Department of Surgery, Center for Liver Disease and Transplantation, Columbia University Medical Center, New York, NY 10032, USA.
Surgery. 2011 Aug;150(2):352-60. doi: 10.1016/j.surg.2011.06.003.
Hypothermic machine perfusion (HMP) mitigates the effects of ischemia/reperfusion injury (IRI) in renal transplantation and preclinical work with livers. In liver transplantation, IRI increases the likelihood of primary graft dysfunction and is associated with significant morbidity. We recently completely the first phase 1 clinical trial of liver HMP at our center, and demonstrated improved clinical parameters and shorter duration of stay for patients who received grafts stored by HMP than patients who received grafts preserved in cold storage. Biomarker analysis of venous effluent collected from the hepatic veins during HMP may yield predictive information reflecting the condition of the donor liver, such as graft injury sustained during brain death and graft preservation. The aim of this study was to characterize biomarkers released into the effluent during HMP.
Effluent was collected every 30 minutes during liver HMP during our phase 1 clinical trial. Serum was extracted from blood samples obtained at incision, before explantation, and at 1, 2, and 3 hours after reperfusion. The effluent and serum samples were assayed in multiplex to determine the concentration of inflammatory cytokines and growth factors. Tissue obtained from liver biopsies was processed for either downstream reverse transcription-polymerase chain reaction or immunofluorescence. Statistical significance was determined by a two-tailed t-test.
Growth factors and most cytokines were not readily detectable in levels above baseline with this technique; however, interleukin-1 (IL-1) receptor antagonist and monocyte chemotactic protein-1 were present in significant concentrations in the effluent at all time points. This finding was confirmed with serum samples and mRNA expression obtained from liver biopsies. The concentrations of these proteins decreased from their initial values over the course of HMP, and mRNA expression levels were decreased by the use of HMP.
IL-1β and tumor necrosis factor (TNF)-α are key mediators of inflammation in IRI. Although difficult to measure because of short half-lives, their downstream effectors indicate their levels of activity. IL-1 receptor antagonist is secreted in response to IL-1β, and monocyte chemotactic protein in response to TNF-α. Their decreased production over the course of HMP suggests that interruption of acute-phase inflammation in the graft may attenuate reperfusion-related graft injury. Further cDNA studies and effluent analyses are required to confirm this hypothesis.
低温机器灌注（HMP）减轻了肾移植和肝脏的缺血/再灌注损伤（IRI）的影响。在肝移植中，IRI 增加了原发性移植物功能障碍的可能性，并与显著的发病率相关。我们最近在我们的中心完成了肝脏 HMP 的首次 1 期临床试验，并且证明了接受 HMP 保存的移植物的患者的临床参数得到改善，并且住院时间更短，而接受冷藏保存的移植物的患者则没有。从 HMP 期间从肝静脉收集的静脉流出物中进行的生物标志物分析可能会产生反映供体肝状况的预测信息，例如脑死亡期间和供体保存期间发生的移植物损伤。本研究的目的是描述在 HMP 期间释放到流出物中的生物标志物。
在我们的 1 期临床试验中，在肝脏 HMP 期间每 30 分钟收集一次流出物。在切开时、在取出之前以及在再灌注后 1、2 和 3 小时从血液样本中提取血清。用多重测定法测定流出物和血清样本中炎症细胞因子和生长因子的浓度。对肝活检组织进行下游逆转录-聚合酶链反应或免疫荧光分析。通过双侧 t 检验确定统计学意义。
生长因子和大多数细胞因子用这种技术在基线以上不易检测到；然而，白细胞介素-1（IL-1）受体拮抗剂和单核细胞趋化蛋白-1在所有时间点均以显著浓度存在于流出物中。这一发现通过血清样本和从肝活检获得的 mRNA 表达得到了证实。这些蛋白质的浓度从 HMP 的初始值开始降低，并且 HMP 的使用降低了 mRNA 表达水平。
IL-1β 和肿瘤坏死因子（TNF）-α是 IRI 中炎症的关键介质。尽管由于半衰期短而难以测量，但它们的下游效应物表明其活性水平。IL-1 受体拮抗剂是针对 IL-1β 分泌的，而单核细胞趋化蛋白是针对 TNF-α 分泌的。它们在 HMP 过程中的产量减少表明，阻断移植物中的急性炎症可能会减轻再灌注相关的移植物损伤。需要进一步的 cDNA 研究和流出物分析来证实这一假设。
