Gene expression profiling of human livers during normothermic machine perfusion. Gene expression profiling of human livers during normothermic machine perfusion

Normothermic machine perfusion (NMP) has been successfully implemented in clinical routine of liver transplantation over the past years. However, little is known about the mechanisms how NMP impacts on the transcriptome of a human donor liver. We herein examined gene expression profiles in transplanted and non-transplanted livers over NMP time. 50 livers subjected to NMP were included in this study. 30 were transplanted after a maximum of 20 hours (h) perfusion, while 15 were discarded due to poor performance. Biopsies were collected befor eNP (PRE), 1h, 6h, 12h, 20h of NMP and after reperfusion. Next-generation sequencing was applied in liver biopsies to assess differential gene expression over perfusion time. Perfusate samples were collected regularly to monitor liver function. Comparison in differential gene expression between PRE and 20h NMP showed 415 upregulated and 727 downregulated genes. Most significantly upregulated genes were associated with extra cellular matrix organization, cell growth/differentiation processes and cytokine signaling. A set of genes were identified which were significantly differentially expressed and important for classification of non-transplanted vs transplanted biopsies, especially at 12 and 20h of NMP. A 7-gene-signature showed good separation already at 6H NMP, thereby CD274 (PD-L1) expression was ponted out as most important. Overall design: Gene expression profiling of human livers were performed at several timepoints during normothermic machine perfusion (NMP) (PRE (baseline before NMP), 1H, 6H, 12H,20H, POST (post transplant)) using RNA sequencing analyses. Differentially expressed genes were identified at each timepoint compared to baseline (PRE) in paired analyses and at each timepoint between nontransplanetd livers (NTP) and transplanted livers (TP).

近年来，常温机械灌注（Normothermic machine perfusion, NMP）已成功应用于肝移植的临床常规实践中。然而，目前关于常温机械灌注如何影响人类供肝转录组的具体机制仍知之甚少。本研究针对经历不同时长常温机械灌注的移植肝与非移植肝，检测其基因表达谱特征。本研究共纳入50例接受常温机械灌注的供肝：其中30例在灌注最长20小时后完成肝移植，另有15例因灌注效果不佳被弃用。分别于常温机械灌注前（PRE）、灌注1小时、6小时、12小时、20小时以及再灌注后采集肝活检样本。采用下一代测序（Next-generation sequencing, NGS）技术对肝活检样本进行检测，以评估灌注过程中的差异基因表达情况。同时定期收集灌注液样本，以监测肝脏功能状态。对比基线（PRE）与20小时常温机械灌注后的差异基因表达情况，共鉴定出415个上调基因与727个下调基因。上调幅度最显著的基因主要富集于细胞外基质组织、细胞生长/分化过程以及细胞因子信号通路相关功能。研究还鉴定出一组显著差异表达的基因，其可有效区分非移植肝（nontransplanted livers, NTP）与移植肝（transplanted livers, TP）的活检样本，尤其在常温机械灌注12小时和20小时时区分效果最佳。其中一组7基因特征在常温机械灌注6小时时即可实现良好的样本区分，其中CD274（PD-L1）的表达被确定为最关键的标志物。研究整体设计：采用RNA测序（RNA sequencing, RNA-seq）技术，对人类供肝在常温机械灌注过程中的多个时间点（PRE：常温机械灌注前基线、1小时、6小时、12小时、20小时以及POST：移植后）进行基因表达谱分析。通过配对分析，分别对比各时间点与基线（PRE）的差异基因表达情况，并对比各时间点非移植肝（NTP）与移植肝（TP）之间的差异基因表达。