肝脏细胞分化和成熟的分子调控机制

肝脏是重要的代谢调控和药物解毒器官,执行体内多种生理功能。肝脏疾病已经越来越严重地影响着人体健康和生存质量。考虑到临床研究和转化医学的迫切需求,人们必须深入研究肝脏内各种细胞特别是肝实质细胞和胆管细胞的分化成熟过程及分子调控机制。该文概述了肝脏内起源于内胚层的肝实质细胞和胆管分化成熟的发育过程,总结了调控此过程的信号通路和转录因子,并简要介绍了最新技术对于肝脏发育研究的推动作用。这些结果对于人们在体外高效地诱导得到或建立更成熟、结构功能更完善的肝脏样细胞或肝脏类器官以及肝脏疾病的研究与治疗有重要意义。     

小鼠精原细胞分化的蛋白质组学研究

对小鼠睾丸中未分化(Undifferentiated)和分化中(Differentiating)的两类精原细胞进行定量蛋白质组研究,探讨两类精原细胞蛋白质组的表达差异,探索与精原细胞分化相关的蛋白质。利用Thy1和c-Kit两个特异抗体结合的磁珠分选技术,分别将生后7 d雄性小鼠睾丸中的Thy1+细胞和c-Kit+细胞分别作为未分化和分化中的精原细胞分选出来,其中Thy1阳性细胞3组,c-Kit阳性细胞4组,分别代表了未分化精原细胞和分化中的精原细胞。采用高效液相色谱串联质谱方法(LC-MS/MS)分析两类细胞蛋白表达差异。并且对两类细胞的差异蛋白进行基因本体(Gene ontology,GO)功能注释、KEGG代谢通路和聚类分析。质谱分析共鉴定了3228种蛋白,其中有256种蛋白在两类细胞中表达差异。其中,差异蛋白的富集分析发现,在生物过程方面,注释结果显示差异蛋白主要在代谢过程(Primary metabolic process),细胞代谢过程(Cellular metabolic process),分子代谢过程(Macromolecule metabolic process)和氮化合物代谢过程(Nitrogen compound metabolic process)中富集;在细胞组分方面,蛋白主要富集在细胞(Cell part)、细胞内组分(Intracellular part)和细胞器(Intracellular organelle)中;在分子功能方面,鉴定到的蛋白主要参与蛋白结合(Protein binding)、核苷结合(Nucleotide binding)、水解酶活性(Hydrolase activity)和核酸结合过程(Nucleic acid binding)。基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)通路注释结果显示:88个蛋白质在KEGG通路分析数据库中有功能注释,共参与了18条代谢通路,其中,最主要的代谢通路是剪接体(Spliceosome)和泛素介导的蛋白水解作用(Ubiquitin mediated proteolysis)。获得小鼠睾丸中未分化和分化中的两类精原细胞蛋白质组表达谱,揭示精原细胞分化的蛋白质组的组成、筛选出差异蛋白。     

小蘖碱抑制游离脂肪酸诱导小鼠肝实质细胞脂肪变性

目的:探讨小蘖碱(Berberine)对游离脂肪酸(free fatty acids,FFAs)诱导的小鼠肝实质细胞脂肪变性的影响。方法:胶原酶灌注分离BALB/c小鼠原代肝实质细胞并体外培养。分对照组,高脂组,高脂加小蘖碱处理组。体外测定细胞内甘油三酯的含量。利用油红染色观察细胞的脂肪样变性。通过Western印迹法检测肝实质细胞内MAPK相关信号通路磷酸化的变化。实时定量PCR检测肝实质细胞中与脂肪化密切相关的mi R-122的表达和相关靶基因的表达改变。结果:与高脂组比较,小蘖碱处理组肝实质细胞内甘油三酯含量降低,脂肪颗粒减少,脂肪变性明显改善,并具有明显的剂量效应,小蘖碱能够抑制FFAs诱导的JNK通路磷酸化。Q-PCR结果表明小蘖碱能够促进肝实质细胞内mi R-122的表达,并降低脂肪化相关基因Dgat2的表达。结论:小蘖碱能够显著改善高脂诱发的肝脂肪变性,抑制JNK通路磷酸化,其机制可能同mi R-122通路相关。     

对去血清后HITASY细胞分子表达及表型分析

以人血管平滑肌细胞克隆株HITASY为实验材料,探讨HITASY细胞分子表达与表型转换间的关系,为阐明血管新生内膜形成及再狭窄病理机制提供实验依据.实验表明,在含血清或去血清培养条件下,平滑肌细胞于体外发生表型转换.去血清后细胞外基质蛋白合成中止,增殖及移行能力趋于降低,细胞特异性标志物平滑肌α肌动蛋白、肌球蛋白重链及钙调结合蛋白等的表达随去血清时间延长而增加.进一步实验证实,在血管活性介质作用下,胞液钙离子浓度骤增产生膜信号级联反应,引发细胞面积减小而显示收缩功能.上述处于分化表型的细胞经补加血清后其表型特征又恢复到原有去分化型,提示体外培养人平滑肌细胞可发生表型转换.为验证去血清诱导表型转换过程中相关基因的表达变化,用差异显示PCR筛选出E1A激活基因阻遏子,在细胞处于分化表型时表达上调并对细胞增殖伴有较强抑制作用.     

组蛋白去乙酰化酶抑制剂影响的代谢相关基因的组学筛查及验证

组蛋白去乙酰化酶(Histone deacetylases, HDACs)催化组蛋白去乙酰化,与细胞增殖、分化及凋亡等诸多过程密切相关。HDAC抑制剂(HADC inhibitors, HADCIs)具有潜在的抗肿瘤作用,是近年药物筛查的热点之一。近期研究提示HDAC2可通过影响细胞代谢过程发挥抗肿瘤作用,但各类HDACIs调控代谢过程的机制尚待研究。本研究以肝细胞系(HepG2)为研究对象,整合比较了两种HDACIs(TSA和SAHA)的表达谱数据。在TSA处理组中,筛查到380个差异表达基因(DEGs)及35个DEGs富集的KEGG通路;SAHA处理组的表达分析印证了大多数DEGs(177/380)和富集通路(23/35)。比较分析发现,在这两类HDACIs共同影响的通路中,近一半通路(9/23)与代谢有关;近1/3共享DEGs(66/177)参与代谢过程。通过HDAC2 siRNA细胞实验证实了TSA和SAHA对代谢基因的影响。本研究结果显示HDACIs在治疗肿瘤等代谢性疾病方面具有潜在的应用 价值。     

M-CSF诱导人骨髓间充质干细胞分化为肝样细胞的分子机制

本研究主要目的是明确M-CSF诱导骨髓间充质干细胞分化为肝样细胞的分子机制,为临床中的肝移植和治疗肝病提供新思路。对取自于本院骨科治疗的患者的股骨骨髓间充质干细胞进行提取、分离、传代培养及鉴定。流式细胞仪检测BMSCs的表面表型。为了诱导BMSCs的肝分化,本研究将BMSCs加入到培养基中。骨髓间充质干细胞诱导21 d后,BMSCs表达了肝细胞特异性标志物a-蛋白(AFP)和细胞角蛋白18(CK18),通过免疫荧光染色证实了分化与为分化的BMSCs表达的差异性。分化的BMSCs还显示了肝细胞的体外功能特征,包括白蛋白产生、尿素分泌和糖原储存。本研究结果表明,BMSCs在M-CSF诱导下可分化为功能性肝细胞样细胞,可作为肝病治疗的细胞来源。     

人胚胎干细胞和拟胚体基因表达谱的初步分析

利用人类全基因组Affymetrix芯片检测人胚胎干细胞与其自发分化7d的拟胚体之间的差异表达基因.结果显示:与未分化的人胚胎干细胞相比.在分化7d的拟胚体中表达下调2倍及以上的已知和未知基因共有1100个,表达上调2倍及以上的已知或未知基因共有2283个.利用Gostat对这些差异表达基因进行功能分析,发现它们分别与细胞的生物代谢过程、信号传导通路、系统发育、细胞分化、分子功能及亚细胞组分相关.胚胎干细胞具有自我更新能力,是研究早期胚胎发育理想的细胞模型,因此对差异表达基因的功能研究有助于了解维持人胚胎干细胞自我更新的分子机制以及胚胎发育早期的分子事件.     

少突胶质细胞发育分化的转录调控

少突胶质细胞(OL)的成熟分化是中枢神经髓鞘形成的关键环节.在少突胶质细胞形成、增殖、分化、成熟的发育过程中,骨形态发生蛋白(BMP)、sonic hedgehog (Shh)、Notch等三种信号通路发挥了重要的调控作用.这些通路最终激活或抑制下游一系列特异性转录因子(TFs)而完成对OL谱系特异性标志基因表达的激活.各种TFs在特定条件下的时空表达和相互作用是OL发生、发育、分化的重要调控方式.     

牛精原干细胞体外分化潜能的分析

家畜精原干细胞操作的核心技术是精原干细胞(spermatogonial stem cells,SSCs)的长期培养。至今家畜精原干细胞的长期培养方法还没有完善。其中的一个原因是缺乏简便有效的家畜精原干细胞生物活性鉴定手段。该研究的目的是建立一种牛精原干细胞生理潜能的体外分析方法,用于体外培养的牛精原干细胞的生物活性鉴定。首先培养牛精原干细胞,进而用干细胞因子(stem cell factor,SCF)对体外长期培养的牛精原干细胞进行诱导分化。在诱导分化过程中对细胞进行显微观察,并且用免疫荧光染色法鉴定分化的细胞。观察结果显示,经过诱导培养8 d后,牛精原干细胞形态发生了明显改变;细胞的运动行为显示出精母细胞特有的特征。免疫荧光染色结果显示,分化的细胞表达精母细胞的标记基因Scp3。上述结果例证了体外培养的牛精原干细胞具有分化为精母细胞的潜能。该研究建立了牛SSCs体外诱导分化的分析方法,用于鉴定体外培养的牛SSCs的生理潜能。但是体外诱导培养条件不能满足牛SSCs减数分裂的全部要求,导致出现一些不完全符合精母细胞特征的现象。诱导分化培养液尚需改进。     

Role of glucocorticoids and resident liver macrophages in induction of tyrosine aminotransferase

Administration of cortisol to an animal induces tyrosine aminotransferase (TAT) in the liver. A similar effect was observed after stimulation of resident liver macrophages (Kupffer cells) by dextran sulfate. Actinomycin D completely blocks enzyme induction both by cortisol and dextran sulfate, whereas their combined effect gives an additive result. In primary culture of hepatocytes, dextran sulfate inhibits TAT activity, but conditioned macrophage medium reliably increases enzyme activity in hepatocytes. However, incubation of isolated macrophages in the presence of dextran sulfate and such medium transfer into hepatocyte culture results in even more pronounced increase in TAT activity. In a combined culture of hepatocytes and non-parenchymal liver cells, reproducing intercellular interactions in vitro, cortisol and non-parenchymal cells exhibit an additive effect on TAT activity. These results show that liver macrophages release a factor of unknown nature launching the mechanism of TAT induction independently of cortisol, a classic TAT inducer.     

Stimulative effect of non-parenchymal liver cells on ability of tyrosine aminotransferase induction in hepatocytes

Hepatocytes and non-parenchymal liver cells were isolated from adult rat liver and co-cultured for 48 hours as a monolayer on polystyrene culture dishes. The ability of tyrosine aminotransferase (TAT) induction in hepatocytes was examined in the presence of dexamethasone and dibutyryl cAMP. Non-parenchymal cells greatly enhance the ability of TAT induction of hepatocytes. A soluble factor with molecular weight of more than 10,000 is responsible for this enhancement, because conditioned medium prepared from non-parenchymal cells is also stimulatory. Non-parenchymal cells restored the ability in hepatocytes damaged with the addition of D-galactosamine. Conditioned medium prepared from non-parenchymal cells treated with D-galactosamine had higher activity of enhancement than the medium from normal cells. The soluble factor might be released in response to some signal of injury. Hepatocytes and non-parenchymal cells were immobilized within Ca-alginate, and although immobilized hepatocytes rapidly lost the ability to induce TAT, hepatocytes co-immobilized with non-parenchymal cells maintained the ability during 4 days of culture. These results indicated that non-parenchymal liver cells, as well as hepatocytes, could be used to construct a bioartificial liver support system.     

Synthesis and degradation of eicosanoids in primary rat hepatocyte cultures

Arachidonic acid metabolites may play an important role in liver physiology, yet hepatocyte prostaglandin synthesis has not been characterized extensively. We used RIA to study production and clearance of several eicosanoids in confluent primary cultures of rat hepatocytes in serum-free, hormonally-defined medium. Under basal, unstimulated conditions 6-keto-PGF1 alpha (spontaneous breakdown product of prostacyclin) and 13,14-dihydro-15-keto-PGE (DHK-PGE, a metabolite of PGE) accumulated in the culture medium. Hepatocytes cleared 6-keto-PGF1 alpha, thromboxane B2, and DHK-PGE from the medium. Production of eicosanoids by primary cultures appeared resistant to indomethacin and several other cyclooxygenase inhibitors. This apparent resistance to indomethacin was not caused by rapid metabolism of indomethacin, by failure of the drug to enter hepatocytes, or by insensitivity of hepatocyte cyclooxygenase to the drug. Metabolism of PGE to DHK-PGE may be saturated under in vitro conditions. Hepatocytes can synthesize significant amounts of eicosanoids, although they are probably less active in this regard than are non-parenchymal cells.     

Propagation and functional characterization of serum-free cultured porcine hepatocytes for downstream applications

Hepatocyte transplantation is considered an alternative to whole organ transplantation. However, the availability of human cadaveric livers for the isolation of transplantation-quality hepatocytes is increasingly restricted. Xenogeneic porcine hepatocytes may therefore serve as an alternate cell ressource. The propagation of hepatocytes is often necessary to yield a sufficient cell number for downstream applications in xenotransplantation and in, for example, bioartificial liver support or pharmacological and toxicological studies. Our goal has been to propagate primary porcine hepatocytes in vitro and to determine the functional maintenance of the propagated cells. Porcine hepatocytes were cultured under serum-free conditions in the presence of hepatocyte growth factor and epidermal growth factor and passaged several times. The viability, proliferation and maintenance of liver-specific functions were determined as culture proceeded. Total cell number increased by 12-fold during four sequential passages, although the proliferative capacity was higher in primary cells and early passages as compared with late passages. Xenobiotics metabolism and urea synthesis gradually decreased with ongoing culture but could be restored by treatment with appropriate stimuli such, as β-naphthoflavone and cAMP. The expression of hepatocyte-specific genes was generally lower at the beginning than at later time-points of culture of individual passages. Porcine hepatocytes can thus be propagated in vitro. The partial loss of hepatocyte function may be restored in vitro by appropriate stimuli. This may also be achieved in a recipient liver after hepatocyte transplantation provided that the proper physiological environment for the maintenance of the differentiated hepatocyte phenotype is present. This study was supported by grants to B. Christ from the German Ministry of Education and Research (01 ZZ 0109 and NBL3-NG4) as well as by grants from the Federal State of Saxonia-Anhalt through the Wilhelm-Roux-Program at the Medical Faculty of the Martin-Luther-University of Halle-Wittenberg to B. Christ (09/07 and 04/03).     

A stable long-term hepatocyte culture system for studies of physiologic processes: cytokine stimulation of the acute phase response in rat and human hepatocytes.

Prior studies on the in vitro hepatic acute phase response have involved either hepatoma cell lines or conventional short-term cultures of primary hepatocytes. No data are available on the response of primary hepatocytes in stable long-term culture systems. In this study, the acute phase response of rat and human hepatocytes in a new long-term culture system was examined in response to interleukin-6 (IL-6), interleukin-1 beta (IL-1 beta), and tumor necrosis factor alpha (TNF-alpha). The cultured cells were sandwiched between two layers of collagen in a (double-gel) configuration which has been shown to preserve both hepatocyte function and morphology over prolonged periods of time. The stability of this culture configuration enabled us to investigate, for the first time, the temporal aspects of the response in addition to the effects of the mediators on protein secretion. Exposure of rat hepatocytes to IL-6 after culture for 16 days resulted in a 2-fold reduction of albumin secretion and a 15-fold increase in the secretion rates of fibrinogen and alpha 2-macroglobulin. In all instances, the peak response occurred at 48 h after IL-6 exposure, and all protein secretion rates returned to pretreatment values within 5 days posttreatment. Changes in the mRNA levels of these proteins in response to IL-6 corresponded with those changes seen with the secreted products, indicating pretranslational regulation. Administration of IL-1 beta to rat hepatocyte produced a similar decline of albumin secretion and a 5-fold increase of fibrinogen secretion, whereas alpha 2-macroglobulin secretion remained undisturbed.(ABSTRACT TRUNCATED AT 250 WORDS)     

3D hepatic cultures simultaneously maintain primary hepatocyte and liver sinusoidal endothelial cell phenotypes

Developing in vitro engineered hepatic tissues that exhibit stable phenotype is a major challenge in the field of hepatic tissue engineering. However, the rapid dedifferentiation of hepatic parenchymal (hepatocytes) and non-parenchymal (liver sinusoidal endothelial, LSEC) cell types when removed from their natural environment in vivo remains a major obstacle. The primary goal of this study was to demonstrate that hepatic cells cultured in layered architectures could preserve or potentially enhance liver-specific behavior of both cell types. Primary rat hepatocytes and rat LSECs (rLSECs) were cultured in a layered three-dimensional (3D) configuration. The cell layers were separated by a chitosan-hyaluronic acid polyelectrolyte multilayer (PEM), which served to mimic the Space of Disse. Hepatocytes and rLSECs exhibited several key phenotypic characteristics over a twelve day culture period. Immunostaining for the sinusoidal endothelial 1 antibody (SE-1) demonstrated that rLSECs cultured in the 3D hepatic model maintained this unique feature over twelve days. In contrast, rLSECs cultured in monolayers lost their phenotype within three days. The unique stratified structure of the 3D culture resulted in enhanced heterotypic cell-cell interactions, which led to improvements in hepatocyte functions. Albumin production increased three to six fold in the rLSEC-PEM-Hepatocyte cultures. Only rLSEC-PEM-Hepatocyte cultures exhibited increasing CYP1A1/2 and CYP3A activity. Well-defined bile canaliculi were observed only in the rLSEC-PEM-Hepatocyte cultures. Together, these data suggest that rLSEC-PEM-Hepatocyte cultures are highly suitable models to monitor the transformation of toxins in the liver and their transport out of this organ. In summary, these results indicate that the layered rLSEC-PEM-hepatocyte model, which recapitulates key features of hepatic sinusoids, is a potentially powerful medium for obtaining comprehensive knowledge on liver metabolism, detoxification and signaling pathways in vitro.     

Acetaminophen-induced hepatotoxicity of gel entrapped rat hepatocytes in hollow fibers

An important application of primary hepatocyte cultures is for hepatotoxicity research. In this paper, gel entrapment culture of rat hepatocytes in miniaturized BAL system were evaluated as a potential in vitro model for hepatotoxicity studies in comparison to monolayer cultures. After exposure for 24 and 48 h to acetaminophen (2.5 mM), gel entrapped hepatocytes were more severely damaged than hepatocyte monolayer detected by methyl thiazolyl tetrazolium (MTT) reduction, intracellular glutathione (GSH) content, reactive oxygen species (ROS) levels, urea genesis and albumin synthesis. CYP 2E1 activities detected by 4-nitrocatechol (4-NC) formation were higher in gel entrapped hepatocytes than in hepatocyte monolayers while the addition of CYP 2E1 inhibitor, diethyl-dithiocarbamate (DDC), more significantly reduced acetaminophen-induced toxicity in gel entrapped hepatocytes. In addition, protective effects of GSH, liquorice extract and glycyrrhizic acid against acetaminophen hepatotoxicity were clearly observed in gel entrapped hepatocytes but not in hepatocyte monolayer at an incubation time of 48 h. Overall, gel entrapped hepatocytes showed higher sensitivities to acetaminophen-induced hepatotoxicity than hepatocyte monolayer by a mechanism that higher CYP 2E1 activities of gel entrapped hepatocytes could induce more severe acetaminophen toxicity. This indicates that gel entrapped hepatocytes in hollow fiber system could be a promising model for toxicological study in vitro.     

The role of non-parenchymal cells in liver growth

The main non-parenchymal cells of the liver, Kupffer cells, sinusoidal endothelial cells and stellate cells, participate in liver growth with respect to both their own proliferation, and effects on hepatocyte proliferation. In the well-characterised paradigm of 70% partial hepatectomy, they undergo DNA synthesis and cell division 20-24h later than the hepatocyte population. They exert both positive and negative influences on hepatocyte proliferation, including provision of an extracellular matrix-bound reservoir of hepatocyte growth factor that is activated after damage; priming of hepatocytes for DNA synthesis through rapid generation of TNF-alpha and IL-6; and generation of factors at later time points that curb hepatocyte DNA synthesis (IL-1, TGF-beta) and initiate reconstruction and reformation of matrix proteins.