动态监测脓毒症患者血清miRNA表达对脓毒症诊断价值的实验研究

发布时间：2018-04-06 03:04

本文选题：miR-146a　切入点：miR-193b*　出处：《中国人民解放军医学院》2013年硕士论文

【摘要】：目的：本实验旨在应用实时荧光定量PCR技术筛选并验证可用于诊断、预测脓毒症预后的血清miRNA，并观察血清miRNA在疾病发生发展过程中的动态变化及与疾病的关系。方法：以2011年9月10日至2012年12月30日期间在中国人民解放军总医院呼吸重症监护病房、急诊重症监护病房和外科重症监护病房的住院患者为研究对象，收集满足脓毒症两项或两项以上条件感染患者的血标本，共66例。按照28天疾病转归情况，分为生存患者33例，死亡患者33例，并以24名健康人血清作为对照。采集第1、3、5、7、10、14天的血清样本，并记录对应时间的生命体征、CRP、PCT、血常规、血生化、凝血功能及动脉血气分析结果。利用qRT-PCR验证前期测序筛选出的差异表达miRNA分子，对有差异表达的miRNA进行ROC曲线分析，比较曲线下面积。最后将上述筛选出的miRNA和常用指标包括基础疾病、WBC、CRP、APECHEⅡ评分和SOFA评分均纳入二分类变量logistic回归分析，筛选同脓毒症预后相关的因素。对最终筛选的miRNAs进行靶基因预测，探讨其参与脓毒症发病的机制。结果：选取同脓毒症相关的7种miRNA(miR-16，miR-122，miR-146a，miR-150，，miR-193b*，miR-483-5p和miR-574-5p)进行qRT-PCR验证。验证后本研究的主要结果如下：1）66例脓毒症患者与24例健康对照组在各个观测时间点的组间比较，miR-483-5p和miR-574-5p差异无统计学意义（p0.05）, miR-16、miR-122、miR-146a、miR-150和miR-193b*的表达差异有统计学意义（p0.001）；2）66例脓毒症患者中的33例生存组和33例死亡组比较， miR-193b*、miR-574-5p在各时间点上p值均小于0.001，miR-16在第1天（p=0.041）、miR-122在第1天（p=0.026）和第10天(p=0.040)，miR-483-5p在第1天(p=0.009)、第5天(p=0.014)第14天(p=0.042)差异有统计学意义，其余miRNAs在各时间点的组间比较差异无统计学意义（p0.05）；3）ROC曲线分析得到miR-146a在各个观测时间点诊断脓毒症的AUC最大（AUC90%），敏感性和特异性都较高；4）将生存组和死亡组存在差异表达的4种miRNA入组第1天的表达量水平同对应时间点的SOFA评分、APACHEⅡ评分、WBC和CRP做预测病死率的ROC受试曲线，结果显示，miR-193b*的AUC最大为0.858(0.763,0.952)，其次是miR-574-5p和miR-483-5p，其AUC分别是0.711(0.573,0.850)和0.681(0.545,0.817)；5）利用二分类变量logistic回归分析评价脓毒症的危险因子与保护因子，结果显示年龄、SOFA评分,miR-122、miR-193b*、miR-483-5p和miR-574-5p在评估脓毒症预后方面有重要的价值（p0.05），但只有miR-193b*进入最后的回归方程，OR是0.769(95%CI，0.667-0.887)，提示miR-193b*是脓毒症预后独立的危险因子；6）利用miRanda和targetscan这两个靶基因数据库对miR-193b*进行靶基因预测，共预测得到18个在两个数据库中有交集的靶基因，并通过KEGG数据库对靶基因进行通路注释，其中7个靶基因可能作用于脓毒症的相关通路，为下一步的研究建立了良好的基础。结论：1.证实了miR-16、miR-122、miR-146a、miR-150在脓毒症病程中表达稳定，可作为诊断脓毒症的生物标记物，其中miR-146a的诊断效果最好；2.MiR-193b*是一种可用于脓毒症诊断的生物标记物，并可以用来评价脓毒症的预后，是脓毒症的独立危险因子；3.经预测，miR-193b*可调控18个靶基因，其中7个靶基因可能作用于脓毒症的相关通路。
[Abstract]:Objective: the aim of this experiment is to screen and verify the serum miRNA that can be used for diagnosis and predict the prognosis of sepsis, and observe the dynamic changes of serum miRNA in the course of disease development and its relationship with disease by real-time fluorescence quantitative PCR.
Methods: from September 10, 2011 to December 30, 2012 in General Hospital of PLA respiratory intensive care ward, emergency ICU and surgical ICU hospitalized patients as the research object, collecting satisfied sepsis in two or two of the above conditions infection of blood specimens, a total of 66 cases. According to the 28 days of disease prognosis, divided into 33 cases the survival of patients, 33 patients died, and 24 healthy people as control group. Serum collected serum samples in 1,3,5,7,10,14 days, and record the corresponding time of vital signs, CRP, PCT, blood routine, blood biochemistry, blood coagulation function and arterial blood gas analysis results. The expression of miRNA between pre screened by qRT-PCR sequencing verification. The different expression of miRNA by ROC curve analysis, comparison of the area under the curve. Finally, the selected miRNA and common indicators including basic disease, WBC, CRP, APECH E II score and SOFA score were all included in the two categorical variable logistic regression analysis. The factors related to the prognosis of sepsis were screened. Target genes of the final screened miRNAs were predicted, and the mechanism of their involvement in the pathogenesis of sepsis was discussed.
Results: select the same 7 sepsis associated with miRNA (miR-16, miR-122, miR-146a, miR-150, miR-193b*, miR-483-5p and miR-574-5p) qRT-PCR verification. The main results of this study are as follows: 1) after verification of 66 cases of patients with sepsis and 24 healthy control group at each observation time point between the two groups miR-483-5p and miR-574-5p, there was no significant difference (P0.05), miR-16, miR-122, miR-146a, there was significant difference in the expression of miR-150 and miR-193b* (p0.001); 2) 66 cases of miR-193b* patients with sepsis in 33 cases of survival group and 33 cases of death group, miR-574-5p at each time point, P values were less than in 0.001, miR-16 in first days (p=0.041), miR-122 (p=0.026) in first days and tenth days (p=0.040), miR-483-5p (p=0.009) in first days, fifth days and fourteenth days (p=0.014) (p=0.042) the difference was statistically significant, no significant remaining miRNAs at each time point between group differences The significance (P0.05); 3) ROC miR-146a curve analysis in various time points in diagnosis of sepsis AUC (AUC90%), high sensitivity and specificity; 4) the survival group and death group, there are 4 miRNA in group first days the expression level of differentially expressed with corresponding time points SOFA score, APACHE score, WBC and CRP to predict mortality rate of ROC test results show that the miR-193b* curve, the maximum is 0.858 AUC (0.763,0.952), followed by miR-574-5p and miR-483-5p, the AUC were 0.711 (0.573,0.850) and 0.681 (0.545,0.817); 5) regression analysis of risk factors and protective factors of sepsis sepsis using two classification variables logistic, the results showed that age, SOFA score, miR-122, miR-193b*, miR-483-5p and miR-574-5p have important value in assessing the prognosis of sepsis (P0.05), but only miR-193b* into the final regression equation, OR is 0.769 (95%CI, 0.6 67-0.887), suggesting that miR-193b* is a risk factor for sepsis prognosis; 6) potential target genes of miR-193b* were predicted by miRanda and targetscan of the two target genes were predicted by the database, 18 of the two databases have target genes overlap, and the database by KEGG pathway annotation of target genes, including related pathways 7 target genes may play a role in sepsis, established a good foundation for the next research.
Conclusion: 1. confirmed miR-16, miR-122, miR-146a, miR-150 expression in sepsis patients, can be used as a biological marker for the diagnosis of sepsis, the diagnosis of miR-146a is the best; 2.MiR-193b* is a kind of biomarkers for the diagnosis of sepsis, and can be used to evaluate the prognosis of sepsis that is the independent risk factor of sepsis; 3. after the forecast, miR-193b* can regulate the expression of 18 target genes, 7 genes related to the pathway may play a role in sepsis.

【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R459.7

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