Inter-patient heterogeneity in the hepatic ischemia-reperfusion injury transcriptome: implications for research and diagnostics [MUG data]. Inter-patient heterogeneity in the hepatic ischemia-reperfusion injury transcriptome: implications for research and diagnostics [MUG data]

Background & Aims: Cellular responses induced by surgical procedure or ischemia-reperfusion injury (IRI) may severely alter transcriptome profiles. We have investigated this effect to obtain insight into molecular ischemia responses during surgical procedures and characterize pre-analytical effects impacting on molecular analyses. Methods: 143 non-malignant liver samples were obtained from 30 patients at different time points of ischemia during surgery from two individual cohorts, treated either with the Pringle maneuver or total vascular exclusion. Transcriptomics profiles were analyzed by Affymetrix microarrays and expression of selected mRNAs was validated by RT-qPCR. Results: Transcriptional profiles of both cohorts displayed 179 genes that were mutually deregulated confirming elevated cytokine signaling, and NFkB as the dominant pathways in ischemia response. Contrary to ischemia, reperfusion induced pro-apoptotic and pro-inflammatory cascades involving TNF, NFkB and MAPK pathways. FOS and JUN were down-regulated in steatosis compared to their up-regulation in normal livers. Surprisingly, molecular signatures of underlying primary and secondary cancers were clearly present in the non-tumor tissue. Conclusions: We identified transcripts mutually deregulated during ischemia and reperfusion injury in both cohorts that can be used to monitor ischemia during liver surgery and highlight the importance of pre-analytical quality control. The marked inter-patient variability might reflect differences in individual stress responses and impact of underlying disease conditions. Furthermore, we provide a comprehensive and pre-analytically highly standardized in vivo transcriptome profile of histologically normal liver and identified 230 genes with substantial pre-analytical robustness (<2 % covariation across both cohorts) that might serve as reference genes and could be particularly suited for future diagnostic applications. Conclusions: We identified transcripts mutually deregulated during ischemia and reperfusion injury in both cohorts that can be used to monitor ischemia during liver surgery and highlight the importance of pre-analytical quality control. The marked inter-patient variability might reflect differences in individual stress responses and impact of underlying disease conditions. Furthermore, we provide a comprehensive and pre-analytically highly standardized in vivo transcriptome profile of histologically normal liver and identified 230 genes with substantial pre-analytical robustness (<2 % covariation across both cohorts) that might serve as reference genes and could be particularly suited for future diagnostic applications. Overall design: Ischemia related changes of RNA profiles obtained from frozen liver tissue samples collected before, during and after routine surgery and compared between the different time points. Medical University of Graz, 83 samples of a total of 21 patients at 4 different time points per patient.

背景与目的：手术操作或缺血再灌注损伤（ischemia-reperfusion injury, IRI）所诱导的细胞应答，可显著改变转录组表达谱。本研究旨在探究该效应，以深入解析外科手术过程中的分子缺血应答，并阐明影响分子分析的前分析因素。
方法：本研究从两个独立队列的30例患者的术中不同缺血时相获取了143份非恶性肝脏样本，两组患者分别采用普林格尔手法（Pringle maneuver）或全血管阻断术进行处理。通过Affymetrix微阵列分析转录组谱，并通过逆转录实时定量聚合酶链反应（Reverse Transcription quantitative PCR, RT-qPCR）验证选定mRNA的表达水平。
结果：两个队列的转录谱均鉴定出179个共同失调基因，证实细胞因子信号通路、核因子κB（nuclear factor kappa B, NFκB）为缺血应答中的核心通路。与缺血阶段相反，再灌注阶段可诱导涉及肿瘤坏死因子（tumor necrosis factor, TNF）、NFκB及丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）通路的促凋亡与促炎症级联反应。相较于正常肝脏中FOS与JUN的上调表达，脂肪变性组织中这两个基因呈下调状态。令人意外的是，非肿瘤组织中可清晰检测到原发性及继发性癌症的分子特征谱。
结论：本研究在两个队列中均鉴定出缺血再灌注损伤过程中共同失调的转录本，可用于监测肝脏外科手术中的缺血状态，并凸显了前分析质量控制的重要性。显著的患者间差异可能反映了个体应激应答的差异及基础疾病状态的影响。此外，本研究提供了一套全面且经严格前分析标准化的组织学正常肝脏的体内转录组谱，并鉴定出230个具有优异前分析稳定性的基因（两个队列间的协变量变异<2%），这些基因可作为参考基因，尤其适用于未来的诊断应用。
结论：本研究在两个队列中均鉴定出缺血再灌注损伤过程中共同失调的转录本，可用于监测肝脏外科手术中的缺血状态，并凸显了前分析质量控制的重要性。显著的患者间差异可能反映了个体应激应答的差异及基础疾病状态的影响。此外，本研究提供了一套全面且经严格前分析标准化的组织学正常肝脏的体内转录组谱，并鉴定出230个具有优异前分析稳定性的基因（两个队列间的协变量变异<2%），这些基因可作为参考基因，尤其适用于未来的诊断应用。
整体设计：采集常规手术前后及术中的冰冻肝脏组织样本，分析其RNA谱的缺血相关变化，并对不同时相进行比较。格拉茨医科大学（Medical University of Graz）队列：纳入21例患者，每例患者采集4个不同时相的样本，共计83份组织样本。