發(fā)布時間：2019-07-03 12:48

【摘要】：研究背景：具有高敏感性和特異性的診斷指標對于肝硬化的診斷、病情進展的隨訪和治療起著十分重要的作用。大量的研究表明循環(huán)中miRNA可作為許多疾病,包括慢性肝病的診斷指標。本研究的目的是對肝硬化患者血漿miRNA的表達水平進行檢查,探討血漿miRNA作為肝硬化無創(chuàng)診斷標志物的意義。實驗方法：該研究為多階段的病例對照研究,過程包括以下幾部分：第一部分,為研究的篩選階段(primary exploration phase),80個血漿樣本(其中25個乙肝肝硬化、22個慢性肝炎和33個正常對照)處理后進行芯片全基因組miRNA表達檢測。該芯片共包含有723個常見的miRNA。初篩結(jié)果經(jīng)分析后,挑選出6個miRNA進行第二部分的驗證工作。第二部分為研究的驗證和建模階段(trainning phase),首先,6個miRNA在41個樣本中(20個乙肝肝硬化、9個慢性乙肝和12分正常對照)運用qPCR的方法進行驗證,挑選出2個具有較高穩(wěn)定性的miRNA,進一步在其余141個樣本(70個乙肝肝硬化、23個慢性乙肝和48個正常對照)中運用qPCR的方法進行驗證,結(jié)果應(yīng)用Logistic回歸進行整合。第三部分為診斷模型的驗證階段(validation phase),在第二部分得到的miRNA診斷模型在兩個獨立隊列中分別進行驗證,以保證該指標的穩(wěn)定性。其中一個隊列有44個樣本,包括13個無臨床表現(xiàn)的早期肝硬化、25個慢性肝炎和6個正常對照,所有樣本都有肝穿病理結(jié)果明確診斷；另一個隊列有92個樣本,包括47個非乙肝肝硬化、7個非乙肝相關(guān)的慢性肝炎和38個正常對照。實驗結(jié)果：1.通過全基因組miRNA表達芯片檢測,6個miRNA (miR-106b、miR-122、 miR-144、miR-181d、miR-181b、和 miR-584)被挑選出來進行下一步qPCR的驗證。3個miRNA(包括niR-106b、miR-122和 miR-144)在乙肝肝硬化患者中明顯低于慢性乙型肝炎患者；而與正常對照相比,乙肝肝硬化病人血漿中miR-181d和miR-181b水平明顯升高,miR-584的水平明顯降低。2.驗證階段對6個miRNA進行驗證,其中2個miRNA(miR-106b和miR-181b)具有較高的穩(wěn)定性,乙肝肝硬化中miR-106b明顯下調(diào),而n miR-181b發(fā)上了明顯的上調(diào)。3. miR-106b和miR-181b對乙肝肝硬化具有較高的診斷價值,ROC曲線下面積分別為0.715(95%CI,0.641-0.789；敏感性=0.804,特異性=0.522)和0.833(95%CI,0.775-0.892；敏感性=0.678,特異性=0.870)。4.對miR-106b和miR-181b進行整合建模建立肝硬化診斷模型：1.763×miR181b-14.622×miR106b-0.367。整合后miRNA診斷模型ROC曲線下面積為0.882(95%CI,0.834,0.929；敏感性=0.856,特異性=0.750),具有較高的診斷價值。5.在早期肝硬化組中對診斷模型進行進一步驗證發(fā)現(xiàn),miRNA診斷模型對早期肝硬化具有較高的診斷價值,ROC曲線下面積為0.774(95%CI,0.589-0.959；敏感性=0.615,特異性=0.935)。6. miRNA診斷模型與肝硬化的纖維增生相關(guān),但與病因無關(guān)。非乙肝肝硬化組驗證結(jié)果：ROC曲線下面積為0.915(95%CI,0.854-0.976；敏感性=0.787,特異性=0.932),說明該模型同樣適用于其它類型的肝硬化的診斷。7. miR-106b和miR-181b與肝臟炎癥程度無關(guān),與其它臨床指標,包括總膽紅素、白蛋白、ALT、凝血酶原時問和國際標準化比值也無線性相。說明miR-106b和miR-181b為纖維形成特異性的指標,與肝臟炎癥程度、肝功能和肝細胞損害無直接相關(guān)。結(jié)論：我們的研究發(fā)現(xiàn)miR-106b和miR-181b可對臨床肝硬化的患者有較高的診斷價值,特別適用于診斷早期肝硬化的患者。
[Abstract]:Background: The diagnostic index with high sensitivity and specificity plays an important role in the diagnosis of liver cirrhosis and the follow-up and treatment of disease progression. A large number of studies have shown that miRNAs in the cycle can be used as a diagnostic index for many diseases, including chronic liver disease. The purpose of this study was to check the expression level of plasma miRNA in patients with liver cirrhosis and to study the significance of plasma miRNA as a non-invasive diagnostic marker of liver cirrhosis. experimental method: the study is a multi-stage case-control study, including the following parts: first part, The whole-genome miRNA expression was tested after treatment with primary expression phase,80 plasma samples (25 of which were hepatitis B cirrhosis,22 chronic hepatitis, and 33 normal controls). The chip contains 723 common miRNAs. After the results of the primary screen were analyzed, the validation of the second part of the six miRNAs was selected. The second part was the validation and modeling phase of the study. First, six miRNAs were tested by qPCR in 41 samples (20 hepatitis B cirrhosis,9 chronic hepatitis B and 12 normal controls) and two miRNAs with higher stability were selected. The results were verified by using qPCR in the remaining 141 samples (70 hepatitis B liver cirrhosis,23 chronic hepatitis B and 48 normal controls). The third part is the validation phase of the diagnostic model, and the miRNA diagnosis model obtained in the second part is respectively verified in two independent queues to ensure the stability of the index. One of the cohort had 44 samples, including 13 early cirrhosis with no clinical manifestations,25 chronic hepatitis and 6 normal controls, all of which had a clear diagnosis of the pathological findings of the liver, and 92 samples in the other cohort, Including 47 non-hepatitis B cirrhosis,7 non-hepatitis B-related chronic hepatitis and 38 normal controls. Experimental results:1. Six miRNAs (miR-106b, miR-122, miR-144, miR-181d, miR-181b, and miR-584) were selected for the next qPCR validation by full-genome miRNA expression chip detection. Three miRNAs (including niR-106b, miR-122, and miR-144) were significantly lower in patients with hepatitis B liver cirrhosis than in patients with chronic hepatitis B; and compared to normal controls, The levels of miR-181d and miR-181b in the plasma of patients with hepatitis B cirrhosis were significantly increased, and the level of miR-584 was significantly reduced. In the validation phase,6 miRNAs were validated, of which 2 miRNAs (miR-106b and miR-181b) had higher stability, and miR-106b was significantly down-regulated in hepatitis B liver cirrhosis, while n-miR-181b was significantly up-regulated. The diagnostic value of miR-106b and miR-181b for hepatitis B liver cirrhosis was 0.715 (95% CI, 0.641-0.789; sensitivity = 0.804, specificity = 0.522) and 0.833 (95% CI, 0.775-0.892; sensitivity = 0.678, specificity = 0.870). Whole-time modeling of miR-106b and miR-181b was performed to establish a liver cirrhosis diagnosis model: 1.763-miR181b-14.622-miR106b-0.367. The area of the ROC curve was 0.882 (95% CI, 0.834, 0.929; sensitivity = 0.856, specificity = 0.750), with high diagnostic value. Further validation of the diagnostic model in the early cirrhosis group found that the miRNA diagnostic model had a high diagnostic value for early cirrhosis and the area under the ROC curve was 0.774 (95% CI, 0.589-0.959; sensitivity = 0.615, specificity = 0.935). The miRNA diagnosis model is related to the fibroplasia of the liver cirrhosis, but is not related to the cause of the liver cirrhosis. The results of the non-hepatitis B liver cirrhosis group showed that the area under the ROC curve was 0.915 (95% CI, 0.854-0.976; sensitivity = 0.787, specificity = 0.932), indicating that the model was also applicable to the diagnosis of other types of liver cirrhosis. The miR-106b and miR-181b are not related to the degree of inflammation of the liver, and have no linear phase with other clinical indicators, including total bilirubin, albumin, ALT, prothrombin time and international normalized ratio. It is indicated that miR-106b and miR-181b are specific indicators for fiber formation, and are not directly related to the degree of liver inflammation, liver function and liver cell damage. Conclusion: Our study found that miR-106b and miR-181b can have high diagnostic value for patients with cirrhosis, and is especially suitable for patients with early cirrhosis.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R575.2

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