基于微囊化猪肝细胞的生物人工肝治疗食蟹猴急性肝衰竭的实验研究

发布时间：2018-03-05 16:19

本文选题：生物人工肝　切入点：急性肝功能衰竭　出处：《南方医科大学》2017年博士论文　论文类型：学位论文

【摘要】：第一章食蟹猴急性肝功能衰竭模型的建立及评价目的:稳定的、重复性好的大动物急性肝衰竭(ALF)模型是人工肝研究不可或缺的平台。本章研究旨在建立一种药物和外科食蟹猴ALF模型。方法:1、16只食蟹猴随机分为4组(A,B,C,D),分别经外周静脉注射0.30,0.25,0.20 + 0.05(间隔 24 h),0.20 g/kg 的 D-氨基半乳糖(D-gal);给药后观察受试猴的临床表现,生存时间,生化、凝血、颅内压(ICP)和肝脏的病理变化;2、6只食蟹猴行袖套式插管门静脉-右肾静脉转流联合胆总管结扎、切断术(PRRS+CBDLT),术后观察动物的临床表现,生化、凝血和肝脏的病理和组织学变化。结果:1、给药后受试猴均出现不同程度的精神萎靡,进食减少、黄疽等表现,生存时间ABC三组分别为56±8.7h、95±5.5h和99±2.2h,D组除1只136h死亡外,其余猴全部存活。血清肝酶、TBiL、Cr、BUN、血Amm等明显升高,ALB水平降低,PT明显延长,ICP增高等,病理学鉴定有明显肝细胞片状坏死、伴出血。2、术后实验猴血清AST、ALT、CK、LDH、ALP、TBiL、DBiL等明显升高,PT明显延长,ALB水平降低,术后第10天肝标本病理学和组织学检测出明显的肝细胞坏死、变性、凋亡等炎症反应。结论:成功构建了 D-gal诱导的药物性食蟹猴ALF模型和PRRS + CBDLT诱导的简化外科食蟹猴ALF模型。第二章基于微囊化猪肝细胞的生物人工肝安全性及有效性的评估目的:以具有自主知识产权的往返翻转灌注型生物反应器为核心,装载微囊化的猪肝细胞,组建生物人工肝(BAL),通过对食蟹猴ALF模型的救治实验对其安全性和有效性进行评价,为其进一步临床试验奠定基础。方法:采用D-gal分次诱导建立食蟹猴药物性ALF模型,离体胶原酶两步灌流法分离西藏小型猪原代肝细胞,制备海藻酸钠-聚赖氨酸(APA)裹肝细胞微囊,装入往返翻转灌注型生物反应器中,构建BAL,用于ALF食蟹猴的治疗。15只ALF食蟹猴随机分为三组:(1)ALF组(空白对照):模型猴仅观察不治疗;(2)BAL组:模型猴接受装载微囊化猪肝细胞的BAL治疗;(3)Sham组(设备对照):模型猴接入BAL系统体外循环6小时,反应器内装载不含细胞的APA空微囊。比较三组实验猴治疗前后的生存时间、生化指标、PT及ICP等变化。结果:治疗过程中实验猴均耐受良好。与两个对照组相比,BAL组受试猴生存时间分别延长14小时、16小时。BAL可以显著降低受试猴血清肝酶、TBiL、血Amm等浓度和ICP,具有明显的肝支持作用。结论:基于微囊化猪肝细胞的生物人工肝系统能够改善ALF食蟹猴的血生化指标和ICP,延长生存时间。
[Abstract]:Chapter 1 Establishment and evaluation of acute hepatic failure model of crab eating monkey objective: stable, The animal model of acute liver failure (ARF) with good reproducibility is an indispensable platform for the study of artificial liver. This chapter aims to establish a drug and surgical ALF model of crab-eating monkey. The clinical manifestations of the tested monkeys were observed after intravenously injection of 0.30g / kg D-galactosamine (0.20g / kg D galactosamine at 24 h interval). Survival time, Biochemistry, Blood Coagulation, Intracranial pressure (ICP) and pathological changes of liver. Six crab-eating monkeys were treated with cuff intubation of portal vein and right renal vein bypass combined with common bile duct ligation. PRRS CBDLTT was performed after operation. The clinical manifestations and biochemistry of the animals were observed after operation. Results the pathological and histological changes of blood coagulation and liver were observed in all the monkeys treated with different degrees of mental retardation, decreased food intake and yellow gangrene. The survival time of ABC group was 56 卤8.7 h, 95 卤5.5 h and 99 卤2.2 h respectively, except that 1 monkey died at 136h. All of the other monkeys survived. The serum TBiLX, Amm, etc., and the levels of serum Amm were significantly increased and PT was significantly prolonged. The histopathological identification showed that there was obvious hepatocyte necrosis. After operation, the levels of ALB in the serum of experimental monkeys were significantly increased, and the levels of ALB were significantly prolonged and decreased. On the 10th day after operation, liver necrosis and degeneration were observed in liver specimens. Conclusion: the ALF model induced by D-gal and the simplified ALF model induced by PRRS CBDLT were successfully constructed. Chapter 2 is based on the safety and safety of bioartificial liver of microencapsulated porcine hepatocytes. Effectiveness assessment purpose: to focus on round-trip reverse perfusion bioreactors with autonomous intellectual property rights, The microencapsulated porcine hepatocytes were loaded into the bioartificial liver BALA. The safety and efficacy of the bioartificial liver were evaluated by the treatment experiment of the ALF model of the crab monkey. Methods: the drug-induced ALF model of crab monkey was established by D-gal fractionation, and the primary liver cells of Tibetan miniature pig were isolated by two steps perfusion of collagenase in vitro. Sodium alginate-polylysine (APA) encapsulated hepatocyte microcapsules were prepared and loaded into a reverse perfusion bioreactor. BAL.15 ALF capped monkeys were randomly divided into three groups: control group (blank control group: model monkey only observed and not treated). Model monkey was treated with BAL loaded with microencapsulated porcine hepatocytes. The model monkey was connected to the BAL system for 6 hours after cardiopulmonary bypass. APA empty microcapsules without cells were loaded in the reactor. The survival time before and after treatment was compared in three groups of experimental monkeys. Results: the experimental monkeys were well tolerated in the course of treatment. Compared with the two control groups, the survival time was prolonged by 14 hours or 16 hours. Conclusion: the bioartificial liver system based on microencapsulated porcine hepatocytes can improve the blood biochemical index and the survival time of ALF monkey.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R575.3

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