【作者】 赵立夫；

【导师】 李兰娟；

【作者基本信息】 浙江大学 ， 内科学， 2012， 博士

【摘要】 研究背景：肝衰竭发生时,肝脏会丧失其正常的解毒、生物合成和生物转化功能,其临床特征包括：凝血酶原时间延长、肝性脑病和黄疸。根据是否存在已知的或未知的慢性肝病病史,不同致病因素所引起的肝衰竭可被划分为两大类：急性肝衰竭和慢加急性肝衰竭,两者均伴随着高病死率。迄今为止,肝脏移植仍然是治疗终末期肝衰竭公认的最终解决方案,但世界性的供体短缺使其在临床实际应用中受到限制。在这种背景下,有研究者致力于体外人工肝支持系统的研发,以期为肝衰竭患者提供一种肝移植前的过渡治疗手段,或者为自体肝脏的恢复赢得机会。根据是否装载具有代谢活性的肝细胞,人工肝支持系统可分为非生物人工肝和生物人工肝。尽管非生物人工肝有改善肝衰竭患者的生化指标和临床症状的作用,但其在改善患者预后方面尚未达到预期的效果。目前公认的观点是,仅具备解毒功能的非生物人工肝不足以对肝衰竭患者提供全面的支持治疗,而在理论上,以载细胞生物反应器为核心的生物人工肝可提供大部分乃至全部的肝功能支持[7,8,10-16]。然而,我们不得不承认,生物人工肝的发展还不成熟,距离常规临床应用仍有距离。迄今,只有两篇文献报道了生物人工肝治疗治疗的随机对照临床试验,结果不如人意。在这种背景下,有学者认为理想的体外人工肝支持系统应该是载细胞生物反应器和多种非生物人工肝治疗手段的有机整合。为研发基于这种理念的体外人工肝支持系统,我们必须从以下三方面着手：1)选择合适的细胞源；2)设计制造能够为肝细胞提供近似体内环境的生物反应器；3)研发新的体外循环装置,该装置不仅可以控制载细胞的生物反应器,还可控制现有的大多数非生物人工肝治疗模式。最近,本研究团队设计制造了一种以装载海藻酸钠-壳聚糖(AC)微球包裹的原代猪肝细胞的漏斗形流化床式生物反应器为核心的生物人工肝支持系统,并证明该系统可显著延长D-氨基半乳糖诱导的急性肝衰竭猪的生存时间。然而,原代猪肝细胞有使患者罹患动物源性传染病的潜在风险。这促使我们去寻找一种更加适用于生物人工肝临床治疗的肝细胞源。近年来,本实验室致力于永生化肝细胞的研究。尽管这类细胞具有体外扩增能力,但猿猴病毒40大T抗原基因(SV40LT)的持续表达具有潜在的致瘤风险。为了解决这个问题,本团队应用他莫西芬介导的Cre/LoxP位点特异性重组,建立了一株可逆性永生化人肝细胞系(HepLi-4), SV40LT可以被程序性的切除。在此基础上,以可逆性永生化人肝细胞为细胞源对大型动物进行生物人工肝治疗的首次试验得以开展。困扰我们的另一个问题是,研究所用的人工肝治疗用体外循环装置大多依赖进口。例如,评价漏斗形流化床式生物反应器和HepLi-4细胞所用的体外循环装置是日本旭化成株式会社制造的Plasauto-iQ。而且,目前国外相关设备的功能较单一。在这种背景下,本团队进行人工肝治疗用体外循环装置的研发,该装置应不仅能够控制血浆置换(PE)、血浆吸附(PA)、血液透析(HD)等目前临床常规应用的非生物人工肝治疗模式和尚处于科研阶段的生物人工肝治疗(BAL)模式,而且,以上各种模式均可便捷的进行联合或序贯应用。本研究拟初步构建具备上述功能的原理机,为下一步的人工肝治疗研究提供一个可靠的平台。第一部分基于可逆性永生化人肝细胞的生物人工肝系统的动物实验评估目的：通过中国实验小型猪急性肝衰竭模型,对以可逆性永生化人肝细胞(HepLi-4)为肝细胞源的生物人工肝系统进行评估。方法：表达他莫昔芬依赖性Cre重组酶的HepLi-4细胞在转瓶中扩增后,通过在含有500nM的4-羟基他莫昔芬的培养液中培养5～7切除SV40LT基因。猪急性肝衰竭模型通过在无麻醉情况下静脉推注D-氨基半乳糖(1.5g/kg)诱导。包裹HepLi-4细胞的海藻酸钠-壳聚糖(AC)微球通过一步法制备。15只急性肝衰竭猪分为以下三组：1)生物人工肝治疗(BAL)组：实验动物接受装载微囊化的HepLi-4细胞的生物人工肝支持系统治疗(n=5)；2)假治疗(Sham BAL)组：即设备对照,实验动物接入生物人工肝支持系统进行体外循环,但反应器内仅装入无细胞AC微囊(n=5)；3)急性肝衰竭(ALF)组：即基线对照,实验动物仅接受监护但不接受治疗(n=5)。记录以上三组实验动物的生存时间以及三种干预过程前后实验动物的血液生化参数。生物人工肝治疗前后,进行微球完整性和细胞活力检测,同时,进行微囊化HepLi-4细胞内肝脏特异性基因的转录水平分析,正常成人肝组织作为对昭结果：干预后,相对于Sham BAL组和ALF组,BAL组动物的Fischer指数较高,血清间接胆红素相对低,差异具有统计学意义。BAL组动物的平均生存时间长于两个对照组,但差异不具有统计学意义。经历人工肝治疗后,进行微球完整性和细胞活力没有出现显著的下；同时,微囊化HepLi-4细胞内肝脏特异性基因的转录水平得以保持,但其相对于正常成人肝组织存在明显的变异。结论：尽管HepLi-4细胞在生物人工肝治疗过程中发挥了一些对急性肝衰竭猪有益的代谢功能,但HepLi-4细胞尚不能作为人工肝治疗用的理想细胞源。我们需要在维持肝细胞分化的研究中付出更多的努力。第二部分人工肝体外循环装置的研发目的：构建可以控制现有的大部分非生物人工肝治疗模式和生物人工肝治疗模式的人工肝体外循环装置的原理机。方法：人工肝体外循环装置的原理机包含非生物部分和生物部分。体外循环装置的控制中枢由的工业用个人计算机,两个串行通讯端口和一个外设部件互连标准(PCI)数字输入/输出(DIO)卡组成。体外循环装置的大多数部件可通过RS485总线得到整合。该装置的软件为用户提供了操作指导和人机交流界面。同时,基于硬件和软件设计,该装置在运行过程中可实现实时状态监测和调节。此外,体外循环装置运行过程中的所有参数值都会被自动记录,便于事后分析。为了验证我们的设计中,我们对该原理机进行了体外测试和动物实验测试。结果：在体外测试和动物实验测试中,体外循环装置原理机的硬件和软件在所有治疗模式下均运行正常。该装置中所有部件的功能都得到了验证。此外,该装置可以实时监测到治疗中的异常情况。结论：体外循环装置的原理机具有良好的安全性和人性化的设计。可以为这种医疗设备的商品化提供可靠的研究平台,也可为培养相关技术人员提供一个模拟培训的平台。更多还原

【Abstract】 Background:Liver failure is the inability of the liver to perform its normal detoxification, biosynthesis, and/or biotransformation functions. The clinical presentation of liver failure includes a prolonged prothrombin time, encephalopathy, and jaundice. Regardless of the etiology, liver failure can be divided into two categories:acute liver failure (ALF) or acute on chronic liver failure (AoCLF). Both are accompanied by high mortality. Liver transplantation is still the only ultimate solution for end stage liver failure, but its application is hampered by a world-wide scarcity of donor organs. In this context, extracorporeal artificial liver support systems have been expected to be a bridge to transplantation or to provide an opportunity for the native liver to regenerate.Depending on whether they are loaded with metabolically active hepatocytes or not, these systems can be roughly classified into two types:non-biological liver (NBL) or bioartificial liver (BAL). NBLs have proven to be useful for improving biochemical parameters and clinical symptoms in many cases, but the prognostic benefits have not yet been fully reflected. It is widely accepted that a NBL, which can only detoxify, is insufficient to support liver failure patients, while in theory an ideal hepatocyte-filled BAL based on hepatocyte-filled bioreactor could provide most or even all normal liver functions. However, until now, only two randomized controlled clinical trials exploring the effectiveness of BALs have been reported, and the results were not encouraging. Probably, BALs alone can not be competent for the job at the present level of technology. Future extracorporeal artificial liver support systems should be the combination of cell-filled bioreactors and NBL components.To develop such an extracorporeal artificial liver support system, three things are essential:1) an appropriate cell source;2) a bioreactor capable of providing in vivo-like environments for cells; and3) an extracorporeal circulation device which can control not only the cell-filled bioreactor, but also most types of existing NBL components.We recently designed a choanoid fluidized bed bioreactor filled with alginate-chitosan (AC) encapsulated primary porcine hepatocytes and proved it could prolong the survival of pigs with acute liver failure (ALF) induced by D-galactosamine injection. The potential risk of zoonotic transmission, however, might exist, which limits its clinical application. Therefore, developing a safe cell source for BAL is necessary. For several years, we have been dedicated to establishing immortalized hepatocyte line. Although these cell types have unlimited expansion capabilities in vitro, continuous expression of simian virus40large T antigen (SV40LT) might be tumorigenic. To solve this problem, we established a reversibly immortalized cell line (HepLi-4) by transfection of primary human hepatocytes with drug-medicated Cre/LoxP recombination. By doing so, the immortalizing Oncogene (SV40LT) can be excised programly. In order to demonstrate the clinical potential of HepLi-4cells in BAL, we carried out experiments on large animal models.Another problem facing us is all extracorporeal circulation device applied in our research were not domestic products. For example, the extracorporeal circulation device used to evaluate our choanoid fluidized bed bioreactor and HepLi-4cells is Plasauto-iQ (Asahi Medical, Japan). Also, functions of these commercial devices are rather limited. This is our motivation to develop a novel extracorporeal circulation device which can control not only most of the existing modes of NBL, such as plasma exchange (PE), hemodialysis (HD) and plasma adsorption (PA),but also BAL treatment modes. Also, all treatment modes can be applied either jointly or sequentially. To build the prototype of such a device is the first step. Part I Evaluation of a bioartificial liver system loaded with reversibly immortalized human hepatocytes in pigsObjective:To evaluate the bioartificial (BAL) system loaded with our newly established reversibly immortalized cell line (HepLi-4) in Chinese experiment miniature pigs with acute liver failure (ALF).Methods:HepLi-4cells expressing Tamoxifen-dependent Cre recombinase were expanded in roller bottles and SV40LT genes were removed by keeping the cells in culture media containing500nM4-hydroxytamoxifen for5-7days. Alginate-chitosan (AC) microbeads containing HepLi-4cells were produced via single-stage procedure. ALF was induced in Chinese experiment miniature pigs with intravenous injection of D-galactosamine at a dose of1.5g/kg body weight. Fifteen ALF pigs were allocated to three groups:a BAL group, receiving BAL treatment with encapsulated HepLi-4cells (n=5); a sham BAL group (device control), receiving cell-free BAL treatment (n=5); a ALF group (baseline control), receiving intensive care only (n=5). Survival time and biochemical parameters of pigs were measured. Before and after BAL, microbead integrity and cell viability were tested, also, expression of liver-specific genes in HepLi-4cells was analyzed, adult human liver acted as a reference.Results:In BAL group, Fischer index was higher and serum indirect bilirubin level was lower compared with two control groups. Survival time in BAL group is longer than that in two control groups, but the difference is not statistically significant. After BAL, microbead integrity and cell viability did not decrease significantly. Gene expression analysis showed that the transcript levels of liver-specific genes in HepLi-4were retained after BAL, but significant variations were observed between HepLi-4and adult human liver.Conclusion:HepLi-4showed beneficial metabolic effects on ALF pigs in BAL, but is still not an appropriate cell source for BAL. More insights into interpreting the conditions for hepatocyte differentiation are needed. Part Ⅱ Development of an extracorporeal circulation device of artificial liver systemObjective:To develop the prototype of an extracorporeal circulation device of artificial liver system which can control most of the existing modes of non-biological liver (NBL) and bioarticial liver (BAL) support treatment.Methods:The prototype of the extracorporeal circulation device consisted of a nonbiological section and a biological section. The control center was composed of an industrial personal computer, two serial communication ports and a peripheral component interconnect (PCI) digital input and output (DIO) card. Most components are integrated via the RS485buses. The software provided users an operation guide and a human-machine communication interface. Based on hardware and software design, real time status monitoring and regulation could be realized. Also, all parameter values during the treatment can be recorded for post hoc analysis. To verify our design, we tested the prototype of extracorporeal circulation device both in vitro and on miniature pigs.Results:The hardware and software of the prototype of the extracorporeal circulation device runned normally in all treatment modes. Functions of all components were verified. Also, timely detection of abnormal conditions in the clinical treatment could be fulfilled.Conclusion:The prototype of the extracorporeal circulation device has a good safety and user-friendly design.It can provide a reliable research platform for commercialization of the device. Also, it can act as a simulation training platform for relevant technical staff.更多还原