肝组织工程研究中生物反应器的研究进展

文章就生物反应器的特点、生物反应器在肝组织工程中的应用（肝组织重建、生物人工肝脏、药物筛选、生物反应器在肝组织工程其他相关方面的应用）进行综述,论述目前生物反应器亟待解决的问题是供氧问题、细胞密度和分布及微型化,指出生物反应器作为一个重要的媒介促进肝组织工程及生物人工肝的研究发展：同时肝细胞反应器可有效提高培养肝细胞的生物功能,其也将为肝再生医学的理论研究、肝脏的药物代谢及功能评价等提供有效的技术手段,肝细胞生物反应器的研发具有重要的理论意义和巨大的经济价值。     

生物型人工肝中的生物成份研究进展

暴发型肝功能衰竭病死率极高,肝移植是其首选治疗方法,但是由于肝供体缺乏和时间关系,故应用人工肝脏对病人进行支持治疗作为肝移植过渡的桥梁非常必要.人工肝脏的研究从最初的非生物型发展到现在的生物型,各项技术都有了很大的发展并日趋成熟,其中最引人注目的就是生物反应器的构建,而生物成份则是其替代肝脏功能的核心.从最早应用肝脏组织匀浆、肝脏组织薄片、冻干的肝脏组织颗粒到体外全肝脏交叉灌流,再到分离的肝细胞、肝肿瘤细胞株.生物成份的发展非常迅速,并且一期临床应用试验已经取得成功.本文回顾了生物成份的新进展,并展望其发展前景.     

肝脏干细胞研究及应用前景展望****○

背景：近年肝脏干细胞移植治疗终末期肝病得到人们的广泛关注,但临床应用中却面临来源有限,细胞数量不足,移植后干细胞往往弥散、流失,且分化率低,无法有效发挥其修复功能等技术问题制约。 目的：总结肝脏干细胞组织工程的研究进展,并展望其应用前景。 方法：应用计算机检索1999-01/2010-12 PubMed数据库相关文献,英文检索词“liver stem cell tissue engineering”分别与“three dimensional culture,biodegradable materials,biological reactor”组合,并限定文献语言种类为English。共检索到文献241篇,最终纳入符合标准的文献43篇。 结果与结论：肝脏干细胞来源有限,而肝脏组织工程需要大量可靠的种子干细胞,三维培养是目前发现行之有效的体外扩增手段。在三维培养条件下,使用不同支架材料和反应器扩增效率及移植效果存在巨大差异,如何确定具有肝脏干细胞特异性的理想扩增条件、改善支架生物相容性以提高移植效率是关键技术问题。未来的研究需要进一步探讨肝脏干细胞特异性扩增生物反应器及肝脏干细胞特异性支架构建等技术难点。     

组织工程生物反应器的生物力学

<正>组织工程,是指用工程科学和生命科学的原理和方法,制备组织和器官替代物,以恢复、维持或改善人体组织、器官的功能,是一个发展迅速、意义深远的生物医学工程应用领域。目前,组织工程化皮肤产品已正式进入临床应用,培育的组织工程骨骼、软骨、血管、皮肤以及神经组织正在进行体内实验,组织工程肝脏、胰脏、乳房、心脏、手指、角膜等也可以在实验室里构建生长。组织工程已形成一个发展中的产业。但是目前组织工程距离广泛应用于临床、成为社会经济新的增长点还有相当长的路要走。阻碍组织工程发展和临床应用的主要因素至少包括两点:(1)对调控组织的功能化培养的特定物理-生物化学因素知之还少;(2)高昂的生产成本和缺乏商业化的功能性组织工程产品。     

基于专利分析看肝脏组织工程领域的竞争态势

文题释义：肝脏组织工程：肝脏组织工程基于组织工程概念,应用生命科学与工程学的原理与技术,利用分离的肝细胞复合特定的支架材料,制造出完整的、可植入、且具有更全面功能、可用于临床肝移植的器官。 专利分析：对专利说明书、专利公报中大量零碎的专利信息进行分析、加工、组合, 并利用统计学方法和技巧使这些信息转化为具有总揽全局及预测功能的竞争情报, 从而为技术、产品及服务开发中的决策提供参考。背景：中国是肝病高发国家,肝脏组织工程为治疗带来了新的希望,但目前此技术仍处于探索阶段。 目的：对肝脏组织工程领域的专利进行分析,揭示全球技术竞争态势,为中国肝脏组织工程的发展与创新提供借鉴和参考。 方法：对近20年肝脏组织工程领域的专利数量与发展趋势、技术发源地、目标市场、申请机构、发明人和技术分布等进行分析,全文检索关键词包括组织工程(tissue engineering)、组织修复或组织再生 (tissue regenerate/repair)和肝(hepatic/liver)等。检索截止时间为2018年12月28日。结果与结论：①全球肝脏组织工程领域发展速度较快,近10年复合增长率8.64%,其中发明专利占84.93%。②中国是全球肝脏组织工程最主要的技术发源地。但三方专利数量远不及美国和日本。③中国和美国是肝脏组织工程领域的最受关注的两大目标市场,但申请人多集中于本国,国际授权数量较少。④在专利权人和发明人排名中,中国分别有8家机构和14位申请人跻身全球前20。⑤结果证实,从专利分析结果来看,中国肝脏组织工程技术发展迅猛,跻身国际前列,但缺乏核心技术,各研发主体还应不断提升创新能力,挖掘高价值技术,从而提高国际竞争力。 ORCID: 0000-0002-8864-5449(王婷婷) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

肝衰竭修复替代治疗的现状与发展对策

文题释义：肝衰竭：是指各种原因引起的肝细胞大面积坏死或严重肝功能损害,出现以黄疸、腹水、肝性脑病和凝血功能障碍等为主要表现的一种临床综合征。 组织工程肝脏：利用组织工程新技术构建的由生物材料和种子细胞组成的,可以在一定程度上模拟肝脏的合成、解毒、代谢和分泌等生理性功能的类肝脏组织器官。 背景：为解决肝移植的临床应用受供肝来源短缺限制的问题,世界各国科学家正在积极探索,相继研究发展了人工肝、组织工程肝脏、异种器官移植等技术手段以期解决缓解器官短缺的问题,从而对肝衰竭起到一个修复或替代的治疗作用。 目的：阐述肝衰竭修复替代治疗的发展历程、研究现状和未来预期。 方法：检索web of science、万方数据库2000至2019年发表的文献,检索关键词为“artificial liver,liver tissue engineering,hepatic failure,肝衰竭,肝移植”。 结果与结论：针对肝衰竭等晚期肝脏疾病,主要有原位肝移植、细胞移植、人工肝系统和组织工程肝脏等修复替代治疗手段。国内目前已有多家医院和机构自主研发了人工肝装置,尽管生物型人工肝和混合型人工肝在肝衰竭治疗上展现出很好的发展前景,但大多仍处于动物实验阶段,未来应加大生物反应器的研发力度,增强支架内的细胞活率。利用生物材料和种子细胞构建的组织工程肝脏可以在一定程度上模拟肝脏的合成、解毒、代谢和分泌等生理性功能,可经体内移植治疗终末期肝病,是组织功能领域的研究热点,而解决生物支架体内移植的凝血问题、促进支架的血管化形成是该领域的需要努力的方向。异种移植是解决人类器官供体严重短缺的最佳途径,但该技术距离临床应用依然还有很长的路要走。 ORCID: 0000-0002-7727-550X(郭伟) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

组织工程与生物材料

介绍了组织工程的原理、研究现状,以及相关生物材料的基本概念和生物材料的发展概况.指出目前组织工程的研究为生物材料提供了极大的发展机会,认为可降解生物材料是组织工程用支架材料的研究重点,未来组织工程相关生物材料的发展方向是仿生化和智能化,组织工程学的发展将会促进材料的发展,并将由此产生巨大的社会效益和经济效益.     

肝干细胞分离培养技术的研究进展

干细胞是一种具有自我更新能力和多向分化潜能的细胞,存在于许多组织中;肝干细胞在肝脏的形成和再生中有重要作用,可以分化为肝细胞、胆管上皮细胞和胰腺上皮细胞等.随着对其特性的深入了解,它将代替肝细胞移植和肝脏移植成为治疗器官衰竭的一种重要细胞来源,在体外对肝干细胞进行分离纯化和培养扩增,将对肝的细胞移植、组织工程和基因治疗等有重要作用.     

生物反应器在血管组织工程中的应用及进展

论述生物反应器的种类与发展,以及其在血管组织工程种子细胞培养和组织工程血管构建方面的主要研究进展。根据生物反应器领域的发展,分析了生物反应器对种子细胞培养、扩增的影响,尤其是对干细胞培养、定向分化方面的影响;阐述生物反应器内种植细胞的方法,以及机械力学对细胞生长、黏附的影响;探讨生物力学与血管构建的关系。最后提出生物反应器未来的发展趋势。     

修复运动性肌腱损伤组织工程化肌腱及种子细胞和支架材料

背景：获得大规模、具有再生活力的种子细胞以及具有与正常人体肌腱组织相接近的力学性能的理想支架材料是当前组织工程化肌腱研究面临的最为关键的限制性因素。 目的：总结和分析组织工程肌腱研究中的种子细胞和支架材料的研究进展。 方法：查阅近年来肌腱组织工程研究的相关文献,综合国内外最新研究成果,就肌腱组织工程中合适的种子细胞来源、研究更为理想的支架材料及组织相容性等方面的进展进行概述。 结果与结论：肌腱组织工程中常用的种子细胞有间充质干细胞、肌腱干细胞及胚胎干细胞等,可以向骨、软骨和脂肪分化,修复肌腱损伤的理想细胞。肌腱组织工程支架材料有天然材料及人工合成材料等,肌腱组织工程支架材料应有良好的生物相容性和适度的机械性能,复合材料将是肌腱组织工程支架材料研究的重点。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程      

Hepatic fibrosis: physiopathology and biological diagnosis

Hepatic fibrosis, of which cirrhosis is the most advanced stage, can result from any chronic liver disease due to any cause. Normal extracellular matrix components accumulate in the liver as a result of imbalances in their production, deposition, and breakdown. These matrix components are produced primarily by myofibroblastic hepatic cells whose main cellular source in the liver is the stellate cell (Ito cell). Histopathological examination of a liver biopsy specimen is currently the gold-standard investigation for estimating the severity of fibrosis in patients with chronic liver disease. However, the recent development of treatments with activity against the fibrosing process requires evaluation of fibrosis at closely spaced intervals and, therefore, the development of tests that do not require a liver biopsy. Advances in our understanding of the mechanisms underlying hepatic fibrogenesis have allowed to identify several substances of potential clinical usefulness. Serum assays of extracellular matrix components, their breakdown products, or enzymes involved in their metabolism have been suggested for the noninvasive evaluation of hepatic fibrosis. Clinical studies have shown that serum hyaluronate is to date the marker with the closest correlations to hepatic fibrosis and the noninvasive parameter with the highest sensitivity for cirrhosis.     

受体相互作用蛋白激酶调控非酒精性脂肪肝中细胞程序性坏死的研究进展

非酒精性脂肪性肝病（NAFLD）是一种常见的慢性肝病,如果得不到有效控制,则会进一步发展为非酒精性脂肪性肝炎（NASH）,进而引起肝纤维化、肝硬化,甚至癌变。程序性坏死是近年来发现的一种新型细胞程序性死亡方式,由受体相互作用蛋白激酶（RIPK）介导所致,最终可以导致细胞膜溶解破裂,引发炎症。RIPK家族作为细胞内和细胞外应激的重要传感器,诱导调控程序性坏死的发生,并参与炎症及其他免疫反应。近年来研究表明,RIPK调控的程序性坏死在非酒精性脂肪性肝病的发生发展中具有重要作用,在动物NAFLD/NASH模型中,RIPK的表达情况与肝脂肪变性程度密切相关。在一些临床研究中亦观察到,NAFLD/NASH患者比健康人RIPK表达水平上升。但程序性坏死到底是加速肝病进程的因素,还是肝病发展过程中的保护因素,仍然没有定论。有研究表明,RIPK抑制剂可能为NAFLD治疗提供方向。我们综述了程序性坏死的分子机制及与非酒精性脂肪性肝病的关系,以及RIPK在其中扮演的重要角色,并总结了其在NAFLD/NASH治疗方面的研究进展,为进一步探究其机制,探索新的治疗手段提供理论依据。     

MHC-independent genetic regulation of liver damage in a mouse model of autoimmune hepatocellular injury

Autoimmune hepatitis (AIH) is mediated by a T-cell attack upon liver parenchyma. Susceptibility to the development of AIH is genetically determined. While particular MHC haplotypes are known risk factors, it has been widely speculated that autoimmune liver damage can be regulated by additional genetic loci unlinked to MHC. However, evidence for the existence of such loci in humans is scant. We examined the contribution of the MHC in a murine model of autoimmune hepatocellular injury. BALB/c mice lacking the immunoregulatory cytokine transforming growth factor-beta1 (TGF-beta1) rapidly develop autoimmune T-helper 1-mediated necroinflammatory liver disease. Susceptibility to liver damage is strictly regulated by genetic background. Whereas TGF-beta1-deficient mice on the BALB/c background develop necroinflammatory liver disease, TGF-beta1-deficient mice on the 129/CF-1 genetic background do not. We asked whether MHC locus haplotype is the principal determinant of genetic susceptibility to liver disease in this model system. BALB/c mice harbor the H-2d haplotype. We used a 'haplotype swapping' approach to generate H-2b or H-2k congenic BALB-background TGF-beta1-deficient mice. In addition, F1 (BALB/c x 129/CF-1)-TGF-beta1-deficient mice were generated. As determined by plasma transaminase levels and histopathology, severe necroinflammatory liver disease developed in all BALB-background TGF-beta1-deficient mice, regardless of H-2 haplotype, but developed neither in 129/CF-1-TGF-beta1-deficient mice nor in F1 (BALB/c x 129/CF-1)-TGF-beta1-deficient mice. Thus, H-2d is neither necessary nor sufficient for the development of necroinflammatory liver disease in BALB-background TGF-beta1-deficient mice. This constitutes the first direct evidence that susceptibility to autoimmune hepatocellular damage, at least in mice, can be determined by genetic loci distinct from the MHC.     

Hepatitis mouse models: from acute‐to‐chronic autoimmune hepatitis

Autoimmune hepatitis (AIH) is a chronic inflammatory liver disease associated with interface hepatitis, raised plasma liver enzymes, the presence of autoantibodies and regulatory T‐cell (Tregs) dysfunction. The clinical course is heterogeneous, manifested by a fulminant or indolent course. Although genetic predisposition is well accepted, the combination with currently undefined environmental factors is crucial for the development of the disease. Progress in the development of reliable animal models provides added understanding of the pathophysiology of AIH, and these will be very useful in evaluating potential therapeutics. It appears that artificially breaking tolerance in the liver is easy. However, maintaining this state of tolerance breakdown, to get chronic hepatitis, is difficult because liver immune homeostasis is strongly regulated by several immune response inhibitory mechanisms. For example, Tregs are crucial regulators in acute and chronic hepatitis, and C57BL/6 mice are most prone to experimental AIH. Immunization of C57BL/6 mice with liver (AIH) autoantigens (CYP2D6/FTCD or IL‐4R) and the disturbance of liver regulatory mechanism(s), leading to experimental AIH, are likely to be most representative of human AIH pathology.     

Hepatic Fibrosis and the Microenvironment: Fertile Soil for Hepatocellular Carcinoma Development

Hepatocellular carcinoma is an emerging worldwide health threat that has few curative treatment options and poor overall survival. Progressive hepatic fibrosis is a common pathway for all forms of chronic liver disease and is closely linked epidemiologically to hepatocellular carcinoma risk. However, the molecular events that predispose a fibrotic liver to cancer development remain elusive. Nonetheless, a permissive hepatic microenvironment provides fertile soil for transition of damaged hepatocytes into hepatocellular carcinoma. Key predisposing features include alterations in the extracellular matrix, bidirectional signaling pathways between parenchymal and nonparenchymal cells, and immune dysfunction. Emerging research into the contributions of autophagy, tumor-associated fibroblasts, and hepatocellular carcinoma progenitor cells to this dangerous milieu also provides new mechanistic underpinnings to explain the contribution of fibrosis to cancer. As effective antifibrotic therapies are developed, these approaches could attenuate the rising surge of hepatocellular carcinoma associated with chronic liver disease.     

Oval cell numbers in human chronic liver diseases are directly related to disease severity

The risk of developing hepatocellular carcinoma is significantly increased in patients with genetic hemochromatosis, alcoholic liver disease, or chronic hepatitis C infection. The precise mechanisms underlying the development of hepatocellular carcinoma in these conditions are not well understood. Stem cells within the liver, termed oval cells, are involved in the pathogenesis of hepatocellular carcinoma in animal models and may be important in the development of hepatocellular carcinoma in human chronic liver diseases. The aims of this study were to determine whether oval cells could be detected in the liver of patients with genetic hemochromatosis, alcoholic liver disease, or chronic hepatitis C, and whether there is a relationship between the severity of the liver disease and the number of oval cells. Oval cells were detected using histology and immunohistochemistry in liver biopsies from patients with genetic hemochromatosis, alcoholic liver disease, or chronic hepatitis C. Oval cells were not observed in normal liver controls. Oval cell numbers increased significantly with the progression of disease severity from mild to severe in each of the diseases studied. We conclude that oval cells are frequently found in subjects with genetic hemochromatosis, alcoholic liver disease, or chronic hepatitis C. There is an association between severity of liver disease and increase in the number of oval cells consistent with the hypothesis that oval cell proliferation is associated with increased risk for development of hepatocellular carcinoma in chronic liver disease.     

Hepatocellular carcinoma in a mouse model fed a choline‐deficient,L‐amino acid‐defined,high‐fat diet

Hepatocellular carcinoma (HCC) is a common cancer worldwide and represents the outcome of the natural history of chronic liver disease. The growing rates of HCC may be partially attributable to increased numbers of people with non‐alcoholic fatty liver disease (NAFLD) and non‐alcoholic steatohepatitis (NASH). However, details of the liver‐specific molecular mechanisms responsible for the NAFLD–NASH–HCC progression remain unclear, and mouse models that can be used to explore the exact factors that influence the progression of NAFLD/NASH to the more chronic stages of liver disease and subsequent HCC are not yet fully established. We have previously reported a choline‐deficient, L‐amino acid‐defined, high‐fat diet (CDAHFD) as a dietary NASH model with rapidly progressive liver fibrosis in mice. The current study in C57BL/6J mice fed CDAHFD provided evidence for the chronic persistence of advanced hepatic fibrosis in NASH and disease progression towards HCC in a period of 36 weeks. When mice fed CDAHFD were switched back to a standard diet, hepatic steatosis was normalized and NAFLD activity score improved, but HCC incidence increased and the phenotype of fibrosis‐associated HCC development was observed. Moreover, when mice continued to be fed CDAHFD for 60 weeks, HCC further developed without severe body weight loss or carcinogenesis in other organs. The autochthonous tumours showed a variety of histological features and architectural patterns including trabecular, pseudoglandular and solid growth. The CDAHFD mouse model might be a useful tool for studying the development of HCC from NAFLD/NASH, and potentially useful for better understanding pathological changes during hepatocarcinogenesis.     

Research progress on hepatic machine perfusion

Nowadays, liver transplantation is the most effective treatment for end-stage liver disease. However, the increasing imbalance between growing demand for liver transplantation and the shortage of donor pool restricts the development of liver transplantation. How to expand the donor pool is a significant problem to be solved clinically. Many doctors have devoted themselves to marginal grafting, which introduces livers with barely passable quality but a high risk of transplant failure into the donor pool. However, existing common methods of preserving marginal grafts lead to both high risk of postoperative complications and high mortality. The application of machine perfusion allows surgeons to make marginal livers meet the standard criteria for transplant, which shows promising prospect in preserving and repairing donor livers and improving ischemia reperfusion injury. This review summarizes the progress of recent researches on hepatic machine perfusion.     

Treatment of viral hepatitis

The primary goals of treatment for viral hepatitis are to eradicate the infection early in the course of the disease, to prevent progression to end-stage liver disease, and to prevent the development of hepatocellular carcinoma. The secondary goal is to decrease the number of chronic carriers who serve as a reservoir for viral transmission. The major step in the management of hepatitis B was the introduction of successful vaccination. The treatment of hepatitis B includes nucleoside analogues (lamivudine and adefovir) and interferon. The major problem in lamivudine treatment is the development of resistant viral mutants. Hepatitis D can worsen hepatitis B and lead to progressive liver disease. The recommended treatment is interferon. Hepatitis C, due to the lack of vaccine and tendency to develop chronic liver disease, is the major health problem and the leading indication for liver transplantation today. The recommended treatment is the combination of pegylated interferon and ribavirin.     

Comparison of blood parameters in degenerative liver disease and liver neoplasia in dogs

The evaluation of blood parameters is the first step in the diagnosis of liver disease. In many cases the blood parameters indicate only cell destruction or disturbances of liver cell synthesis. It is impossible for one to make a diagnosis by blood parameters only. In our investigation we compared the liver parameters ALT, ALP, GLDH, albumin, bile acid and total bilirubin in dogs with severe degenerative liver disease and in dogs with malignant liver tumours. The aim of our investigation was to find differences between the blood parameters in both liver diseases. We found no significant difference in parameters between degenerative liver disease and liver neoplasia. However, in our investigation, compared with other reports, the mean levels of ALT, ALP, albumin and bilirubin were more often altered in liver neoplasia than in degenerative liver disease. Thus, we conclude that disturbances of liver cell integrity and function occur more often in liver tumours than in severe degenerative liver disease.