生物人工肝研究和应用进展

人工肝支持系统是用来为肝衰竭患者提供体外肝脏功能支持的技术方法.非生物人工肝在已经广泛应用肝衰竭患者的临床治疗,生物人工肝(BAL)以分离的哺乳动物肝细胞构成的生物反应器为解毒系统,可有效替代肝脏的解毒功能和合成功能,并可预防肝性脑病、肝昏迷和脑水肿.可作为肝移植前的过渡辅助,同时改善患者自身肝脏的功能以利于其功能的恢复.本文主要对生物人工肝的研究及应用进展进行综述.生物人工肝研究虽然取得了重大进展,但仍然面临寻找理想肝细胞来源,长期维持肝细胞的活性和功能,进一步优化反应器设计等问题.     

非酒精性脂肪性肝病与肠粘膜屏障关系的研究进展

近年来,非酒精性脂肪性肝病的发病率正呈逐年升高趋势,且可进一步发展为非酒精性肝炎、肝硬化甚至肝癌,但其具体的发病机制目前尚未完全阐明。迄今为止,关于非酒精性脂肪性肝病较为人们所接受的是"两次打击学说",即肝脏的脂肪变性及脂质的过氧化反应。自"肠-肝轴"被提出后,关于肠道粘膜屏障功能与非酒精性脂肪性肝病的发生和发展的关系备受研究人员的关注。近些年来,关于非酒精性脂肪性肝病与肠道粘膜的机械屏障、生物屏障、化学屏障、免疫屏障方面的研究越来越多,肠粘膜的四个屏障功能与非酒精性脂肪性肝病密切相关,相互影响共同促进疾病的发生发展。本文就非酒精性脂肪性肝病与肠粘膜屏障关系的研究进展进行了综述。     

终末期肝病细胞治疗供体细胞来源的研究进展

许多因素所致的肝脏疾病都有可能发展为终末期肝病,而终末期肝病目前唯一有效的治疗方法就是原位肝移植。然而,由于受供体肝脏短缺等因素的限制,细胞治疗方法一直被临床上所期待。近年来,基于谱系重编程的诱导型肝干细胞和诱导型肝实质细胞的技术体系的出现,为解决肝脏疾病细胞治疗中细胞来源匮乏的问题提供了新的思路,也加速了肝脏疾病细胞治疗研究向临床转化的进程。该文介绍了肝脏疾病细胞治疗研究中供体细胞来源的现状,并重点介绍了谱系重编程获取肝系细胞的研究进展。     

肝脏移植与肠道微生态变化的研究进展

摘要：肝移植已成为公认的治疗终末期肝疾病的有效手段。由于“肝－肠轴”和“肝脏－微生态轴”的存在,肝移植术后感染和移植肝损伤与肠道微生态变化密切相关。本研究综述了肝脏移植与肠道微生态变化的研究进展,主要包括大鼠肝移植中缺血再灌注损伤与肠道微生态变化、大鼠肝移植后移植排斥损伤与肠道微生态变化、大鼠肝损伤肝移植与肠道微生态变化的互作关系,以及临床肝移植围手术期、术前术后等与肠道微生态变化的研究。同时,指出了临床肝脏移植与肠道微生态变化可能的研究方向,提出了肝移植后微生物标志物的鉴定及潜在的靶向微生物治疗的策略。     

单人直视下改良建立大鼠原位肝移植模型的体会

目的:建立稳定大鼠原位肝移植模型,缩短术中无肝期时间,提高手术成功率及受体存活率。方法:在Kamada"二袖套法"的基础上改进,单人直视下建立大鼠原位肝移植模型,行60例SD大鼠原位肝移植手术。本研究简化供受体麻醉方式,供肝采用经门静脉(必要时配合腹主动脉补救方式)进行冷灌注,缩短修肝时间,提前预置牵引线,固定进针位置,改进植入肝脏肝上下腔静脉吻合、肝下下腔静脉及门静脉套管。观察并记录各组大鼠供体手术、修肝套管、无肝期、受体手术及肝移植手术总时间。术后检测1,7,30天受体大鼠肝功能(血清丙氨酸转氨酶(ALT),天冬氨酸转氨酶(AST)及总胆红素(TB))并分析生存情况。结果:无肝期结束后,供体肝脏灌注良好,受体麻醉移除后较快苏醒。供体手术、修肝套管、无肝期、受体手术及肝移植手术总时间分别为(32.5±1.58)、(7.3±1.43)、(15.6±2.62)、(53.2±3.74)、(108.5±2.34)min。大鼠术后24 h(手术成功率)为95%,1周生存率分别为90%,1月生存率分别为86.7%。大鼠术后短时间内肝功能水平增高,24 h时ALT(228.5±54.5 IU/L),AST(439.3±86.3 IU/L),TB(6.2±0.7μM),1周后逐渐恢复正常。结论:改良后的方法可以简易麻醉流程,缩短无肝期,提高手术成功率及受体的生存率。     

生物人工肝的研究进展

肝衰竭是临床常见的严重肝病症候群,病死率极高,除肝移植外,目前尚无有效的治疗方法。人工肝支持系统是治疗肝功能衰竭的重要方法之一,主要包括非生物型人工肝和生物型人工肝。结合了功能性肝细胞的生物型人工肝装置可发挥肝脏解毒、合成、代谢等功能,并且可以部分替代人体肝脏功能。生物型人工肝研发的关键在于种子细胞的筛选和生物反应器的构建。具有良好特性种子细胞的生物人工肝对于肝衰竭的疗效已在一些临床前研究中得以体现,并且目前已启动了多个生物人工肝的临床研究。该文从种子细胞的来源、生物反应器结构分类和临床应用等不同方面,对目前生物型人工肝的发展现状进行了综述。     

彩色多普勒超声对原位肝移植术后血流动力学研究

目的:探讨彩色多普勒超声(CDI)在原位肝移植术后对移植肝血流动力学变化的诊断价值。方法:应用CDI连续观察6例原位肝移植患者移植肝脏形态学变化,肝动脉峰值流速(HAmax)、门静脉及肝静脉平均流速(PVmean,HVmean)等指标,并以30例健康成人男性肝脏的血流动力学参数作为正常对照组。结果:在术后2周内肝脏移植组的HAmax低于正常对照组,1周内表现更为明显;术后1周内移植肝脏组的PVmean高于正常对照组,1周后表现不明显;移植肝脏组的Hvmean与正常对照组相比无明显差异。2周后,HA、PV和HV的血流速度基本上趋于正常。结论:CDI技术对了解肝移植术后移植肝的灌注情况,及早发现肝移植术后早期的并发症有重要意义。     

彩色多普勒超声对原位肝移植术后血流动力学研究

目的：探讨彩色多普勒超声(CDI)在原位肝移植术后对移植肝血流动力学变化的诊断价值。方法：应用CDI连续观察6例原位肝移植患者移植肝脏形态学变化,肝动脉峰值流速(HAmax)、门静脉及肝静脉平均流速(PVmean,HVmean)等指标,并以30例健康成人男性肝脏的血流动力学参数作为正常对照组。结果：在术后2周内肝脏移植组的HAmax低于正常对照组,1周内表现更为明显;术后1周内移植肝脏组的PVmean高于正常对照组,1周后表现不明显;移植肝脏组的Hvmean与正常对照组相比无明显差异。2周后,HA、PV和HV的血流速度基本上趋于正常。结论：CDI技术对了解肝移植术后移植肝的灌注情况,及早发现肝移植术后早期的并发症有重要意义。     

肠道菌群在慢性肝脏疾病中作用的研究进展

随着肠-肝轴机制研究的不断深入,肠道菌群与多种慢性肝脏疾病如非酒精性脂肪性肝病、酒精性肝病、肝硬化等相关性研究日益增多。肠道菌群通过肠道菌群失调、物质能量代谢改变及免疫反应激活等机制在多种肝脏疾病发生发展中发挥重要作用。本文对肠道菌群与慢性肝脏疾病关系的研究进展进行综述。     

CT全肝灌注的临床应用及进展

随着影像技术的发展和后处理功能的完善,肝灌注成像以研究组织、器官血流动力学变化已成为影像界关注的热点,然而CT灌注成像在肝脏等实质性器官中的应用尚处于探索阶段。肝脏具有双重血供,在各种病理生理情况下,肝脏动静脉之间及门静脉之间的血流动力学发生着复杂的变化。CT被认为是诊断肝脏病变最有价值的影像学方法,CT灌注成像能反映组织器官微循环内的血流动力学变化,是一种快速、准确、无创的功能成像方法。肝脏灌注CT检查可以同时获得形态和功能两方面的信息,使我们有可能早于形态学变化之前发现肝脏病变,有助于肝脏疾病的早期诊断和治疗,并能评价各种治疗手段对肝脏血流动力学变化的影响。本文针对全肝灌注CT扫描在临床的应用进行综述。     

间充质干细胞的来源及其对肝脏损伤及修复的研究进展

全球终末期肝病、肝衰竭的发病率和死亡率逐年升高,且目前肝移植是唯一疗效确切的治疗选择,但是,肝移植的使用受到肝源供体严重不足,长期存活率低,医疗费用昂贵等缺点使得原位肝移植的应用受限,绝大多数患者无法受益。为了克服肝脏器官短缺,干细胞替代治疗策略逐渐成为另一个肝病治疗的重要选择,干细胞治疗,特别是间充质干细胞（MSC）提供了一个新的肝病治疗选择。MSC是一群贴壁生长的成纤维细胞样细胞,由于MSC能够分化为多种类型的细胞,能够产生多种的细胞因子和生长因子,具有造血支持和免疫调节和抗炎功能,MSC被认为在再生医学领域具有重大的科学和实用价值。另外,由于MSC应用于治疗实验性肝损伤能明显提高动物存活率,明显改善肝功能。此外,一些临床前研究和临床研究也表明MSC对肝损伤性疾病具有显著地疗效。因此MSC在损伤性和退行性肝脏疾病的治疗具有广阔的应用前景。本文综述了MSC在肝损伤疾病治疗应用的进展,并对MSC在肝病治疗中的应用前景进行了展望。     

肝脏类器官的研究及应用进展

肝脏疾病易感性差异大且个体间的肝脏细胞存在明显的异质性,因此开发体外能够长期存活并具有代谢功能的人体类肝组织细胞模型,对治疗终末期肝病、开展肝脏致病机理研究及药物筛选具有重要意义。过去十年中,体外三维类器官模型发展迅猛,为疾病模拟、精准化治疗领域的研究提供了新的工具,显示出巨大潜力。肝脏类器官具有患者的基因表达与突变特征,在体外能够较长时间地保持肝脏细胞功能,已被应用于疾病模拟及药物有效性研究,并具有进行原位或异位移植发挥治疗作用的应用潜能。就干细胞、肝脏原代细胞等不同来源的肝脏类器官的发展及近年的研究进展作了综述,以期为肝脏类器官在疾病建模、药物发现和器官移植领域的研究和应用提供新的思路。     

胎肝造血干细胞的移植特性

胚胎发育中,肝脏是一个重要的造血器官。近年来胎肝移植的临床应用重新引起了人们的关注。本文应用染色体的 C-带染色法研究了小鼠骨髓和胎肝造血干细胞在照射受体小鼠中的增殖能力与相互间的竞争作用。实验结果表明胎肝造血干细胞在成年骨髓中的植入率比较同样条件下的成年骨髓造血干细胞低,但胎肝造血干细胞比较成年骨髓造血干细胞具有更强的自我更新或增殖能力。在同种胎肝造血干细胞移植中,为了降低同种移植抗力,提高移植的胎肝造血干细胞在受体中的耐受性,移植前对受体作适当的免疫抑制处理是必要的。因此,克服个体发育屏障和移植免疫屏障是提高同种胎肝造血干细胞移植效果中两个重要的研究课题。     

Improved survival of porcine acute liver failure by a bioartificial liver device implanted with induced human functional hepatocytes

Acute liver failure (ALF) is a life-threatening illness. The extracorporeal cell-based bioartificial liver (BAL) system could bridge liver transplantation and facilitate liver regeneration for ALF patients by providing metabolic detoxification and synthetic functions. Previous BAL systems, based on hepatoma cells and non-human hepatocytes, achieved limited clinical advances, largely due to poor hepatic functions, cumbersome preparation or safety concerns of these cells. We previously generated human functional hepatocytes by lineage conversion (hiHeps). Here, by improving functional maturity of hiHeps and producing hiHeps at clinical scales (3 billion cells), we developed a hiHep-based BAL system (hiHep-BAL). In a porcine ALF model, hiHep-BAL treatment restored liver functions, corrected blood levels of ammonia and bilirubin, and prolonged survival. Importantly, human albumin and α-1-antitrypsin were detectable in hiHep-BAL-treated ALF pigs. Moreover, hiHep-BAL treatment led to attenuated liver damage, resolved inflammation and enhanced liver regeneration. Our findings indicate a promising clinical application of the hiHep-BAL system.     

Hepatic tissue engineering: from transplantation to customized cell-based liver directed therapies from the laboratory

Today, liver transplantation is still the only curative treatment for liver failure due to end-stages liver diseases. Donor organ shortage, high cost and the need of immunosuppressive medications are still the major limitations in the field of liver transplantation. Thus, alternative innovative cell-based liver directed therapies, e.g. liver tissue engineering, are under investigation with the aim, that in future an artificial liver tissue could be created and be used for the replacement of the liver function in patients. Using cells instead of organs in this setting should permit (i) expansion of cells in an in vitro phase, (ii) genetic or immunological manipulation of cells for transplantation, (iii) tissue typing and cryopreservation in a cell bank, and (iv) the ex vivo genetic modification of patient's own cells prior re-implantation. Function and differentiation of liver cells are influenced by the three-dimensional organ architecture. The use of polymeric matrices permits the three dimensional formation of a neo-tissue and specific stimulation by adequate modification of the matrix-surface which might be essential for appropriate differentiation of transplanted cells. Additionally, culturing hepatocytes on three dimensional matrices permits culture in a flow bioreactor system with increased function and survival of the cultured cells. Based on bioreactor technology, bioartificial liver devices (BAL) are developed for extracorporeal liver support. Although BALs improved clinical and metabolic conditions, increased patient survival rates have not been proven yet. For intra-corporeal liver replacement, a concept which combines Tissue Engineering using three-dimensional, highly porous matrices with cell transplantation could be useful. In such a concept, whole liver mass transplantation, long term engraftment and function as well as correction of a metabolic defect in animal models could be achieved with a principally reversible procedure. Future studies have to investigate, which environmental conditions and transplantation system would be most suitable for the development of artificial functional liver tissue including blood supply for a potential use in a clinical setting.     

The perspectives of treatment of liver insufficiency by stem cells

The insufficiency of liver functions remains one of the major causes of death. The liver transplantation is the most effective method for treating severe liver diseases. The shortage of donor organs and high risk of graft rejection are the main problems for liver transplantation. Stem cells and isolated hepatocytes are the alternative means for repopulating liver after various injuries instead of liver transplantation. This review analyses achievements in therapy of liver insufficiency by means of stem cells in model experiments on animals as well as in clinical practice and also perspectives of employment of stem cells for treatment of liver insufficiency.     

Mammalian hepatocytes as a foundation for treatment in human liver failure.

Technological advances in the separation and culture of mammalian hepatocytes have facilitated the use of these cells as the foundation for either hepatocyte transplantation or hepatocyte-seeded hollow fiber liver assist devices (LAD). To fully appreciate the practical applications of these tissue engineering solutions, it is necessary to understand the types of human liver failure as well as the corresponding animal models. The most immediate application of this type of technology is the treatment of hepatic encephalopathy (HE), an acute and highly fatal complication of fulminant hepatic failure. Although the pathogenesis of HE is unknown, failure of the detoxification function of the liver is accepted as playing an important role in this disorder. Consequently, the assaying and preservation of P450 activity in the grafted cells or in the LAD must be among the main targets of this research. This review explores the problems in hepatocyte transplantation and culture that deserve special consideration and emphasizes the conditions contributing to the in vitro maintenance of phenotypic expression of these cells.     

Fetal and adult liver stem cells for liver regeneration and tissue engineering

For the development of innovative cell-based liver directed therapies, e.g. liver tissue engineering, the use of stem cells might be very attractive to overcome the limitation of donor liver tissue. Liver specific differentiation of embryonic, fetal or adult stem cells is currently under investigation. Different types of fetal liver (stem) cells during development were identified, and their advantageous growth potential and bipotential differentiation capacity were shown. However, ethical and legal issues have to be addressed before using fetal cells. Use of adult stem cells is clinically established, e.g. transplantation of hematopoietic stem cells. Other bone marrow derived liver stem cells might be mesenchymal stem cells (MSC). However, the transdifferentiation potential is still in question due to the observation of cellular fusion in several in vivo experiments. In vitro experiments revealed a crucial role of the environment (e.g. growth factors and extracellular matrix) for specific differentiation of stem cells. Co-cultured liver cells also seemed to be important for hepatic gene expression of MSC. For successful liver cell transplantation, a novel approach of tissue engineering by orthotopic transplantation of gel-immobilized cells could be promising, providing optimal environment for the injected cells. Moreover, an orthotopic tissue engineering approach using bipotential stem cells could lead to a repopulation of the recipients liver with healthy liver and biliary cells, thus providing both hepatic functions and biliary excretion. Future studies have to investigate, which stem cell and environmental conditions would be most suitable for the use of stem cells for liver regeneration or tissue engineering approaches.     

Positive effects of cryopreserved adult or fetal liver cell transplants on hypercholesterolemia and hepatic antioxidant defenses in cholesterol-fed rabbits

The liver plays a central role in lipid metabolism and the pathophysiology of many lipid disorders leads in turn to liver cell injury. Adult hepatocyte transplants provide well-recognized metabolic support, whilst hepatic stem cells may promote liver regeneration and repair, but in both cases, any clinical application would require low temperature banking of the cells. A model of dietary hypercholesterolemia was established in rabbits over 5 months, and transplants of cryopreserved adult hepatocytes (CH) and cryopreserved fetal liver cells (CFLC) were compared to Sham transplants. Cryopreservation was performed by a two-step freezing protocol using 1.5mol/l dimethyl sulfoxide (Me(2)SO). Serum contents of cholesterol lipid classes were measured during the subsequent 4 weeks, in addition to markers of serum and liver oxidative stress. Both CH and CFLC transplantation resulted in a decrease of serum lipids during the 1st week after transplantation. The effect of CH was limited to the 1st week, but CFLC provided a sustained lipid-lowering effect over the 4 weeks. The ultimate outcome of CFLC transplantation by the end of 4 weeks was more pronounced and statistically significant for both serum total cholesterol (0.15+/-0.05 versus 3.65+/-1.4mmol/l) and high-density lipoprotein-cholesterol (0.04+/-0.01 versus 0.56+/-0.06mmol/l) compared to Sham transplants (p<0.05 in both cases). CFLC transplantation also normalized hepatic tissue antioxidant defenses, namely an increase in reduced glutathione content, and enzyme activities for catalase and glutathione reductase (all significantly higher at p<0.05 than in Sham transplants) by 4 weeks.