脓毒症患儿血浆微RNA表达及其与炎症细胞因子的相关性

Relationship between expression of microRNA and inflammatory cytokines plasma level in pediatric patients with sepsis

目的探讨儿童脓毒症患者血浆微RNA（miRNA）的表达及其与炎症细胞因子的相关性和临床意义。方法选取2012年4月至2013年3月深圳市儿童医院儿童重症监护病房（PICU）住院的40例脓毒症患儿为研究对象，其中男27例、女13例，中位年龄0．75（0．52，1．90）岁，其中严重脓毒症16例。选取20例手术后全身性炎症反应综合征（SIRS）患儿和15名健康儿童分别作为SIRS组和对照组，通过实时荧光定量PCR（qRT—PCR）方法检测血浆miR-21、miR-125b、miR-132、miR-146a、miR-155、miR-223表达量。受试者工作特征曲线（ROC）评价表达差异的血浆miRNA、降钙素原（PCT）和c反应蛋白（CRP）等指标对脓毒症的预测价值。利用流式液相多重蛋白定量技术（CBA）检测患儿血浆肿瘤坏死因子（TNF）-α和白细胞介素（IL）-10水平。正态分布的多组计量资料采用单因素方差分析，以LSD-t检验进行组间两两比较。非正态分布的资料多组间比较采用Kmskal—Wallis日检验，两组问采用Mann—WhitneyU检验。结果各组间年龄、性别差异均无统计学意义，具有可比性。各组血浆miR-21、miR-12513、miR-132、miR-155表达水平差异均无统计学意义（均P〉0．05）。脓毒症组miR-146a、miR-223表达水平均高于SIRS组和对照组[（5．7±3．5）×10^-5比（2．4±1．6）×10^-5和（2．6±1．2）×10^-5，（12．5±7．7）×10^-4比（8．3±3．4）×10^-4和（5．3±2．2）×10^-4，均P〈0．01]，SIRS组miR-223水平高于对照组（P〈0．01）。严重脓毒症组miR-146a表达水平较-般脓毒症组高[（7．1±3．3）×10^-5比（4．6±2．6）×10^-5，P〈0．01]。脓毒症组和SIRS组CRP、PCT水平均高于对照组，脓毒症组PCT高于SIRS组（均P〈0．05）。miR-146a、miR-223和PCT、CRP预测脓毒症的ROE曲线下面积（AUC）分别为0．815（95％C1：0．708-0．922），0．678（95％CI：0．537～0．818），0．706（95％CI：0．571～0．842）和0．588（95％CI：0．427～0．748）。脓毒症组血浆IL-10、IL-10／TNF—α水平均高于SIRS组和对照组（均P〈0．01），且IL-10、IL-10／TNF-α和miR-146a、miR-223水平均呈正相关（r=0．545、0．305和0．562、0．373，P=0．000、0．008和0．000、0．001）。结论脓毒症患儿血浆miR-146a、miR-223表达上调，与IL—10、IL-10／TNF—α水平正相关，有望作为脓毒症早期诊断和判断病情严重程度的标志物。

Objective Sepsis is the major cause of death in pediatric intensive care unit （HCU）. The clinical manifestations of early sepsis is very similar to systemic inflammatory response syndrome （SIRS） caused by non-infectious reason. This study aimed to investigate the expression of miRNA and inflammatory cytokines in plasma in pediatric sepsis patients and its clinical significance. Method Forty children withsepsis seen in Shenzhen children＇s hospital PICU from April 2012 to March 2013 were enrolled in this study, the median age was 0. 75 （0. 52, 1.90） years; 27 were males and 13 females, of whom 16 had severe sepsis. We selected 20 postsurgical patients with SIRS and 15 healthy children as a control group. The expression levels of plasma miR-21, miR-125b, miR-132, miR-146a, miR-155 and miR-223 were detected by real-time quantitative PCR （qRT-PCR）. The predictive value of miRNA, PCT and CRP for sepsis were evaluated by Receiver operating characteristic curve （ROC）. TNF-α and IL-10 levels in plasma detected by Cytometric Beads Array （CBA）. Quantitative data of normal distribution was compared with ANOVA among the three groups and LSD-t test between two groups. To non-normal distribution of data, multiple comparisons among three groups were conducted by Kruskal-Wallis H test and differences between two groups were assessed by Mann-Whitney U test for post hoc analysis. Result There were no significant differences between the age and gender of each group. Expression of miR-21, miR-125b, miR-132 and miR-155 in plasma had no significant difference in each group （all P 〉0. 05）. MiR-146a and miR-223 levels in sepsis were upregulated compared with SIRS group and control group [ （5.7 ±3.5） ×10 ^-5 vs. （2.4 ± 1.6） × 10^-5 and （2.6±1.2） ×10^-5, （12.5±7.7） ×10^-4 vs. （8.3±3.4） ×10^-4 and （5.3±2.2）×10^-4, allP〈 0. 01 ] , expression levels of miR-223 in SIRS increased as compared to control group （P 〈 0. 01 ）. MiR- 146a levels in severe sepsis were higher than those of the general sepsis [ （7. 1 ±3.3）× 10^-5 vs. （4. 6 ± 2.6） ×10^-5 , P 〈0. 01 ]. CRP and PCT levels are all higher in sepsis and SIRS groups than control group （ all P 〈0. 01 ）. The area under ROC curve （AUC） of miR-146a, miR-223, PCT and CRP to predict sepsis were 0. 815 （95%CI： 0. 708-0.922）, 0. 678 （95%CI： 0. 537 -0.818）, 0.706 （95%CI： 0.571 - 0. 842） and 0. 588 （95% CI： 0. 427 -0. 748）. Expression levels of IL-10 and IL-10/TNF-α in sepsis were upregulated compared with and SIRS group and the control group （ all P 〈 0. 01 ）. There was a positive correlation between miR-146a, miR-223 and IL-10 and IL-10/TNF-α （r = 0. 545, 0. 305, 0. 562, 0. 373, all P 〈 0. 01 ）, Conclusion The expression levels of miR-146a and miR-223 in plasma in pediatric patients with sepsis was significantly upregulated, and had a positive correlation with IL-10 and IL-10/TNF-α, which may be used as early diagnostic markers and can reflect the severity of condition to a certain degree.