JNK信号通路调控大鼠再生肝8种细胞的增殖和凋亡

目的 从基因转录水平了解JNK信号通路在大鼠再生肝8种细胞中的作用。方法 用密度梯度离心和免疫磁珠等方法分离肝细胞（HC）、胆管上皮细胞（BEC）、卵圆细胞（OC）、肝星形细胞（HSC）、窦内皮细胞（SEC）、库普弗细胞（KC）、陷窝细胞（PC）、树突状细胞（DC）等8种肝脏细胞,用大鼠Genome 230 2.0芯片检测大鼠再生肝8种细胞的基因表达谱,用生物信息学和系统生物学等方法分析基因表达变化预示的JNK信号通路在调控大鼠再生肝8种细胞增殖、凋亡中的作用。用实时荧光定量PCR方法验证了芯片结果的可靠性。结果 JNK信号通路涉及240个基因和42条途径,其中,225个基因与大鼠肝再生相关。基因协同作用分析显示,在大鼠肝再生启动阶段,JNK信号通路启动HC和KC增殖,促进OC凋亡,启动部分PC和SEC增殖和促进部分PC和SEC凋亡;在进展阶段,JNK信号通路促进HC、BEC、KC和DC增殖,促进部分PC增殖、部分PC凋亡。在终止阶段,JNK信号通路促进HC、OC和PC凋亡,促进部分KC增殖、部分KC凋亡。结论 大鼠肝再生中JNK信号通路的42条途径和225个基因参与调控大鼠再生肝8种细胞的增殖和凋亡。     

大鼠肝再生中肝脏细胞的基因转录谱预示EM>AI408225/EM>促进抗原肽与TCR结合

目的 了解新基因AI408225与大鼠肝再生涉及的体液免疫相关性。 方法 大鼠再生肝8种细胞的分离,采用Percoll密度梯度离心结合免疫磁珠分选方法进行,用Microsoft Excel、BLAST等软件分析基因的序列同源性及共表达关系,用生物信息学和系统生物学等方法分析新基因参与的生理活动。 结果 93个参与体液免疫的已知基因与肝再生相关,肝细胞、胆管上皮细胞、卵圆细胞、肝星形细胞、窦内皮细胞、库普弗细胞、陷窝细胞、树突状细胞的体液免疫相关基因数为33、46、17、59、48、35、53、68。其中,cd4与AI408225同源和共表达。 结论 大鼠肝再生与体液免疫相关,AI408225参与MHC II-抗原肽-CD4-TCR复合物形成。     

大鼠肝再生中8种肝脏细胞的凝血反应相关基因转录谱预示的生理活动

目的 了解大鼠肝再生中8种肝脏细胞的凝血反应相关基因转录谱及其预示的生理活动。 方法 大鼠再生肝8种细胞的分离,基因表达变化检测,预示的生理活动分析用Cluster等软件及生物信息学和系统生物等方法进行,用Microsoft Excel等软件分析基因的表达模式。 结果 40个参与凝血反应的基因与肝再生相关,肝细胞、胆管上皮细胞、卵圆细胞、星形细胞、窦内皮细胞、库普弗细胞、陷窝细胞、树突状细胞的相应基因数为13、31、12、33、32、9、34、33。serpine1、a2m 在上述8种细胞,vwf、klkb1 在除库普弗细胞之外的7种细胞,其他基因在两种或两种以上细胞中发生有意义表达变化。上述基因转录谱预示,肝再生启动和进展阶段激肽释放酶和凝血酶原合成,凝血酶形成等活动增强。终止阶段纤维蛋白单体形成纤维蛋白聚合体等活动增强。 结论 大鼠肝再生与凝血反应密切相关。     

大鼠肝再生中8种肝脏细胞的细胞免疫相关基因转录谱预示的生理活动

目的 了解大鼠肝再生中8种肝脏细胞的细胞免疫相关基因转录谱,及其预示的细胞免疫活动。 方法 用Percoll密度梯度离心结合免疫磁珠分选方法分离大鼠再生肝的肝细胞、胆管上皮细胞、卵圆细胞、星形细胞、窦内皮细胞、库普弗细胞、陷窝细胞和树突状细胞等8种细胞,用Rat Genome 230 2.0芯片等检测细胞免疫相关基因在上述细胞中表达变化,用Cluster等软件及生物信息学和系统学生物等方法分析它们的表达模式及预示的生理活动。 结果 大鼠肝再生中40个细胞免疫相关基因发生了表达变化,相应细胞的基因数为19、19、9、19、19、21、22、21。肝再生启动阶段和进展阶段抗原肽MHC复合物形成,NF-κB激酶活性和IL-2等细胞因子合成增加,终止阶段NF-κB促进细胞分化活动和caspase诱导T细胞凋亡活动增强。 结论 大鼠肝再生与细胞免疫相关。     

Caspase信号通路调控大鼠再生肝肝细胞的凋亡

目的 从基因转录水平了解Caspase信号通路各途径对大鼠再生肝肝细胞凋亡的调节作用。方法 将114只大鼠随机分为19组,包括9个部分肝切除组,9个假手术组和1个正常对照组。于手术后10个不同时间点用常规的两步灌流法和Percoll密度梯度离心法分离大鼠肝细胞,用大鼠Genome 230 2.0芯片检测在大鼠肝再生（LR）中Caspase信号通路相关基因表达变化,用荧光定量PCR确定芯片结果的可靠性,用生物信息学和系统生物学等方法分析基因表达变化预示的Caspase信号通路在调控大鼠再生肝肝细胞凋亡中的作用。结果Caspase信号通路涉及38条途径和123个基因,大鼠Genome 230 2.0芯片含其中的106个基因,38个基因在大鼠肝再生中与肝细胞相关。Caspase信号通路抑制细胞凋亡的21条途径中,途径1、2和11在大鼠部分肝切除（PH）后的30h,途径27、29和31在72h,途径15和16在2h和30h,途径3和4在30h和72h抑制肝细胞凋亡。促进细胞凋亡的17条途径中,途径14在2h,途径34在6h,途径7在30h,途径13在2h和30h,途径36在6h和30h促进肝细胞凋亡。同时,尚未发现其他途径参与肝细胞增殖和（或）凋亡调控。结论 Caspase信号通路的15条途径和38个基因调控大鼠再生肝的肝细胞凋亡。     

Comparative analysis of the role of JNK signaling pathway in cell proliferation and apoptosis of rat liver regeneration and cirrhosis

目的 从基因转录水平了解JNK信号通路在大鼠肝再生(LR)和肝硬化(LC)中的作用异同. 方法 采用2/3部分肝切除手术制备大鼠肝再生模型,以腹腔注射CCl4中性菜籽油溶液法建立大鼠肝硬化模型,采用大鼠Genome 230 2.0芯片检测不同时间点再生肝和肝硬化组织中JNK信号通路的基因表达谱,用生物信息学和系统生物学等方法分析基因表达谱预示的增殖和凋亡活动. 结果 JNK信号通路涉及302个基因和42条途径,大鼠Genome 230 2.0芯片含上述基因中的240个基因,其中,79个基因发生有意义表达变化,涉及LR的52个基因,LC的5个基因,有22个基因与两者相关.在大鼠LR启动阶段,途径1和16促进细胞增殖及途径22 ～ 33促进细胞凋亡作用强于对照;在进展阶段,途径1～17、34和35促进细胞增殖及途径22 ～33促进细胞凋亡作用强于对照,途径37～41抑制细胞凋亡作用弱于对照;在终止阶段,途径37、39、41和42诱导细胞凋亡作用弱于对照,同时,尚未发现途径18 ～ 21和36参与大鼠LR.而在LC发生中,JNK信号通路中的这些途径与对照相比均无显著差异.结论 JNK信号通路的37条途径调控大鼠肝再生的细胞增殖和凋亡,对大鼠肝硬化的调控作用则不显著.     

肝细胞生长和分化相关基因在大鼠肝再生中的表达方式及作用分析

目的在基因转录水平了解肝再生中肝细胞的生长和分化情况。方法用搜集网站资料和查阅相关论文等方法获得参与肝细胞生长和分化基因,用大鼠基因组230 2.0芯片检测它们在大鼠肝再生(LR)中的表达情况,通过比较手术组和假手术组中基因表达差异性以确定上述基因中的肝再生相关基因。结果初步证实上述基因中110个基因与肝再生相关。肝再生启动(PH后0.5~4h)、G0/G1过渡(PH后4~6h)、细胞增殖(PH后6~66h)、细胞分化和组织结构功能重建(PH后72~168h)等4个阶段起始表达的基因数为63、11、43和3,基因总表达的次数为63、43、101和80,表明相关基因主要在肝再生启动阶段起始表达,在不同阶段发挥作用。它们共表达上调488次,下调248次,分为6种表达方式,表明肝再生中细胞生理生化活动的多样性和复杂性。结论肝细胞生长和分化贯穿于整个肝再生中。     

大部分肝切除模型大鼠肝再生组织中CD34蛋白及Delta-like-1蛋白的表达

背景：当肝脏受到严重损伤或肝脏大部分缺失时,肝干细胞迅速分裂增殖向成熟肝细胞分化修复肝组织,但肝再生是一个复杂的多信号分子调控的过程,参与调控的相关基因及蛋白质目前尚不十分清楚。 目的：观察大部分肝切除后大鼠肝再生组织中CD34蛋白、Delta-like-1蛋白的表达变化。 方法：切除2/3肝脏建立大鼠肝再生动物模型,应用苏木精-伊红染色、酶组织化学染色、免疫组织化学染色方法在不同时间点检测肝再生组织中ATP酶、增殖细胞核抗原、CD34、Delta-like-1蛋白的表达情况。 结果与结论：在肝再生的启动阶段ATP酶表达下调,CD34、Delta-like-1表达上调;在肝再生的持续增殖阶段,ATP酶表达上调到正常水平,CD34、Delta-like-1表达下调;增殖细胞核抗原表达为持续上调。说明ATP酶、CD34、Delta-like-1在不同阶段表达不同,提示在肝再生过程中各信号分子的协同作用及Notch- Delta信号通路参与其作用机制。     

肝陷窝细胞的研究进展

肝陷窝细胞（PCs）是肝脏的自然杀伤细胞（HNKCs），具有抗肿瘤、抗病毒、调节肝细胞的生长、分化、抑制肝纤维化等作用。本文简要地总结了近几年肝陷窝细胞的研究进展。     

GADD45α对大鼠部分肝切除后肝脏再生的调控作用

目的探讨生长阻滞和DNA损伤诱导基因GADD45α在大鼠部分肝切除(PH)后肝再生中的作用及其调节机制。方法将114只大鼠随机分为19组,2/3肝切除9组,手术对照9组,正常对照1组,制作大鼠部分肝切除模型,然后用大鼠基因组芯片Rat Genome 230 2.0检测部分肝切除后再生肝的基因表达变化,采用实时定量PCR技术验证芯片检测结果。利用谱函数(Ep)分析基因表达变化预示的生理活动改变,进而通过Ingenuity Pathway Analysis(IPA)汇总GADD45α调控肝再生的信号通路并分析其可能的分子机制。结果 GADD45α在PH后2~6h、24h和36~72h均表达上调,GADD45α通过NF-κB、p38PRAK、p53、STAT3-p21、STAT3-Bcl-2、STAT3-c Myc等6条途径参与大鼠部分肝切除后的肝脏再生调控。谱函数分析发现,GADD45α调控的生理活动的变化情况基本与大鼠肝再生进程相符。结论 GADD45α在大鼠肝再生中可能通过上述信号通路调控细胞增殖、细胞周期和细胞存活等生理活动,进而调节肝脏的再生。     

High-Throughput Analysis of Tumor Necrosis Factor Signaling Pathways in Eight Cell Types during Rat Hepatic Regeneration

This study aims to clarify the relevance of tumor necrosis factor (TNFs) signaling pathways and liver regeneration (LR) at the cellular level. Eight liver cell types were isolated using Percoll density gradient centrifugation and immunomagnetic beads methods. Expressions of TNF signaling pathway-involved genes in each cell type after 2/3 hepatectomy (PH) were detected using gene chip. Results show the following: gene TNFα was upregulated in most cell types, especially in Kupffer cells (KC); TNFβ expression was insignificantly changed in eight liver cell types; the majority of genes involved in four TNFα signaling pathways showed increased expression during LR in hepatocytes (HC); TNFα-induced NFκB pathway-involved genes were upregulated preferentially between 2 and 24 h during LR in biliary epithelial cells (BECs); and TNFα-induced apoptotic pathway genes were downregulated preferentially at progressing phase of LR in dendritic cells (DCs). Referring to the above results, TNFα-mediated signaling pathways, in contrast to TNFβ, play the more proactive role in LR, and four TNFα-mediated signaling pathways seem helpful to regulate biological events in HC; BEC proliferation was partly controlled by TNFα-mediated NFκB pathway; and the impaired TNFα-mediated apoptotic pathway in DCs might contribute to the restoration of DC mass after PH. Briefly, the comparative analysis of genomewide expression profiles of TNF signaling pathways between different cell types is helpful in understanding the implication of TNF signaling in LR at the cellular level.     

New insights into functional aspects of liver morphology

The liver is structurally and functionally complex and has been considered second only to brain in its complexity. Many mysteries still exist in this heterogeneous tissue whose functional unit of the lobule has continued to stump morphologists for over 300 years. The primary lobule, proposed by Matsumoto in 1979, has been gaining acceptance as the functional unit of the liver over other conceptual views because it's based on vessel architecture and includes the classic lobule as a secondary feature. Although hepatocytes comprise almost 80% of the liver, there are at least another dozen cell types, many of which provide "cross-talk" and play important functional roles in the normal and diseased liver. The distribution and functional roles of all cells in the liver must be carefully considered in both the analysis and interpretation of research data, particularly data in the area of genomics and "phenotypic anchoring" of gene expression results. Discoveries regarding the functional heterogeneity of the various liver cell types, including hepatocytes, hepatic stellate cells, sinusoidal endothelia, and Kupffer cells, are providing new insights into our understanding of the development, prevention and treatment of liver disease. For example, functional differences along zonal patterns (centrilobular or periportal) have been demonstrated for sinusoidal endothelium, Kupffer cells, and hepatocytes and can explain the gradients and manifestations of disease observed within lobules. Intralobular gradients of bile uptake, glycogen depletion, glutamine synthetase, and carboxylesterase by hepatocytes; widened fenestrations in centrilobular sinusoidal lining cells; and differences in the components of centrilobular extracellular matrix or function of Kupffer cells have been demonstrated. Awareness of the complexities and heterogeneity of the liver will add to a greater understanding of liver function and disease processes that lead to toxicity, cancer, and other diseases.     

先天免疫反应与非酒精性脂肪性肝病

非酒精性脂肪性肝病(non-alcoholic fatty liver disease,NAFLD)是世界上最常见的肝脏疾病之一。其特点是脂质异常聚集于肝细胞,即肝脂肪变性,继而发展为有或没有纤维化的非酒精性脂肪性肝炎(nonalcoholic steatohepatitis,NASH)。肝脏可看做一个“免疫器官”,它可调节非淋巴细胞,如巨噬枯氏细胞、星状细胞以及淋巴细胞。这些细胞组成了经典的先天免疫系统,从而使肝脏能够更好地抵抗病原体。尽管肝脏提供了耐受性的环境,但先天免疫信号通路的异常激活可诱发炎症,导致组织损伤、纤维化以及致癌作用。此外,细胞因子能够通过诱发并参与免疫反应,激发肝脏细胞内的信号通路,在肝脏炎症反应中也起着重要作用。本文总结各类先天免疫细胞、以及细胞因子对非酒精性脂肪性肝病的影响。     

Immune functioning in non lymphoid organs: the liver

The liver is the primary hematopoietic organ of the mammalian body during the fetal stage. The postnatal liver retains immunologically important functions and contains a substantial population of immunologically active cells, including T and B lymphocytes, Kupffer cells, liver-adapted natural killer (NK) cells (pit cells), natural killer cells expressing T cell receptor (NKT cells), stellate cells, and dendritic cells. The liver is the major site of production of the acute phase proteins that are associated with acute inflammatory reactions. Kupffer cells have an important role in the nonspecific phagocytosis that comprises a major component of the barrier to invasion of pathogenic organisms from the intestine. Hepatic NK and NKT cells are important in the nonspecific cell killing that is important in resistance to tumor cell invasion. The liver has a major role in deletion of activated T cells and induction of tolerance to ingested and self-antigens. Disposal of waste molecules generated through inflammatory, immunologic, or general homeostatic processes is accomplished via the action of specific endocytic receptors on sinusoidal endothelial cells of the liver. Age-related changes in sinusoids (pseudocapillarization), autophagy, and functions of various hepatic cell populations result in substantial alterations in many of these immunologically important functions.     

Local control of the immune response in the liver

Summary: The physiological function of the liver – such as removal of pathogens and antigens from the blood, protein synthesis and metabolism – requires an immune response that is adapted to these tasks and is locally regulated. Pathogenic microorganisms must be efficiently eliminated while the large number of antigens derived from the gastrointestinal tract must be tolerized. From experimental observations it is evident that the liver favours the induction of tolerance rather than the induction of immunity. The liver probably not only is involved in transplantation tolerance but contributes as well to tolerance to orally ingested antigens (entering the liver with portal‐venous blood) and to containment of systemic immune responses (antigen from the systemic circulation entering the liver with arterial blood). This review summarizes the experimental data that shed light on the molecular mechanisms and the cell populations of the liver involved in local immune regulation in the liver. Although hepatocytes constitute the major cell population of the liver, direct interaction of hepatocytes with leukocytes in the blood is unlikely. Sinusoidal endothelial cells, which line the hepatic sinusoids and separate hepatocytes from leukocytes in the sinusoidal lumen, and Kupffer cells, the resident macrophage population of the liver, can directly interact with passenger leukocytes. In the liver, clearance of antigen from the blood occurs mainly by sinusoidal endothelial cells through very efficient receptor‐mediated endocytosis. Liver sinusoidal endothelial cells constitutively express all molecules necessary for antigen presentation (CD54, CD80, CD86, MHC class I and class II and CD40) and can function as antigen‐presenting cells for CD4+ and CD8+ T cells. Thus, these cells probably contribute to hepatic immune surveillance by activation of effector T cells. Antigen‐specific T‐cell activation is influenced by the local microenvironment. This microenvironment is characterized by the physiological presence of bacterial constituents such as endotoxin and by the local release of immunosuppressive mediators such as interleukin‐10, prostaglandin E2 and transforming growth factor‐b. Different hepatic cell populations may contribute in different ways to tolerance induction in the liver. In vitro experiments revealed that naive T cells are activated by resident sinusoidal endothelial cells but do not differentiate into effector T cells. These T cells show a cytokine profile and a functional phenotype that is compatible with the induction of tolerance. Besides sinusoidal endothelial cells, other cell populations of the liver, such as dendritic cells, Kupffer cells and perhaps also hepatocytes, may contribute to tolerance induction by deletion of T cells through induction of apoptosis.