人肝干细胞来源的细胞外囊泡减轻离体常温缺氧大鼠肝脏灌流模型中的损伤。

Extracellular Vesicles from Human Liver Stem Cells Reduce Injury in an Ex Vivo Normothermic Hypoxic Rat Liver Perfusion Model.

机构信息

General Surgery 2U, Liver Transplantation Center, AOU Città della Salute e della Scienza di Torino, University of Turin, Turin, Italy.

2i3T, Società per La Gestione Dell'incubatore Di Imprese e Per Il Trasferimento Tecnologico Dell'Università degli Studi di Torino, Scarl., Molecular Biotechnology Center (MBC), Turin, Italy.

出版信息

Transplantation. 2018 May;102(5):e205-e210. doi: 10.1097/TP.0000000000002123.

DOI:10.1097/TP.0000000000002123

PMID:29424767

Abstract

BACKGROUND

The gold standard for organ preservation before transplantation is static cold storage, which is unable to fully protect suboptimal livers from ischemia/reperfusion injury. An emerging alternative is normothermic machine perfusion (NMP), which permits organ reconditioning. Here, we aimed to explore the feasibility of a pharmacological intervention on isolated rat livers by using a combination of NMP and human liver stem cells-derived extracellular vesicles (HLSC-EV).

METHODS

We established an ex vivo murine model of NMP capable to maintain liver function despite an ongoing hypoxic injury induced by hemodilution. Livers were perfused for 4 hours without (control group, n = 10) or with HLSC-EV (treated group, n = 9). Bile production was quantified; perfusate samples were collected hourly to measure metabolic (pH, pO2, pCO2) and cytolysis parameters (AST, alanine aminotransferase, lactate dehydrogenase). At the end of perfusion, we assessed HLSC-EV engraftment by immunofluorescence, tissue injury by histology, apoptosis by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, tissue hypoxia-inducible factor 1-α, and transforming growth factor-beta 1 RNA expression by quantitative reverse transcription-polymerase chain reaction.

RESULTS

During hypoxic NMP, livers were able to maintain homeostasis and produce bile. In the treated group, AST (P = 0.018) and lactate dehydrogenase (P = 0.032) levels were significantly lower than those of the control group at 3 hours of perfusion, and AST levels persisted lower at 4 hours (P = 0.003). By the end of NMP, HLSC-EV had been uptaken by hepatocytes, and EV treatment significantly reduced histological damage (P = 0.030), apoptosis (P = 0.049), and RNA overexpression of hypoxia-inducible factor 1-α (P < 0.0001) and transforming growth factor-beta 1 (P = 0.014).

CONCLUSIONS

HLSC-EV treatment, even in a short-duration model, was feasible and effectively reduced liver injury during hypoxic NMP.

摘要

背景

器官移植前的金标准是静态冷保存，但它无法完全保护不理想的肝脏免受缺血/再灌注损伤。一种新兴的替代方法是常温机器灌注（NMP），它可以使器官再适应。在这里，我们旨在通过使用 NMP 和人肝干细胞衍生的细胞外囊泡（HLSC-EV）的组合，探索对离体大鼠肝脏进行药理学干预的可行性。

方法

我们建立了一种体外鼠 NMP 模型，即使在由血液稀释引起的持续缺氧损伤下，该模型也能够维持肝功能。肝脏在没有（对照组，n=10）或有 HLSC-EV（治疗组，n=9）的情况下灌注 4 小时。定量胆汁生成；每小时收集一次灌注液样本，以测量代谢（pH 值、pO2、pCO2）和细胞溶解参数（AST、丙氨酸氨基转移酶、乳酸脱氢酶）。在灌注结束时，我们通过免疫荧光评估 HLSC-EV 移植，通过组织学评估组织损伤，通过末端脱氧核苷酸转移酶 dUTP 末端标记测定法评估细胞凋亡，通过定量逆转录-聚合酶链反应评估组织缺氧诱导因子 1-α 和转化生长因子-β 1 RNA 表达。

结果

在缺氧 NMP 期间，肝脏能够维持内稳态并产生胆汁。在治疗组中，与对照组相比，在灌注 3 小时时 AST（P=0.018）和乳酸脱氢酶（P=0.032）水平显著降低，在 4 小时时 AST 水平持续降低（P=0.003）。在 NMP 结束时，HLSC-EV 已被肝细胞摄取，EV 治疗可显著减轻组织损伤（P=0.030）、细胞凋亡（P=0.049）以及缺氧诱导因子 1-α（P＜0.0001）和转化生长因子-β 1（P=0.014）的 RNA 过表达。