Neutrophil Gene expression in Acute-on-chronic liver failure (ACLF), Chronic liver disease (CLD) and Healthy Controls_AIIMSND_Aug2020. Neutrophil Gene expression in Acute-on-chronic liver failure (ACLF), Chronic liver disease (CLD) and Healthy Controls_AIIMSND_Aug2020

To address the molecular basis of immune-dysfunction in Acute-on-chronic liver failure (ACLF), we carried out gene expression profiling of blood derived neutrophil from ACLF, belonging to both sterile inflammatory and sepsis conditions. Peripheral whole blood was subjected to PMN enrichment by double gradient centrifugation, and RNA isolation was done by TRIZOL method, followed by microarray experiment using Agilent one-color platform. We compared the gene expression of these neutrophils with that of Chronic liver disease (CLD) patients and healthy controls (HC) for baseline relative quantification, and found unique set of upregulated and downregulated genes in ACLF. We validated the expression of the most differentially expressed genes by quantitative RT-PCR and also stratified the patients into survivors and non-survivors, sepsis and sterile-inflammation. We found an upregulated 3-gene signature of ELANE-MPO-CD177 to be associated with 28-day mortality, irrespective of presence or absence of sepsis. Overall design: Total RNA from blood enriched neutrophils was subjected to one-color microarray experiment for the following study groups: ACLF sepsis (n=6), ACLF sterile inflammation (n=6), CLD (n=6), Healthy controls HC (n=6). Differential gene expression analysis was done for CLD vs HC, ACLF overall (n=12) vs CLD, and ACLF sepsis vs sterile inflammation.

为解析慢加急性肝衰竭（Acute-on-chronic liver failure, ACLF）免疫功能异常的分子基础，我们针对涵盖无菌性炎症与脓毒症两类表型的慢加急性肝衰竭患者的外周血来源中性粒细胞，开展了基因表达谱分析。
我们采用双梯度离心法对外周全血进行中性粒细胞（polymorphonuclear neutrophil, PMN）富集，通过TRIZOL法完成总RNA提取，随后使用安捷伦（Agilent）单通道平台开展基因芯片实验。
我们以慢性肝病（Chronic liver disease, CLD）患者与健康对照（Healthy controls, HC）的基因表达谱作为基线对照进行相对定量分析，发现慢加急性肝衰竭患者体内存在一组独特的上调与下调基因集。
我们通过定量逆转录聚合酶链反应（quantitative RT-PCR, qRT-PCR）对差异表达最显著的基因进行了表达验证，并按照患者生存结局、脓毒症与无菌性炎症表型完成分层分析。
我们发现ELANE-MPO-CD177三基因表达特征的上调与28天死亡率显著相关，且不受脓毒症存在与否的影响。
整体实验设计：对来自各研究组的血液富集中性粒细胞的总RNA开展单通道芯片实验，各组样本量如下：慢加急性肝衰竭脓毒症组（n=6）、慢加急性肝衰竭无菌性炎症组（n=6）、慢性肝病组（n=6）、健康对照组（n=6）。我们针对以下三组开展差异基因表达分析：慢性肝病组vs健康对照组、整体慢加急性肝衰竭组（n=12）vs慢性肝病组，以及慢加急性肝衰竭脓毒症组vs无菌性炎症组。