血浆microRNA检测在肝硬化早期诊断中的价值

发布时间：2019-07-03 12:48

【摘要】：研究背景：具有高敏感性和特异性的诊断指标对于肝硬化的诊断、病情进展的随访和治疗起着十分重要的作用。大量的研究表明循环中miRNA可作为许多疾病,包括慢性肝病的诊断指标。本研究的目的是对肝硬化患者血浆miRNA的表达水平进行检查,探讨血浆miRNA作为肝硬化无创诊断标志物的意义。实验方法：该研究为多阶段的病例对照研究,过程包括以下几部分：第一部分,为研究的筛选阶段(primary exploration phase),80个血浆样本(其中25个乙肝肝硬化、22个慢性肝炎和33个正常对照)处理后进行芯片全基因组miRNA表达检测。该芯片共包含有723个常见的miRNA。初筛结果经分析后,挑选出6个miRNA进行第二部分的验证工作。第二部分为研究的验证和建模阶段(trainning phase),首先,6个miRNA在41个样本中(20个乙肝肝硬化、9个慢性乙肝和12分正常对照)运用qPCR的方法进行验证,挑选出2个具有较高稳定性的miRNA,进一步在其余141个样本(70个乙肝肝硬化、23个慢性乙肝和48个正常对照)中运用qPCR的方法进行验证,结果应用Logistic回归进行整合。第三部分为诊断模型的验证阶段(validation phase),在第二部分得到的miRNA诊断模型在两个独立队列中分别进行验证,以保证该指标的稳定性。其中一个队列有44个样本,包括13个无临床表现的早期肝硬化、25个慢性肝炎和6个正常对照,所有样本都有肝穿病理结果明确诊断；另一个队列有92个样本,包括47个非乙肝肝硬化、7个非乙肝相关的慢性肝炎和38个正常对照。实验结果：1.通过全基因组miRNA表达芯片检测,6个miRNA (miR-106b、miR-122、 miR-144、miR-181d、miR-181b、和 miR-584)被挑选出来进行下一步qPCR的验证。3个miRNA(包括niR-106b、miR-122和 miR-144)在乙肝肝硬化患者中明显低于慢性乙型肝炎患者；而与正常对照相比,乙肝肝硬化病人血浆中miR-181d和miR-181b水平明显升高,miR-584的水平明显降低。2.验证阶段对6个miRNA进行验证,其中2个miRNA(miR-106b和miR-181b)具有较高的稳定性,乙肝肝硬化中miR-106b明显下调,而n miR-181b发上了明显的上调。3. miR-106b和miR-181b对乙肝肝硬化具有较高的诊断价值,ROC曲线下面积分别为0.715(95%CI,0.641-0.789；敏感性=0.804,特异性=0.522)和0.833(95%CI,0.775-0.892；敏感性=0.678,特异性=0.870)。4.对miR-106b和miR-181b进行整合建模建立肝硬化诊断模型：1.763×miR181b-14.622×miR106b-0.367。整合后miRNA诊断模型ROC曲线下面积为0.882(95%CI,0.834,0.929；敏感性=0.856,特异性=0.750),具有较高的诊断价值。5.在早期肝硬化组中对诊断模型进行进一步验证发现,miRNA诊断模型对早期肝硬化具有较高的诊断价值,ROC曲线下面积为0.774(95%CI,0.589-0.959；敏感性=0.615,特异性=0.935)。6. miRNA诊断模型与肝硬化的纤维增生相关,但与病因无关。非乙肝肝硬化组验证结果：ROC曲线下面积为0.915(95%CI,0.854-0.976；敏感性=0.787,特异性=0.932),说明该模型同样适用于其它类型的肝硬化的诊断。7. miR-106b和miR-181b与肝脏炎症程度无关,与其它临床指标,包括总胆红素、白蛋白、ALT、凝血酶原时问和国际标准化比值也无线性相。说明miR-106b和miR-181b为纤维形成特异性的指标,与肝脏炎症程度、肝功能和肝细胞损害无直接相关。结论：我们的研究发现miR-106b和miR-181b可对临床肝硬化的患者有较高的诊断价值,特别适用于诊断早期肝硬化的患者。
[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.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R575.2

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