体外培养人胎肝细胞生物学功能及免疫细胞的测定

目前,人胎肝细胞已用于治疗肝病,但存在着肝细胞来源困难和免疫排斥等问题。为此我们对人胎肝细胞进行了体外培养观察,并对其生长及生物活性的变化进行了测定。 取4～6个月胎龄水囊引产胎儿肝脏,机械研磨法制备单个胎肝细胞悬液,用完全1640     

Toll样受体在肝脏疾病中的功能

Toll样受体(Toll-like receptors,TLR)是一类可以识别病原体并迅速启动天然免疫反应的跨膜蛋白,它们也可以调节机体的获得性免疫及组织的炎症反应,是机体感知、抵御及清除病原体的关键分子。近来发现TLR在多种肝脏疾病的发生、发展及恢复过程中起着重要的调节作用,这方面的研究为许多慢性肝病的治疗提供了新的线索。该文综述了TLR在酒精性肝病、脂肪肝、病毒性肝炎、肝硬化以及肝细胞癌的病理生理学中的作用,展望了将来需重点研究的问题。     

血管新生在肝脏疾病发生与发展中作用的研究进展

血管新生是在现有血管的基础上,通过多种细胞因子的调控进而形成新的毛细血管的过程。近年来研究发现,血管新生不仅在组织修复过程中发挥其重要作用,而且在肝脏疾病的发生发展中扮演着重要的角色。本文就血管新生的概念以及其在各类肝脏疾病,如:酒精性肝病、非酒精性脂肪肝炎、病毒性肝炎、肝纤维化、肝细胞癌中的作用机制及研究进展作一综述,旨在为肝病的预防及治疗提供新的思路,并且为药物的开发提供一定的理论支持。     

极化上皮细胞中转运蛋白的分选

上皮组织细胞必须极化其表面区域以执行其转运生理功能。不同膜转运蛋白定位于细胞膜的不同区域,而细胞与细胞之间则须通过紧密连接复合体紧密连接成极化区域,并调节旁细胞途径的通透性。精密的机体要求上皮细胞具备一个筛选装置,用于将新合成的转运蛋白定位于合适的表面区域;转运蛋白本身也必须内含规定其功能位置的分选信号。目前上皮细胞蛋白分选和蛋白质之间相互作用已被逐渐阐明。上皮细胞通过细胞信号转导途径形成极化初始状态,将自己定位于特定位置,调节细胞与细胞之间、细胞与基质之问的相互作用。最近研究发现其信号转导通路的一个成员是一种AMP激活的蛋白激酶（AMP-stimulated protein kinase．AMPK）,它也是细胞能量感受器。     

肝细胞的异质性：葡萄糖和酮体在大鼠肝脏的代谢

用大鼠肝脏门静脉或肝静脉周围的肝细胞来研究葡萄糖和酮体生成的区域分布。肝细胞通过毛地黄皂苷－胶原酶灌流技术分离。门静脉周围肝细胞的ｒ谷氨酰转肽酶的活性比肝静脉周围肝细胞高２．４倍;而谷氨酰胺合成酶的活性则相反,肝静脉周围肝细胞高出５６倍。门静脉周围肝细胞的内源性葡萄糖合成比肝静脉周围肝细胞高１．５７倍。给予刺激葡萄糖异性的底物,门静脉周围肝细胞的葡萄糖合成则增加１．７－２．１倍。肝静脉周围肝细胞的     

干细胞在肝脏疾病治疗中的临床应用及分子机制

干细胞是指一群具有自我更新和多向分化潜能的细胞,是最有治疗潜力的细胞资源,已成为再生医学领域的研究热点。目前,已有多种干细胞用于肝脏疾病的治疗,能有效改善患者血清指标,减少并发症发生,并提高生活质量。这些干细胞在细胞来源、移植途径及治疗效果等多个方面各有特点,但其治疗肝脏疾病的机制尚不清楚。本文将对目前已用于肝病治疗的各种干细胞的临床应用以及可能的分子机制进展进行阐述。     

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。     

间充质干细胞在肝脏疾病应用的研究现况

间充质干细胞（MSC）属于成体干细胞的一种,是一类具有自我更新和多向分化能力的多能干细胞。其来源丰富,免疫原性低,目前体内外实验均发现MSC可促进损伤肝脏修复,改善症状,提高存活率。通过调节肝脏局部和全身炎症反应和免疫紊乱发挥治疗作用。本文就MSC治疗肝脏疾病的研究现况进行综述。     

肝细胞的异质性：葡萄糖和酮体在大鼠肝脏的代谢

用大鼠肝脏门静脉或肝静脉周围的肝细胞来研究葡萄糖和酮体生成的区域分布。肝细胞通过毛地黄皂苷－胶原酶灌流技术分离。门静脉周围肝细胞的γ谷氨酰转肽酶的活性比肝静脉周围肝细胞高２．４倍;而谷氨酰胺合成酶的活性则相反,肝静脉周围肝细胞高出５６倍。门静脉周围肝细胞的内源性葡萄糖合成比肝静脉周围肝细胞高１．５７倍。给予刺激葡萄糖异生的底物,门静脉周围肝细胞的葡萄糖合成则增加１．７－２．１倍。肝静脉周围肝细胞的内源性酮体生成比门静脉周围肝细胞高１．３倍。给予能明显刺激酮体生成的辛酸盐,肝静脉周围肝细胞的酮体生成仅略为增加。我们的结果证实,在基础和刺激的条件下,葡萄糖的异生在门静脉周围肝细胞中优先,而酮体生成仅在肝静脉周围肝细胞占微弱的优势。     

肝细胞生长因子Kringle的三维结构模建及与功能的关系

利用同源模建的方法模拟得到了肝细胞生长因子4个Kringle域的三维结构。结果表明,HGFKringle与纤溶酶原Kringle的氨基酸序列具有较高的同源性,其功能区附近的序列比较保守。HGF的Kringlel和3与其它具有Lys结合功能的Kringle相比,功能区的残基发生了变化,可能丧失了结合Lys的功能,而2和4仍具有一定的该功能。根据Kringle 1的模建结构,推测该Kringle功能区的结构为一个通道,该通道的底部和一侧有部分疏水残基,同时两侧还分布着少量酸性或碱性残基,该通道可能具有结合特定肽链的功能,从而与Kringle 2一起实现HGF与受体结合的作用。     

Growth potential of adult hepatocytes in mammals: highly replicative small hepatocytes with liver progenitor-like traits

The liver is one of the few organs that is capable of completely regenerating itself without using a stem cell population. When damaged, growth factors and cytokines are released, stimulating terminally differentiated adult hepatocytes and making them re-enter the cell cycle. We have been developing a series of studies on the growth potential of rat and human hepatocytes to identify a population of hepatocytes that is responsible for the regeneration of the injured liver. For this purpose, we established an appropriate culture method for hepatocytes by which growth and differentiation capacities are practically examined under various experimental conditions. This in vitro assay system allows us to identify small hepatocytes that are prominently replicative compared to large hepatocytes. Non-parenchymal cells play critical roles in the proliferation of small hepatocytes. These hepatocytes are present in both rat and human liver and are located in portal regions there. Phenotypic features were examined at morphological and gene/protein levels in detail, which showed the phenotypic plasticity in vitro. Mammalian liver includes a population of small hepatocytes in normal adults with a minute occupancy rate. We speculate that small hepatocytes play a role in regenerating the injured liver and in compensating for apoptotic hepatocytes in the physiological turnover of hepatocytes.     

Reinterpreting polarity and cancer: The changing landscape from tumor suppression to tumor promotion

Cell polarity is a fundamental property used to generate asymmetry and structure in all cells. Cancer is associated with loss of cell and tissue structure. While observations made in model system such as Drosophila, identify polarity regulators as tumor suppressors that cause inappropriate cell division, studies in mammalian epithelia do not always support such a causative contribution. Our analysis of published cancer dataset shows that many polarity genes, including PARD6B, SCRIB, PRKCI, DLG1, DLG2, DLG5 and LLGL2, are frequently amplified in multiple cancers raising the possibility that mammalian epithelia may have evolved to use polarity proteins in multiple ways where they may have tumor promoting functions. In this review, we reinterpret the published results and propose a modified perspective for the role of polarity regulators in cancer biology. In addition to the traditional form of cell polarity, which is involved establishment of maintenance of normal cell structure and asymmetry, we propose that some mammalian polarity proteins also regulate subcellular polarity (intracellular asymmetry), which can improve cellular fitness to carry out functions such as proliferation, apoptosis, stress adaptation, stemness and organelle biology. Here, we define subcellular polarity and discuss evidence that supports a role for subcellular polarity in biology.     

Hepatocyte culture in bioartificial livers with different membrane characteristics

Rat hepatocytes were cultured in three polysulfone, hollow-fiber cartridges, characterized by two membrane variables: pore size and inner diameter (ID). Hepatocytes entrapped in a micro-filtration (MF) cartridge with the membrane pore size 0.1 microm had twice the production of urea and 4-fold the amount of albumin in comparison to the control cartridge, a ultra-filtration (UF) cartridge with a molecular weight cut-off (MWCO) of 100 kDa. Hepatocytes entrapped in a UF cartridge with ID of 0.5 mm secreted twice the amount of urea and 10-fold the amount of albumin compared with the control UF cartridge.     

Culturing of primary hepatocytes as entrapped aggregates in a packed bed bioreactor: A potential bioartificial liver

Summary Conventional culture systems for hepatocytes generally involve cells cultured as flat, monolayer cells, with limited cell-cell contact, in a static pool of medium, unlike the liver in vivo where the parenchymal cells are cuboidal, with extensive cell-cell contact, and are continuously perfused with blood. We report here a novel bioreactor system for the culturing of primary hepatocytes with cuboidal cell shape, extensive cell-cell contact, and perfusing medium. The hepatocytes were inoculated into the bioreactor and allowed to recirculate at a rate optimal for them to collide and form aggregates. These newly-formed aggregates were subsequently entrapped in a packed bed of glass beads. The bioreactor was perfused with oxygenated nutrient medium, with controlled oxygen tension, pH, and medium perfusion rate. The hepatocytes were viable for up to the longest time point studied of 15 days in culture based on urea synthesis, albumin synthesis and cell morphology. Light microscopy studies of hepatocytes cultured for 15 days in the bioreactor showed interconnecting three-dimensional structures resembling the hepatic cell plate in the liver organ. Electron microscopy studies on the same cells revealed ultrastructure similar to the hepatocytes in vivo, including the presence of plentiful mitochondria, rough and smooth endoplasmic reticulum, glycogen granules, peroxisomes, and desmosomes. We believe that our hepatocyte bioreactor is a major improvement over conventional culture systems, with important industrial applications including toxicology, drug metabolism, and protein/peptide synthesis. The hepatocyte bioreactor concept may also be used as the basis for the development of a bioartificial liver to provide extracorporeal hepatic support to patients with hepatic failure.     

Fractionation of Rat Hepatocyte Subpopulations with Varying Metabolic Potential, Proliferative Capacity, and Retroviral Gene Transfer Efficiency

The liver contains hepatocytes with varying ploidy and gene expression. To isolate cells on the basis of ploidy for analyzing mechanisms concerning cell proliferation and differentiation, we used Percoll gradients to separate F344 rat hepatocyte subpopulations. Specific fractions were enriched in polyploid (H2 fraction) or diploid (H3 and H4 fractions) hepatocytes containing glycogen and glucose-6-phosphatase. H4 cells were relatively smaller with greater nuclear/cytoplasmic ratios, less complex cytoplasm, and higher serum albumin or ceruloplasmin biosynthetic rates. H2 fraction cells were larger with lesser nuclear/cytoplasmic ratio, more complex cytoplasm, and more cytochrome P450 activity. Phenotypic marking showed that H4 cells originated in zone one and H2 cells in zones two or three of the liver lobule. H4 cells showed much greater mitogenic responsiveness to human hepatocyte growth factor. Retroviral gene transfer, which requires both viral receptors and cellular DNA synthesis, was significantly more efficient in H4 cells. The findings indicated thatsmalldiploid andlargepolyploid hepatocytes show unique biological differences. The ability to isolate hepatocytes of varying maturity is relevant for mechanisms concerning liver growth control and hepatic gene expression.     

Role of cell polarity and planar cell polarity (PCP) proteins in spermatogenesis

Abstract

Studies on cell polarity proteins and planar cell polarity (PCP) proteins date back to almost 40?years ago in Drosophila and C. elegans when these proteins were shown to be crucial to support apico-basal polarity and also directional alignment of polarity cells across the plane of an epithelium during morphogenesis. In adult mammals, cell polarity and PCP are most notable in cochlear hair cells. However, the role of these two groups of proteins to support spermatogenesis was not explored until a decade earlier when several proteins that confer cell polarity and PCP proteins were identified in the rat testis. Since then, there are several reports appearing in the literature to examine the role of both cell polarity and PCP in supporting spermatogenesis. Herein, we provide an overview regarding the role of cell polarity and PCP proteins in the testis, evaluating these findings in light of studies in other mammalian epithelial cells/tissues. Our goal is to provide a timely evaluation of these findings, and provide some thought provoking remarks to guide future studies based on an evolving concept in the field.     

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.