肝再生增强因子研究进展

肝脏是人体中少有的能够进行快速再生的器官之一。肝脏的部分切除,化学药物引起的肝损伤以及肝炎病毒感染引起的肝损伤,都能够触发肝脏的再生。肝细胞再生过程中存在着一系列的分子生物学调节事件,近年来包括肝细胞生长因子(HGF)、转化生长因子β(TGFβ)、胰岛素样生长因子Ⅰ、Ⅱ(IGF-Ⅰ、Ⅱ)和胰岛素等生长因子在肝再生过程中的作用与机制逐步得到阐明。与肝细胞再生有关的新的生长因子也不断发现,肝再生增强因子(ALR)的发现即为一例。有关肝再生相关因子的研究不仅有助于阐明肝再生过程中的分子生物学凋节机制,而且还可据此研制出促进肝再生的药物,以解决各种重症肝病及暴发性肝衰竭的治疗问题。     

人肝再生增强因子在肝衰竭患者血清及肝组织中的表达及其意义

目的 探讨人肝再生增强因子(ALR)在肝衰竭患者血清及肝组织中的表达水平及其意义.方法 制备Balb/c小鼠来源的人ALR蛋白多克隆抗体并纯化其IgG组分,以该抗体通过酶联免疫吸附法建立ALR浓度与吸光值的标准曲线.获取18例肝衰竭患者、24例慢性乙型肝炎患者及10名正常人血清,比较其血清ALR水平差异,实时荧光PCR法比较各组患者肝组织ALR mRNA表达水平差异.据资料不同采用Bonferroni或LSD检验进行统计学分析. 结果 Western blot结果发现人ALR蛋白多克隆抗体能特异性结合人ALR蛋白,酶联免疫法检测结果发现其在一定的ALR浓度范围内有良好的线性关系.18例肝衰竭患者中,6例好转出院的患者血清ALR水平为(1613.5±369.6)pmol/ml,高于12例肝功能恶化、死亡或不得不接受肝移植患者的(462.3±235.8)pmol/ml(t=11.21,P<0.05);慢性乙型肝炎患者为(969.2±332.5)pmol/ml,与正常人的(806.9±240.8)pmol/ml差异无统计学意义(t=2.17,P>0.05).5例接受肝移植的患者肝组织ALR mRNA表达水平为(3.45±0.38)log10拷贝/μl,低于慢性乙型肝炎患者的(4.37±0.15)log10拷贝/μl及正常供体的(4.31±0.10)log10拷贝/μl(P<0.05),而后两者差异无统计学意义(P>0.05).结论 血清ALR水平反映了肝脏ALR mRNA表达水平,其在预测慢加急性肝衰竭患者的预后方面有一定意义.     

肝再生增强因子在肝癌细胞中的表达及意义

目的研究肝再生增强因子(ALR)对肝癌细胞和原代大鼠肝细胞增殖作用的影响及在肝细胞癌(HCC)中的表达。方法将不同种属的ALR分别与原代大鼠肝细胞和人肝癌细胞株QGY和HepG2共同培养后，用^3H-胸腺嘧啶核苷掺入法测定这些细胞的增殖情况。并应用免疫组织化学法对9例正常肝组织和21例HCC组织中ALR的表达进行了研究。结果不同种属的ALR在体外均能刺激人肝癌细胞株QGY和HepG2增殖，并呈剂量依赖关系，但对原代大鼠肝细胞均无刺激增殖作用。在正常肝组织中ALR不表达，但在HCC肝组织中ALR均表达，且ALR的表达程度与HCC分化程度和大小均无关。结论ALR可能在HCC的发生发展中起着重要作用。     

肝细胞生成素的研究

急性肝衰竭的病死率极高,其预后主要取决于病损肝细胞的坏死程度和再生能力,因此,预防肝坏死与促进肝再生是目前各种治疗手段的出发点.以往临床上的治疗措施如支持疗法、活跃微循环、全血及血浆交换、体外吸附剂灌注及人工肝等技术,其目的均是以稳定内环境、延长患者生命、为残存肝细胞再生创造条件为主.这些措施虽有助于清除体内有毒物质,但无助于刺激病损肝脏的再生.因此,探索并找到既能阻止肝细胞坏死、又能促进肝脏再生的物质或药物一直是肝脏病学面临的重大课题[1].     

大鼠肝再生增强因子假基因的克隆化与序列分析

目的 研究大鼠肝再生增强因子（augmenter of liver regeneration,ALR）在大鼠基因组织中的存在方式。方法 以大鼠基因组DNA为模板,根据ALP cDNA序列设计引物,用多聚酶链反应（PCR）扩增产物,连入pGEM Teasy Vector后测序。结果 PCR法扩增出两条产物,其一经测序发现为ALR的假基因,预测氨基酸序列与ALR的同源性为88.8%。结论 作为肝细胞再生过程中重要的生长刺激因子,大鼠ALR存在假基因,提示可能存在ALR多基因家族。为研究ALR分子的进货规律提供了依据。     

人肝再生增强因子表达载体的构建及其在酵母中的表达

肝再生增强因子是一种多肽调节因子,具有重要的生物学功能,与高等生物线粒体的生成、细胞分裂周期调节及肝脏,睾丸等重要脏器发育有关,最主要是能特异的刺激肝源细胞的增殖。对于肝再生增强因子（augmenter of liverregeneration,ALR）的产生、分泌、体内转运的过程都存有诸多疑间。因此为了进一步研究该蛋白质的功能,我们在酵母细胞中表达了ALR。     

肝再生增强因子在HBV相关肝硬化患者中的表达及意义

目的:研究肝再生增强因子(augmenter of liver regeneration,ALR)在乙型肝炎病毒(hepatitis B virus,HBV)相关肝硬化患者中的表达及临床意义.方法:病例来源于我科门诊及住院的慢性乙型肝炎患者,包括性慢性乙型肝炎轻度组(n=196)、代偿期肝硬化组(n=69)、失代偿期肝硬化组(n=148);失代偿肝硬化患者根据并发症进一步分为腹水组(n=51)、消化系出血组(n=32)、肝性脑病组(n=27)和慢性肝衰竭组(n=38),其中腹水组分为原发性腹膜炎组(n=9)与非原发性腹膜炎组(n=42).健康对照组(n=200)来源于输血科健康献血者.收集患者血清及临床资料;采用ELISA法检测各组患者血清ALR浓度;瞬时弹性扫描(FibroScan)检测代偿期肝硬化患者肝脏硬度.比较各组间ALR浓度差异,分析ALR浓度与肝硬化及其并发症的关系.结果:ALR浓度在代偿期肝硬化组和失代偿肝硬化组均明显高于健康对照组和慢性乙型肝炎轻度组(P<0.01).在代偿期肝硬化患者ALR浓度(1.54μg/L±0.53μg/L)与肝脏FibroScan值(16.96 kPa±5.6 kPa)负相关(r2=-0.218,F=15.736,P<0.01).失代偿肝硬化组中慢性肝衰竭患者ALR浓度明显高于代偿期肝硬化组(1.73μg/L±0.23μg/L vs 1.54μg/L±0.53μg/L,P=0.012),腹水组患者ALR浓度明显低于代偿期肝硬化组(1.32μg/L±0.64μg/L vs 1.54μg/L±0.53μg/L,P=0.027),原发性腹膜炎组与非原发性腹膜炎组间ALR浓度无显著性差异.ALR血清浓度与肝硬化患者腹水的发生相关,低ALR血清浓度是肝硬化患者发生腹水的危险因素(P=0.031,OR=0.841).结论:ALR在HBV相关肝硬化患者表达明显升高,其水平与肝脏硬度及腹水发生负相关,提示ALR在肝硬化患者增高可能是机体的一种保护机制.     

人肝再生增强因子抑制层黏连蛋白B1链基因转录活性的研究

肝再生增强因子（ALR）不仅有促进肝细胞再生和抗肝损伤作用，还有一定的抗肝纤维化作用。本研究利用生物信息学技术，对层黏连蛋白B1链（LAMB）基因上游调控序列进行分析克隆，采用基因重组技术构建pCAT3-LAMBp报告基因载体，与pcDNA3.1（-）-ALR表达载体共转染COS7细胞，证明ALR的转基因表达对LAMB启动子具有下调作用，使下游CAT基因的表达减弱。本研究为阐明ALR的生物学作用和抗肝纤维化用用提供新的实验及理论基础。     

肝再生的发生机制及血清标志物的研究现状

肝再生是在肝损伤及坏死后,为维持肝脏体积及功能,一系列因子和信号转导通路参与的重要反应。其过程包括三个主要阶段:某些因子引发有丝分裂的初始阶段,促进肝细胞重新进入细胞周期的增殖阶段,促使肝细胞达到一定数量及肝脏质量恢复的终止阶段。介绍了促使各种肝干细胞分化为肝细胞以恢复肝脏体积和功能的各种因子及多种细胞信号通路,并归纳了本课题组前期研究发现——AFP是肝衰竭后肝再生的重要血清标志物。经分析表明,深入研究肝再生的发生基础及临床应用,有助于进一步增加对肝再生的了解,更好预测急慢性肝病的预后,为各种晚期肝病的临床诊治提供新的思路和方法。     

国外人工肝治疗的现状与评价

人工肝支持系统简称人工肝，是国外20世纪50年代开始逐渐发展起来的为肝衰竭患者提供体外肝功能支持的一种技术。目前国外人工肝治疗的方法主要是借助体外机械、化学或生物性装置暂时及部分替代肝脏功能，在体外采用某种具有解毒、代谢等作用的装置和方法来代偿肝脏功能，从而辅助治疗肝功能不全、肝衰竭或相关疾病；其作用原理是基于肝损伤具有可逆性和肝细胞有强大的再生能力，通过人工肝辅助治疗在内环境改善的情况下，肝功能能够自发恢复，或为肝脏移植和其他特效治疗进行准备。近年来随着材料学、细胞工程学及临床医学的不断发展，人工肝支持系统已成为肝衰竭治疗不可缺少的有效方法之一。     

Augmenter of liver regeneration (ALR) may promote liver regeneration by reducing natural killer (NK) cell activity in human liver diseases

Cytotoxicity of liver natural killer cells against regenerating hepatocytes has been reported as a possible mechanism of regeneration failure in fulminant hepatitis. An augmenter of liver regeneration (ALR) inhibits liver natural killer cell activity in rats. In this study, we measured hepatic expression of ALR mRNA, blood levels of ALR, and peripheral blood natural killer cell activity in patients with various types of acute liver disease to investigate the relationship between failure of liver regeneration and hepatic natural killer cells. Hepatic ALR mRNA expression was higher in liver disease patients than in non-liver disease controls, and a correlation was found between serum ALR values and hepatic levels of ALR mRNA. In acute liver injury, the serum ALR level also showed a negative correlation with NK activity. ALR was produced by and released from the liver at the time of hepatic injury. Our findings suggest that ALR may protect against failure of regeneration by inhibition of hepatic natural killer cell activity in acute liver injury. Received: December 7, 1998 / Accepted: August 27, 1999     

Augmenter of liver regeneration: A key protein in liver regeneration and pathophysiology

Liver is constantly exposed to pathogens, viruses, chemicals, and toxins, and several of them cause injury, leading to the loss of liver mass and sometimes resulting in cirrhosis and cancer. Under physiological conditions, liver can regenerate if the loss of cells is less than the proliferation of hepatocytes. If the loss is more than the proliferation, the radical treatment available is liver transplantation. Due to this reason, the search for an alternative therapeutic agent has been the focus of liver research. Liver regeneration is regulated by several growth factors; one of the key factors is augmenter of liver regeneration (ALR). Involvement of ALR has been reported in crucial processes such as oxidative phosphorylation, maintenance of mitochondria and mitochondrial biogenesis, and regulation of autophagy and cell proliferation. Augmenter of liver regeneration has been observed to be involved in liver regeneration by not only overcoming cell cycle inhibition but by maintaining the stem cell pool as well. These observations have created curiosity regarding the possible role of ALR in maintenance of liver health. Thus, this review brings a concise presentation of the work done in areas exploring the role of ALR in normal liver physiology and in liver health maintenance by fighting liver diseases, such as liver failure, non‐alcoholic fatty liver disease/non‐alcoholic steatohepatitis, viral infections, cirrhosis, and hepatocellular carcinoma.     

Insights on augmenter of liver regeneration cloning and function

Hepatic stimulator substance (HSS) has been referred to as a liver-specific but species non-specific growth factor. Gradient purification and sequence analysis of HSS protein indicated that it contained the augmenter of liver regeneration (ALR), also known as hepatopoietin (HPO). ALR, acting as a hepatotrophic growth factor, specifically stimulated proliferation of cultured hepatocytes as well as hepatoma cells in vitro, promoted liver regeneration and recovery of damaged hepatocytes and rescued acute hepatic failure in vivo. ALR belongs to the new Erv1/Alr protein family, members of which are found in lower and higher eukaryotes from yeast to man and even in some double-stranded DNA viruses. The present review article focuses on the molecular biology of ALR, examining the ALR gene and its expression from yeast to man and the biological function of ALR protein. ALR protein seems to be non-liver-specific as was previously believed, increasing the necessity to extend research on mammalian ALR protein in different tissues, organs and developmental stages in conditions of normal and abnormal cellular growth.     

Autophagy in anti-apoptotic effect of augmenter of liver regeneration in HepG2 cells

AIM:To investigate the role of autophagy in the antiapoptotic effect of augmenter of liver regeneration(ALR).METHODS:Autophagy was induced through serum deprivation.An ALR-expressing plasmid was transfected into HepG2 cells,and autophagic flux was determined using fluorescence microscopy,electron microscopy,Western blot and quantitative polymerase chain reaction(q PCR) assays.After ALR-expressing plasmid transfection,an autophagy inhibitor [3-methyladenine(3-MA)] was added to HepG2 cells,and apoptosis was observed using fluorescence microscopy and flow cytometry.RESULTS:Autophagy was activated in HepG2 cells,peaking at 24 h after serum deprivation.Microtubuleassociated protein light chain three-II levels were higher in HepG2 cells treated with ALR than in control cells,fluorescence microscopy,electron microscopy and q PCR studies showed the similar trend,and p62 levels showed the opposite trend,which indicated that ALR may play an important role in increasing autophagy flux.The numbers of apoptotic cells were substantially higher in HepG2 cells treated with both ALR and 3-MA than in cells treated with ALR alone.Therefore,the protective effect of ALR was significantly attenuated or abolished when autophagy was inhibited,indicating that the anti-apoptotic effect of ALR may be related to autophagy.CONCLUSION:ALR protects cells from apoptosis partly through increased autophagy in HepG2 cells and may be valuable as a new therapeutic treatment for liver disease.     

肝再生增强因子研究进展

肝再生增强因子是一种新型的肝细胞刺激因子。本文从分子生物学特性、生物活性与作用机制等方面对肝再生增强因子的研究进展进行综述。     

人肝再生增强因子基因组DNA的克隆化与序列分析

目的 克隆人肝再生增强因子（ａｕｇｍｅｎｔｅｒ ｏｆ ｌｉｖｅｒ ｇｅｇｅｎｅｒａｔｉｏｎ,ＡＬＲ）的基因组ＤＮＡ,并进行序列分析。方法 采用人肝再生增强因子ｃＤＮＡ及其编码序列, 核苷酸序列数据库（ＧｅｎＢａｎｋ）以Ｂｌａｓｔ为工具进行核苷酸同源性比较分析,寻找相应的ＡＬＲ基因组ＤＮＡ序列。结果 从ＧｅｎＢａｎｋ核苷酸序列数据库中寻找到ＡＬＲ基因组ＤＮＡ全序列,由１８１３个核苷酸组成。     

Augmenter of liver regeneration promotes hepatocyte proliferation induced by Kupffer cells

AIM: To observe the effects of augmenter of liver regeneration (ALR) on Kupffer cells and to determine whether ALR promotes hepatocyte proliferation induced by Kupffer cells. METHODS: Kupffer cells and hepatocytes were cultured in vitro and various concentrations of recombinant rat ALR (rrALR) were added. 3H-thymidine, BrdU and 3H-leucine incorporation was determined in cultured Kupffer cells and hepatocytes, in hepatocytes conditioned by Kupffer cells, and in associated medium. rrALR was labeled by iodination and used to determine its binding activity by Scatchard analysis in Kupffer cells and primarily cultured rat hepatocytes. RESULTS: rrALR stimulated DMA replication in Kupffer cells and protein synthesis both in cells and in medium in a non-concentration-dependent manner. The effect was significant at the concentration of 1μg/L ALR. However, rrALR had no effect on primarily cultured hepatocytes, when hepatocytes were cultured with the Kupffer cell medium conditioned by ALR, DNA replication and protein synthesis in hepatocytes increased significantly at the concentration of 1μg/L ALR. When the ALR concentration was increased, its effect on hepatocyte proliferation decreased to the basal level. Scatchard analysis indicated the presence of a single class of high affinity receptors with a dissociation constant (Kd) of 0.883 nmol/L and a maximum binding capacity (Bmax) of 126.1 pmol/g protein in the rat Kupffer cells. CONCLUSION: ALR can promote hepatocyte proliferation induced by Kupffer cells, which is associated with the concentration of ALR, suggesting that Kupffer cells play a dual role in liver regeneration.     

Effects of augmentation of liver regeneration recombinant plasmid on rat hepatic fibrosis

AIM: To investigate the effects of eukaryotic expression of plasmid on augmentation of liver regeneration (ALR) in rat hepatic fibrosis and to explore their mechanisms. METHODS: Ten rats were randomly selected from 50 Wistar rats as normal control group. The rest were administered intraperitoneally with porcine serum twice weekly. After 8 wk, they were randomly divided into: model control group, colchicine group (Col), first ALR group (ALR1), second ALR group (ALR2). Then colchicine ALR recombinant plasmid were used to treat them respectively. At the end of the 4th wk, rats were killed. Serum indicators were detected and histopathological changes were graded. Expression of type Ⅰ, Ⅲ, collagen and TIMP-1 were detected by immunohisto-chemistry and expression of TIMP-1 mRNA was detected by semi-quantified RT-PCR. RESULTS: The histologic examination showed that the degree of the rat hepatic fibrosis in two ALR groups was lower than those in model control group. Compared with model group, ALR significantly reduced the serum levels of ALT, AST, HA, LN, PCIII and IV (P<0.05). Immunohistochemical staining showed that expression of type Ⅰ, Ⅲ, collagen and TIMP-1 in two ALR groups was ameliorated dramatically compared with model group (I collagen: 6.94±1.42,5.80±1.66 and 10.83±3.58 in ALR1, ALR2 and model groups, respectively; Ⅲ collagen: 7.18±1.95, 4.50±1.67 and 10.25±2.61, respectively; TIMP-1: 0.39±0.05,0.20±0.06 and 0.53±0.12, respectively,P<0.05 or P<0.01). The expression level of TIMP-1 mRNA in the liver tissues was markedly decreased in two ALR groups compared with model group (TIMP-1 mRNA/β-actin: 0.89±0.08, 0.65±0.11 and 1.36±0.11 in ALR1, ALR2 and model groups respectively, P<0.01). CONCLUSION: ALR recombinant plasmid has beneficial effects on rat hepatic fibrosis by enhancing regeneration of injured liver cells and inhibiting TIMP-1 expressions.