常温机械灌注保存DCD小型猪脂肪肝供肝的实验研究

发布时间：2018-03-14 03:01

  本文选题：机械灌注　切入点：器官保存　出处：《天津医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的快速有效建立小型猪脂肪肝动物模型;探讨应用常温机械灌注装置保存对DCD小型猪脂肪肝的保护、修复及评估作用。方法研究分为两部分:第一部分:小型猪脂肪肝模型的建立,选择巴马小型猪10只,随机分为两组,对照组5只,模型组5只(1只造模过程中死亡)。对照组常规基础饲料喂食;模型组采用综合配料喂食,综合配料由基础饲料(50%)+高脂饲料(猪油50%)+酒精饮料+四环素组成。其中猪油、酒精饮料通过胃造瘘管进行管饲。两组动物分别在0、2、4、6周末按编号进行耳缘静脉抽血检测肝脏功能及血脂生化指标(包括AST、ALT、GGT、TG、CHO),并于6周末小切口开腹取肝脏组织进行病理检查。成模标准为肝内TG等血脂含量显著增加,血清ALT等酶学水平明显增高,肝组织学观察见多灶性炎症细胞浸润及肝细胞脂肪变性。第二部分:建立脂肪肝猪DCD及热缺血时间30min动物模型后,切取肝脏约5cm×5cm放入UW液冷藏保存(UW液组),余放入常温机械灌注装置中,与其相应管路连接进行灌注(NMP组)。分别在灌注前,灌注开始后不同时间点(1h、2h、3h、4h、5h、6h)抽取灌注液进行检验,检验指标包括AST、ALT、GGT、TG、CHO;记录灌注开始后在每小时胆汁引流量;灌注前及灌注开始后2h、4h、6h对两组分别切取肝组织进行病理检查及肝匀浆质检验(包括AST、ALT、GGT、TG、CHO)。观察比较两组各时间点生化指标及肝脏病理组织学变化。结果一、1.一只猪胃造瘘术后第1天死亡。2.模型组和对照组在喂饲期间各生化指标比较:在喂饲前(即喂饲0w)、喂饲2w后两组间AST、ALT、GGT、TG、CHO、TG值差异均无统计学意义(P0.05);在喂饲4w后两组间AST、ALT、GGT、TG值差异有显著统计学意义(P0.001),CHO值差异无统计学意义(P=0.056);在喂饲6w后各两组间AST、ALT、GGT、TG、CHO、TG值差异均有显著统计学意义(P0.001)。3.小切口开腹切取肝组织病理检查对照组肝组织病理:肝小叶结构清晰,肝细胞索排列正常,核圆、大、居中,无脂滴和炎症细胞浸润,中央静脉和肝窦内有少量血细胞;模型组(造模6w后)肝组织病理:肝小叶结构模糊,肝细胞胞浆中有大量大、小脂滴空泡,中央静脉和肝血窦内充血,炎性细胞浸润,肝窦受挤压,间隙变窄。二、1.NMP组不同灌注时间灌注液各生化指标比较:灌注2h较开始时AST、ALT差异有统计学意义(P0.05),余时间较开始时差异无统计学意义(P0.05);GGT灌注过程差异无统计学意义(P0.05);灌注5h和6h均较开始时CHO、TG差异有统计学意义(P0.05)。2.NMP组和UW液冷保存组及保存终点肝匀浆质各生化指标比较:经6h保存,NMP组肝匀浆质酶学水平较UW液冷保存组有明显降低(t,P分别为AST:2.827,0.0311;ALT:3.524,0.0073;GGT:3.010,0.0224),而血脂水平降低无显著差异(t,P分别为CHO:2.315,0.0947;TG:2.620,0.0511);NMP保存6h肝匀浆质ALT、CHO、TG水平较0h明显降低(P0.05),AST、GGT差异无统计学意义(P0.05);UW液冷保存6h肝匀浆质GGT水平较0h明显升高(P0.05),而AST、ALT、CHO、TG差异均无统计学意义(P0.05)。3.不同灌注时间NMP组胆汁产量变化:开始灌注后2h胆汁引流量约3.5ml/h,且浑浊粘稠;2h-4h胆汁产量最多,维持在约4ml/h,颜色逐渐变清亮;4h-6h胆汁产量明显下降,约2ml/h,金黄色,清亮。4.不同保存时间UW组和NMP组肝脏组织病理比较:UW组在冷保存6h过程中,肝脏肝窦淤血有所加重,肝脏脂肪变程度无明显变化;NMP组开始灌注后2h中央静脉及肝窦广泛淤血,后逐渐减轻,肝脏脂肪变程度亦逐渐减轻。结论1、通过胃造瘘管管饲高脂、酒精饮料及四环素综合配料喂饲能够快速有效建立小型猪脂肪肝动物模型。2、通过NMP保存小型猪脂肪肝供肝能减轻肝脏脂质蓄积情况,防止肝细胞随保存时间延长而损伤,有效改善边缘供肝质量,从而扩大供体池,缓解供体短缺问题。3、在NMP过程中,能实时对移植术前供肝作出精准评估,优化供肝选择,从而有效改善受体预后。4、随着保存时间延长,NMP较SCS保存供肝的优势将越来越明显。
[Abstract]:Objective to establish effective pig animal model of fatty liver; application of normothermic machine perfusion preservation device of fatty liver of DCD mini pigs, repair and evaluation. Methods the research is divided into two parts: the first part: the establishment of fatty liver model in pigs, Bama small pig 10 type selection, were randomly divided into two groups. 5 rats in the control group, 5 rats in model group (1 rats died in the process of making the model). The control group routine diet; model group with synthetic ingredients fed by comprehensive ingredients (50%) basal diet + high fat diet (50% lard) + alcohol + tetracycline. The lard, alcoholic beverages by gastrostomy tube feeding. Two groups were used respectively in the animal 0,2,4,6 weekend according to the number of ear vein blood detecting liver function and blood lipids (including AST, ALT, GGT, TG, CHO), and the liver tissue in the 6 week minilaparotomy for pathological examination. Become the standard model for TG content of blood in the liver increased significantly, serum ALT enzyme levels were significantly higher, observation of multifocal inflammatory cell infiltration and hepatic steatosis of liver tissue. The second part: to establish the model of fatty liver of pig DCD and 30min warm ischemia time after the animal liver is about 5cm * 5cm into UW solution cold preservation (UW group), more than in the normothermic machine perfusion device, and the corresponding line connection of reperfusion (NMP group) respectively. The perfusion before and after the start of reperfusion at different time points (1H, 2h, 3h, 4h, 5h, 6h) perfusion fluid extraction test, test indexes including AST, ALT, GGT, TG, CHO; perfusion drainage in the record after the start of each hour before perfusion and perfusion bile; after the start of 2h, 4h, 6h of the two groups were cut pathological examination and liver homogenates test livers were (including AST, ALT, GGT, TG, CHO). Biochemical parameters and liver histopathological changes observed comparison of two groups of all time. 鏋滀竴,1.涓，

本文编号：1609308