded to verify the size of the sample, and its potential molecular mechanism still needs to be clarified.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R747.2

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本文編號：2065626