BMMSCs对SD大鼠DCD供肝常温机械灌注脂肪变性的影响机制研究

目的:研究铁死亡在骨髓间充质干细胞(BMMSCs)联合常温机械灌注(NMP)修复SD大鼠心脏死亡器官捐献(DCD)脂肪变性供肝中的作用。方法:提取SD大鼠BMMSCs。建立大鼠脂肪肝DCD模型。24只SD大鼠随机分为单纯脂肪肝组(Sham)、静态冷保存组(SCS)、常温机械灌注保存组(NMP)、BMMSCs联合NMP保...     

BMMSCs对SD大鼠DCD供肝常温机械灌注脂肪变性的影响机制研究

目的研究铁死亡在骨髓间充质干细胞(BMMSCs)联合常温机械灌注(NMP)修复SD大鼠心脏死亡器官捐献(DCD)脂肪变性供肝中的作用。方法提取SD大鼠BMMSCs。建立大鼠脂肪肝DCD模型。24只SD大鼠随机分为单纯脂肪肝组(Sham)、静态冷保存组(SCS)、常温机械灌注保存组(NMP)、BMMSCs联合NMP保存组(BNMP), 不同条件下保存DCD脂肪供肝4 h。通过检测肝脏病理、灌注液肝脏酶学和乳酸含量、胆汁分泌量和炎症因子指标, 对肝脏进行功能质量评估;检测肝组织中Fe2+、丙二醛和还原型谷胱甘肽(GSH)含量, 以及肝脏中环氧合酶-2、前列腺素内过氧化物合酶2(Ptgs2)、谷胱甘肽过氧化物酶4(GPX4)及铁蛋白重链1(FTH1)的mRNA或蛋白表达, 评价肝脏铁死亡发生。结果 BNMP和NMP组的肝脏病理、促炎及抗炎细胞因子的表达均优于SCS组;机械灌注保存期间, BNMP组丙氨酸氨基转移酶、天冬氨酸氨基转移酶和乳酸含量均低于NMP组[灌注4 h:(189.0±12.5)U/L比(227.7±16.2)U/L、(207.3±18.6)U/L比(247.0±11.8)U/...     

大鼠肝移植供肝热缺血时间与胆道损伤的关系

目的 探讨大鼠心源性死亡(DCD)供肝不同的热缺血时间与胆道缺血再灌注损伤的关系.方法 采用无肝素化的大鼠DCD供体肝移植模型,按供肝的热缺血时间分为0 min(WI0),10 min(WI10)、15 min(WI15)3组,每组36对大鼠.术后动态观察大鼠胆道病理改变及其并发症、肝功能指标,最后统计总体生存率.结果 供肝热缺血小于10 min时,术后胆道病理改变较轻且为可逆性改变,肝功能恢复较快;供肝热缺血时间为15 min时,术后胆道病理改变较重且为不可逆性改变,肝功能恢复延迟;3组胆道并发症的发生率差异有统计学意义(5.56%比8.33%比16.67%,P＜0.05).WI0组和WI10组的大鼠术后4周生存率差异无统计学意义(83.33%比77.78%,P＞0.05),而与WI15组比较4周生存率的差异有统计学意义(83.33%比58.33%,77.78%比58.33%,P＜0.05).结论 无肝素化的大鼠DCD供肝热缺血时间超过15 min时,移植术后胆道损伤明显,可导致不可逆改变.     

大鼠DCD供肝原位肝移植术后早期死亡的原因分析

目的建立稳定的大鼠心脏死亡器官捐献(donation after cardiac death,DCD)供肝原位肝移植模型,分析移植后24 h内大鼠死亡原因并探索相应的改进措施。方法所有供肝获取前经历供体心脏停跳10 min,采用改良Kamada的"二袖套"吻合法完成大鼠DCD供肝原位肝移植手术,记录各个手术阶段所用时间及术后大鼠的死亡情况。结果在40 d内完成大鼠DCD供肝原位肝移植手术100例,供体手术时间为(20±5)min,受体手术时间为(55±5)min,无肝期为(20±3)min。移植术后受体一般情况可。术中死亡9例,其中术中大出血4例、麻醉意外1例、无肝期过长1例、套管置入失败1例、空气栓塞2例;术后12 h内死亡22例,其中术后肠道坏死6例、术后吻合口渗血6例、术后肺水肿3例、术中失血量过多4例、术后血管栓塞2例、不明原因死亡1例;12~24 h死亡19例,其中术后肠道坏死9例、术后吻合口渗血3例、术后肺水肿2例、术中失血量过多1例、术后血管栓塞1例、不明原因死亡3例。结论导致大鼠DCD供肝原位肝移植术后早期死亡的原因很多,其中术后肠道坏死、术中及术后出血、术后肺水肿及术后血管栓塞是主要的致死因素。针对术后死亡原因采取相应的预防及改进措施能大大提高大鼠术后存活率,从而建立稳定的大鼠DCD供肝原位肝移植模型。     

幼儿心脏死亡肝脏捐献一例

在供肝日益紧张的情况下,儿童心脏死亡器官捐献(DCD)可以成为供肝来源之一,但关于儿童DCD肝移植的报道较少.2011年11月,我院成功完成1例幼儿心脏死亡肝脏捐献,现报告如下.     

空气氧合常温机械灌注对心死亡大鼠肾缺血损伤的修复作用

目的通过建立大鼠心死亡供肾常温机械灌注(normothermic mechanical perfusion, NMP)模型, 探讨空气氧合NMP和氧气氧合NMP对心死亡供肾损伤修复的影响。方法取12只12～14周龄的健康雄性SD大鼠, 建立大鼠心脏死亡供体器官捐献(donation after cardiac death, DCD)模型。大鼠肾脏在热缺血损伤30 min并冷保存8 h后, 采用随机数字表法随机分为使用氧气氧合NMP(O组, 6只)、空气氧合NMP(A组, 6只)2 h。另取相同数量大鼠, 采用随机数字表法随机平分为开腹即获取肾组织的假手术组(C组, 6只)和心死亡后仅静态冷保存8 h的心死亡肾脏静态保存组(SCS组, 6只)作为O组和A组的对照。微板法检测肾灌注液损伤指标肌酐(Cr)、天冬氨酸氨基转移酶(AST)和乳酸脱氢酶(LDH)水平变化, HE染色评定肾组织中肾损伤指标, 免疫组织化学和蛋白质印迹法检测髓过氧化物酶(MPO)、细胞间黏附分子-1(ICAM-1)表达, 酶联免疫吸附法检测TNF-α和IL-6水平, 硫代巴比妥酸和WST-8法测定脂质过氧化产物丙二醛(...     

体外膜肺氧合在心脏死亡器官捐献供体肝移植中的应用进展

供肝短缺已经成为制约肝移植发展的重要因素,心脏死亡器官捐献(DCD)供体是扩大供体池的一个重要来源。应用体外膜肺氧合(ECMO)技术可提高DCD供肝的质量,增加器官捐献供体池,改善肝移植受体的预后。本文综述了ECMO对DCD供体器官支持的基本原理和操作技术、ECMO用于DCD供体肝移植的研究进展及存在的问题,提示ECMO在我国DCD供体肝移植中具备较大的发展潜力和应用前景。     

DCD供肝术前评估应注意的问题

<正>心脏死亡器官捐献(Donation after cardiacdeath,DCD)已成为我国器官来源的主要途径。除了器官获取和运输过程中通常关注的热缺血、冷缺血时间等因素影响外,DCD捐献者还受到原发病及救治过程中多种不确定因素的影响,国外甚至将DCD供肝作为边缘供肝~([1]),认为其具有较高的移植肝无功能、缺血性胆道病变发生率。因此如何对DCD供肝质量进行准确评估是保证保障较好移植效果的关键     

常温机械灌注保存对DCD小型猪脂肪肝供肝的影响

目的 探讨应用常温机械灌注装置保存对DCD小型猪脂肪肝的降脂及修复作用.方法 选取巴马小型猪4只,建立脂肪肝猪DCD及热缺血时间30main动物模型后,切取肝脏约5cm×5cm放入UW液冷藏保存(SCS组),余放入常温机械灌注装置中进行灌注保存(NMP组).在保存开始后2h、4h、6h分别切取肝组织进行肝匀浆质脂质含量...     

心脏死亡供者供肝肝移植11例疗效观察

目的 观察利用Maastricht分类第Ⅲ型心脏死亡供者(donors after cardiac death,DCD)供肝行原位肝移植的疗效.方法 2011年9月至2012年6月期间,吉林大学白求恩第一医院共有14例DCD供者进行器官捐献,其中11例行原位肝移植,分析总结心脏死亡供肝行肝移植的疗效.结果 本组11例DCD供肝热缺血时间为(21.3 ±2.6)min,冷缺血时间为(2.5±0.8)h,供肝质量为(1245 ±180)g.11例受体行原位肝移植术,术中无肝期42～ 80 min,手术时间380 ～ 740 min,术中出血量600 ～ 3000 ml,平均1750 ml.除l例受体于术后第3天死于原发移植肝无功能外,其余受体术后均恢复良好.在全程随访期间预后较好,均未出现排斥反应及胆管、血管并发症.结论 通过规范捐献流程、尽量减少冷、热缺血时间及加强器官功能维护,利用Maastricht分类第Ⅲ型心脏死亡供者供肝对终末期肝脏疾病患者行肝移植,疗效良好.     

Preliminary Single‐Center Canadian Experience of Human Normothermic Ex Vivo Liver Perfusion: Results of a Clinical Trial

After extensive experimentation, outcomes of a first clinical normothermic machine perfusion (NMP) liver trial in the United Kingdom demonstrated feasibility and clear safety, with improved liver function compared with standard static cold storage (SCS). We present a preliminary single‐center North American experience using identical NMP technology. Ten donor liver grafts were procured, four (40%) from donation after circulatory death (DCD), of which nine were transplanted. One liver did not proceed because of a technical failure with portal cannulation and was discarded. Transplanted NMP grafts were matched 1:3 with transplanted SCS livers. Median NMP was 11.5 h (range 3.3–22.5 h) with one DCD liver perfused for 22.5 h. All transplanted livers functioned, and serum transaminases, bilirubin, international normalized ratio, and lactate levels corrected in NMP recipients similarly to controls. Graft survival at 30 days (primary outcome) was not statistically different between groups on an intent‐to‐treat basis (p = 0.25). Intensive care and hospital stays were significantly more prolonged in the NMP group. This preliminary experience demonstrates feasibility as well as potential technical risks of NMP in a North American setting and highlights a need for larger, randomized studies.     

Impact of Temperature on Porcine Liver Machine Perfusion From Donors After Cardiac Death

Normothermic machine perfusion (NMP) has been introduced as a promising technology to preserve and possibly repair marginal liver grafts. The aim of this study was to compare the effect of temperature on the preservation of donation after cardiac death (DCD) liver grafts in an ex vivo perfusion model after NMP (38.5°C) and subnormothermic machine perfusion (SNMP, 21°C) with a control group preserved by cold storage (CS, 4°C). Fifteen porcine livers with 60 min of warm ischemia were preserved for 10 h by NMP, SNMP or CS (n = 5/group). After the preservation phase all livers were reperfused for 24 h in an isolated perfusion system with whole blood at 38.5°C to simulate transplantation. At the end of transplant simulation, the NMP group showed significantly lower hepatocellular enzyme level (AST: 277 ± 69 U/L; ALT: 22 ± 2 U/L; P < 0.03) compared to both SNMP (AST: 3243 ± 1048 U/L; ALT: 127 ± 70 U/L) and CS (AST: 3150 ± 1546 U/L; ALT: 185 ± 97 U/L). There was no significant difference between SNMP and CS. Bile production was significantly higher in the NMP group (219 ± 43 mL; P < 0.01) compared to both SNMP (49 ± 84 mL) and CS (12 ± 16 mL) with no significant difference between the latter two groups. Histologically, the NMP livers showed preserved cellular architecture compared to the SNMP and CS groups. NMP was able to recover DCD livers showing superior hepatocellular integrity, biliary function, and microcirculation compared to SNMP and CS. SNMP showed some significant benefit over CS, yet has not shown any advantage over NMP.     

常温机械灌注修复长时间冷保存供肝的大动物实验研究

目的探索常温机械灌注(NMP)在挽救大动物边缘供肝中的价值。 方法6只雄性、10～12月龄巴马小型猪分为静态冷保存(SCS)6 h组和SCS 24 h组,每组3只,分别于供肝获取后SCS 6 h、24 h后进行2 h NMP复苏。基于荷兰Organ Assist公司Liver Assist系统和供体猪自身血液,整合四通道生理仪器及自制灌注管路搭建NMP平台。在NMP过程中分别于灌注0、15、60、90、120 min 5个时间点收集灌注液用于肝功能(ALT、AST)检测及血气分析(pH、氧分压)。灌注结束取肝左叶相同位置少许组织,以10%甲醛固定,用于后续HE染色。 结果NMP开始时,SCS 24 h组ALT、AST水平略高于SCS 6 h组,之后AST、ALT水平均缓慢上升。NMP开始时SCS 24 h组pH值中位数为7.28,氧分压中位数为46 mmHg(1 mmHg＝0.133 kPa,下同),均低于SCS 6 h组(7.36,52 mmHg),经过2 h NMP后SCS 24 h组pH值和氧分压逐渐接近甚至优于SCS 6 h组。HE染色发现,灌注前SCS 24 h组肝脏充血严重,炎性细胞浸润明显;NMP 2 h后,肝脏充血明显改善、炎性细胞减少,与SCS 6 h组无明显差异。 结论基于Liver Assist系统和供体猪自身血液成功搭建的NMP平台,可有效改善长时间冷保存的边缘供肝质量,提示NMP在修复边缘供肝、扩展供肝来源方面具有重要临床及基础研究价值。     

Human liver stem cell-derived extracellular vesicles reduce injury in a model of normothermic machine perfusion of rat livers previously exposed to a prolonged warm ischemia

Livers from donors after circulatory death (DCD) are a promising option to increase the donor pool, but their use is associated with higher complication rate and inferior graft survival. Normothermic machine perfusion (NMP) keeps the graft at 37°C, providing nutrients and oxygen supply. Human liver stem cell-derived extracellular vesicles (HLSC-EVs) are able to reduce liver injury and promote regeneration. We investigated the efficacy of a reconditioning strategy with HLSC-EVs in an experimental model of NMP. Following total hepatectomy, rat livers were divided into 4 groups: (i) healthy livers, (ii) warm ischemic livers (60 min of warm ischemia), (iii) warm ischemic livers treated with 5 × 10⁸ HLSC-EVs/g-liver, and (iv) warm ischemic livers treated with a 25 × 10⁸ HLSC-EVs/g-liver. NMP lasted 6 h and HLSC-EVs (Unicyte AG, Germany) were administered within the first 15 min. Compared to controls, HLSC-EV treatment significantly reduced transaminases release. Moreover, HLSC-EVs enhanced liver metabolism by promoting phosphate utilization and pH self-regulation. As compared to controls, the higher dose of HLSC-EV was associated with significantly higher bile production and lower intrahepatic resistance. Histologically, this group showed reduced necrosis and enhanced proliferation. In conclusion, HLSC-EV treatment during NMP was feasible and effective in reducing injury in a DCD model with prolonged warm ischemia.     

两种重症急性胰腺炎造模方式对大鼠胰外器官损害的比较

目的:比较胰胆管逆行注射牛黄胆酸钠与腹腔注射L-精氨酸两种重症急性胰腺炎(SAP)造模方式对胰外器官损害的差异。方法:18只雄性远交群(SD)大鼠经随机数表法分为3组,每组6只：空白对照组(A组),L-精氨酸诱导SAP模型组(B组),牛磺胆酸钠诱导SAP模型组(C组)。造模后,分别于12、24 h割尾采血,检测谷草转氨...     

Liver Transplantation After Ex Vivo Normothermic Machine Preservation: A Phase 1 (First‐in‐Man) Clinical Trial

The number of donor organs suitable for liver transplantation is restricted by cold preservation and ischemia–reperfusion injury. We present the first patients transplanted using a normothermic machine perfusion (NMP) device that transports and stores an organ in a fully functioning state at 37°C. In this Phase 1 trial, organs were retrieved using standard techniques, attached to the perfusion device at the donor hospital, and transported to the implanting center in a functioning state. NMP livers were matched 1:2 to cold‐stored livers. Twenty patients underwent liver transplantation after NMP. Median NMP time was 9.3 (3.5–18.5) h versus median cold ischaemia time of 8.9 (4.2–11.4) h. Thirty‐day graft survival was similar (100% NMP vs. 97.5% control, p = 1.00). Median peak aspartate aminotransferase in the first 7 days was significantly lower in the NMP group (417 IU [84–4681]) versus (902 IU [218–8786], p = 0.03). This first report of liver transplantation using NMP‐preserved livers demonstrates the safety and feasibility of using this technology from retrieval to transplantation, including transportation. NMP may be valuable in increasing the number of donor livers and improving the function of transplantable organs.     

肝脏再移植原因及效果分析

目的探讨再次肝移植的原因及效果,并比较不同供肝来源与再移植的关系。 方法回顾性分析2000年1月至2018年5月四川大学华西医院1 429例肝移植受者临床资料。首次肝移植供肝来源分别为尸体供肝686例、心脏死亡器官捐献(DCD)供肝346例和活体供肝397例。其中31例受者接受再次肝移植(32例次,其中1例受者接受2次再移植),再移植率为2.24%(32/1 429),供肝来源分别为尸体供肝23例、DCD供肝6例、活体供3例。再移植间隔时间中位数为311 d(88～845 d),间隔1～7 d 3例,8～30 d 1例,31～365 d 15例,>1年13例。采用Kaplan-Meier法计算肝脏再移植术后受者生存时间并绘制生存曲线,采用Breslow法比较再移植间隔时间>1年及≤1年的受者1、5和10年生存率,采用Fisher确切概率法比较不同供肝来源的受者再移植率。P<0.05为差异有统计学意义。 结果截至2018年5月,31例肝脏再移植受者术后12例存活(38.7%)、19例死亡(61.3%),中位生存时间为17个月(2～102个月)。尸体供肝、DCD供肝和活体供肝再移植率分别为3.4%(23/686)、1.7%(6/346)和0.8%(3/367)。尸体供肝再移植率高于活体肝移植,差异有统计学意义(P＝0.007),DCD供肝再移植率与尸体供肝、活体供肝再移植率相比,差别均无统计学意义(P＝0.137和0.222)。其中18例再移植间隔时间<1年的受者,6例存活、12例死亡;13例再移植间隔时间≥1年的受者,6例存活、7例死亡。31例肝脏再移植受者术后1、5和10年生存率分别为64.2%、51.2%和46.6%。再移植间隔时间<1年的受者1、3和5年生存率分别为49.4%、41.2%和30.9%,间隔时间≥1年的受者1、3和5年生存率分别为84.6%、65.8%和65.8%,二者差异无统计学意义(χ²＝2.946,P>0.05)。 结论再移植是肝移植术后移植物失功的唯一有效治疗方法,再移植术后受者往往病情危重,围手术期死亡率高,胆道并发症及排斥反应是再次肝移植的主要原因。应该慎重把握再移植手术时机,目前亟待更多的研究对再移植做进一步探讨。     

Donation after cardiac death: the University of Wisconsin experience with liver transplantation

OBJECTIVE: To determine whether the outcomes of liver transplantation (LTx) from donation after cardiac death (DCD) donors are equivalent to those from donation after brain death (DBD) donors. SUMMARY BACKGROUND DATA: Because of the significant donor organ shortage, more transplant centers are using livers recovered from DCD donors. However, long-term, single-center outcomes of liver transplantation from DCD donors are limited. METHODS: From January 1, 1993, to July 31, 2002, 553 liver transplants were performed from DBD donors and 36 were performed from DCD donors. Differences in event rates between the groups were compared with Kaplan-Meier estimates and the log-rank test. Differences in proportion and differences of means between the groups were compared with Fisher exact test and the Wilcoxon rank sum test, respectively. RESULTS: Mean warm ischemic time at recovery in the DCD group was 17.8 +/- 10.6 minutes. The overall rate of biliary strictures was greater in the DCD group at 1 year (33% versus 10%) and 3 years (37% versus 12%; P = 0.0001). The incidence of hepatic artery thrombosis, portal vein stenosis/thrombosis, ischemic-type biliary stricture (ITBS), and primary nonfunction were similar between groups. However, the incidence of both hepatic artery stenosis (16.6% versus 5.4%; P = 0.001) and hepatic abscess and biloma formation (16.7% versus 8.3%; P = 0.04) were greater in the DCD group. Trends toward worse patient and graft survival and increased incidence of ITBS were seen in DCD donors greater than 40 years compared with DCD donors less than 40 years. Overall patient survival at 1 year (DCD, 80%; versus DBD, 91%) and 3 years (DCD, 68%; versus DBD, 84%) was significantly less in the DCD group (P = 0.002). Similarly, graft survival at 1 year (DCD, 67%; versus DBD, 86%) and 3 years (DCD, 56%; versus DBD, 80%) were significantly less in the DCD group (P = 0.0001). CONCLUSIONS: Despite similar rates of primary nonfunction, LTx after controlled DCD resulted in worse patient and graft survival compared with LTx after DBD and increased incidence of biliary complications and hepatic artery stenosis. However, overall results of LTx after controlled DCD are encouraging; and with careful donor and recipient selection, LTx after DCD may successfully increase the donor liver pool.     

Hepatic function in hypothermically stored porcine livers: comparison of hypothermic machine perfusion vs cold storage

Hypothermic machine perfusion (HMP) has a potential to relieve the current donor liver crisis by providing an improved and extended preservation method. This study examined the effect of HMP on hepatocellular functions, using a prototype liver transporter capable of preserving livers for 24 hours. Livers obtained from adult farm pigs (28 to 32 kg body weight) were divided into three groups: fresh control, HMP, and simple cold storage (n = 4 each). A 4-hour normothermic reperfusion of livers was conducted to assess hepato-metabolic and cellular functions. The hepatic transport function, as indicated by canalicular excretion of indocyanine green, was improved in the HMP group than in the SCS group. The overall tissue viability, as indicated by oxygen consumption levels, was notably improved in HMP and control livers as compared to the SCS group. Higher bile production in both the preserved groups as compared to the fresh control livers could be a result of biliary edema and leakage of plasma into the canaliculus. The hepato-cellular injury, measured by ALT, release was significantly greater in the SCS group as compared to the HMP and control groups. These findings suggest that HMP could be a better method to preserve hepatic function and overall tissue viability as compared to SCS. Improved hepatic functions are indirect indicators of superior microcirculation and sinusoidal endothelial cell functions. Further studies in progress will evaluate these functions to confirm the significance of these observations.     

Short- and Long-Term Outcomes with the Use of Kidneys and Livers Donated after Cardiac Death

The shortage of deceased donor kidneys and livers for transplantation has prompted the use of organs from donors deceased after cardiac death (DCD). We used the UNOS database to examine patient and graft survival following transplantation of DCD organs compared to those following grafts from donors deceased after brain death (DBD; for livers, grafts from donors < 60 years old were labeled '< 60 yrs'). Of 44035 deceased donor kidney transplant recipients, 1177 (3%) received a DCD kidney. There was no difference in patient or graft survival at 5 years (DCD vs. DBD: 81.3% vs. 80.8% and 66.9% vs. 66.5%; p = 0.70 and p = 0.52 respectively). Of 24688-deceased donor liver transplant recipients, 345 (1.4%) were from DCD donors and 20289 (82%) were from '< 60 yrs' DBD donors. Three-year patient and graft survival were inferior in the DCD group (DCD vs. '< 60 yrs' DBD: 77% vs. 80% and 65% vs. 75%; p = 0.016 and p < 0.0001 respectively) but were comparable to current alternatives, '>/= 60 yrs' DBD livers (donor age >/= 60) and split livers. DCD livers are a reasonable option when death is imminent. Our study demonstrates good outcomes using DCD kidneys and livers and encourages their use.