Animal models of liver regeneration

Owing to its powers of regeneration, the liver is capable of in vivo "tissue engineering" which enables complete restoration of liver architecture and re-establishment of the specific functions of the liver after various types of liver injury. Our current understanding of liver regeneration forms the basis of modern liver surgery and is now taken into consideration in the treatment of many liver diseases, in liver transplantation and hepatic tissue engineering.These advances have been achieved primarily by studies of liver regeneration in animal models after partial hepatectomy, attention being focused on the general mechanisms of cell proliferation. In recent years, however, toxin-induced models of liver regeneration have assumed growing importance, and by studying the interaction between cell damage and cell regeneration have made possible an investigation of liver regeneration of greater clinical relevance. However, the mechanisms of liver regeneration in patients with pre-existing chronic liver damage such as liver cirrhosis are still largely unexplored.This review examines and critically appraises the various approaches to the study of liver regeneration in animal models, including both surgical and pharmacological approaches.     

生长停滞和DNA损伤诱导蛋白45α在肝再生及肝病中的研究进展

生长停滞和DNA损伤诱导蛋白45α（GADD45α）是一种应激诱导蛋白,它可诱导Gadd45α基因的表达。GADD45α在再生肝及肝硬化、肝癌、肝衰竭等肝病中表达显著上调,与多种肝病发生发展及预后密切相关。GADD45α蛋白通过与其他蛋白相互作用,在调控细胞周期、维持基因组稳定、诱导细胞凋亡等方面发挥重要作用。以下我们综述了GADD45α与肝再生关系的研究进展,为进一步了解肝再生与肝脏疾病的发生机制以及治疗和预防肝病奠定基础。     

Liver repair by intra- and extrahepatic progenitors

Despite its remarkable capacity for endogenous regeneration, the mammalian liver is vulnerable to a number of chronic or acute conditions that exceed or circumvent the proliferative capabilities of its mature cell complement. Bipotential hepatic progenitors, or “oval cells,” have been shown to contribute to organ regeneration under such circumstances, both in human patients and in animal models. These progenitors are attractive agents for cell therapy, but have thus far proven challenging to isolate and manipulate. New reports indicating that transplanted bone marrow cells (BMCs) can also generate hepatocytes and contribute to liver repair have attracted considerable attention, because these cells are familiar and accessible to both clinicians and scientists. Recently, the issue of whether nuclear transfer (via cell fusion between donor BMC and recipient hepatocyte) or previously unrecognized differentiation potential (i.e., plasticity/transdifferentiation of BMC) is the primary origin of donor-derived hepatocytes has generated considerable controversy. In the liver, most evidence supports cell fusion as the key agent in the reversal of hepatopathology. However, regardless of their origin, the frequency of hepatocyte correction events is low. As is the case for the delivery of intrahepatic progenitors, substantial improvements in the understanding of this process will be needed before clinical application becomes practical.     

The effect of preconditioning on liver regeneration after hepatic resection in cirrhotic rats

Background/Aims

Ischemic preconditioning (IP) decreases severity of liver necrosis and has anti-apoptotic effects in previous studies using liver regeneration in normal rats. This study assessed the effect of IP on liver regeneration after hepatic resection in cirrhotic rats.

Methods

To induce liver cirrhosis, thioacetamide (300 mg/kg) was injected intraperitoneally into Sprague-Dawley rats twice per week for 16 weeks. Animals were divided into four groups: non-clamping (NC), total clamping (TC), IP, and intermittent clamping (IC). Ischemic injury was induced by clamping the left portal pedicle including the portal vein and hepatic artery. Liver enzymes alanine transaminase (ALT) and aspartate aminotransferase (AST) were measured to assess liver damage. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining for apoptosis and proliferating cell nuclear antigen (PCNA) staining for cell replication were also performed.

Results

Day-1 ALT and AST were highest in IP, however, levels in NC and IC were comparably low on days 1-7. There was no significant correlation of AST or ALT with experimental groups (P=0.615 and P=0.186). On TUNEL, numbers of apoptotic cells at 100× magnification (cells/field) were 31.8±24.2 in NC, 69.0±72.3 in TC, 80.2±63.1 in IP, and 21.2±20.8 in IC (P<0.05). When regeneration capacity was assessed by PCNA staining, PCNA-positive cells (cells/field) at 400× were 3.4±6.0 in NC, 16.9±69 in TC, 17.0±7.8 in IP and 7.4±7.6 in IC (P<0.05).

Conclusions

Although regeneration capacity in IP is higher than IC, the liver is vulnerable to ischemic damage in cirrhotic rats. Careful consideration is needed in applying IP in the clinical setting.     

Clinical report: Chronic liver dysfunction in an individual with an AMOTL1 variant

AMOTL1 is a member of the Motin protein family and localizes to tight junctions and is involved in cell polarity and paracellular permeability. Pathological variants have been reported in three patients from two separate families in recent years. The clinical spectrum includes cleft lip and palate along with a high incidence of congenital cardiac disease and ear malformations. We report a case of AMOTL1 pathogenic variant in a 11-year-old male patient with nonspecific and chronic liver dysfunction accompanied by persistently elevated liver enzymes since early infancy. Liver biopsy at 8 years of age revealed a mildly dilated central vein and sinusoid with no specific etiology. Liver dysfunction is not a known clinical feature of AMOTL1 malfunction. However, given that the protein is known to be involved in angiogenesis, it may be inferred that abnormalities in this process may lead to liver dysfunction. This is the first report of liver dysfunction identified in a patient with AMOTL1 malfunction, which will shed light on other putative functions of the protein.     

大鼠再生肝对二乙基亚硝胺启动作用的敏感性

目的比较再生肝和正常肝对二乙基亚硝胺（ＤＥＮ）启动作用的敏感性。方法以２／３肝叶切除后８周末的大鼠为实验组,正常大鼠为对照组,作如下比较：肝重、常规组织学检查及３Ｈ－ＴｄＲ掺入试验;用修改的Ｓｏｌｔ－Ｆａｒｂｅｒ模型,通过对ＧＧＴ阳性癌前病灶的体视学测量,观察肝脏对ＤＥＮ的启动效应;在体内和体外（无血清原代培养肝细胞）经ＤＥＮ攻击后,以核酸原位缺口标记方法观察肝细胞ＤＮＡ的损伤程度。结果２／３肝叶切除后８周末的实验组肝脏的修复过程已完成,未见肝细胞继续增生的表现;经ＤＥＮ攻击后,实验组肝癌前病灶在数密度和体积密度上都显著高于对照组;无论在体内或体外接受ＤＥＮ攻击后,实验组肝细胞ＤＮＡ的损伤程度都显著大于对照组。结论即使再生过程已经完成,再生肝仍比正常肝具有较高的致癌敏感性,这与再生肝肝细胞在ＤＥＮ攻击后其ＤＮＡ损伤较重相关。     

Absence of the portal bifurcation at the hilum of the liver due to intrahepatic origin of the left branch of the portal vein

Abstract The authors report a rare anomaly of portal vascularization which was detected by CT-scan and MRI and then confirmed surgically. There was no portal bifurcation at the hilum of the liver. After giving off its right dorsal branch, the portal vein entered the right liver and divided in the parenchyma into the right ventral and left branches. The arterio-biliary distribution was normal. Only a few similar cases have been reported. The left branch of the portal vein is reported to have few variations in contrast with the right one, which has many. The venous structure of the liver varies increasingly with the distance from the left umbilical vein. During a right hepatectomy, the possibility of such a vascularization makes it necessary to ensure that the left branch of the portal vein starts upstream before dividing a portal branch entering the right liver.     

Immunohistochemical study on the liver in autopsy cases with disseminated intravascular coagulation (DIC) with reference to clinicopathological analysis

Summary Immunohistochemical and clinicopathological studies were performed in 27 autopsy cases with indisputable DIC, which had been selected from 1,800 autopsy cases of elderly people based on the following two criteria; 1. presence of fibrin thrombi in glomeruli, and 2. presence of fresh patchy necrotic foci in myocardium and/or fibrin thrombi in splenic sinuses. A high incidence of liver lesions (22/27) was revealed in autopsy cases with indisputable DIC. The liver lesions could be classified into four groups. Group-I (Central degeneration) was characterized by massive precipitation of fibrin irregularly around the central vein, causing parenchymal damage. Group-II (Central necrosis), showed coagulation necrosis in the cental zone due to circulatory disturbance caused by either shock as a cause of DIC or abrupt cessation of blood flow into the lobules following fibrin thrombus formation in vessels of Glisson's sheath. Both group-I and -II showed a short clinical duration of DIC. Group-III (Sinusoidal thrombosis), showed the presence of fibrin thrombi in sinusoids with mild parenchymal damage and long clinical duration of DIC. Group-IV (No thrombosis), showed neither parenchymal damage nor fibrin thrombi in sinusoids, but a long clinical duration of DIC.     

血清腺苷脱氨酶在肝病诊断中的特异性

目的:通过测定血清中腺苷脱氨酶(ADA)的活性,来了解ADA在肝病诊断中的临床价值和特异性。方法:用速率法测定肝功能损伤者和正常健康人组的腺苷脱氨酶及转氨酶(ALT)值。结果:通过ADA及ALT的测定结果,表明77例病人ADA与ALT值明显高于正常人,除黄疸标本升高不明显外,其余各组均有非常显著的升高。结论:ADA对各肝脏疾病的诊断有较高的特异性,对肝脏功能是否损伤有一定的临床意义。     

肝微循环障碍在病毒所致肝损伤中作用的研究

本研究旨在探讨肝微循环障碍在病毒所致肝损伤发生中的作用。实验Ⅰ,麻疹活疫苗同时注入家兔门静脉及耳缘静脉,24小时快速放血处死,立即取肝固定送病理检查。光镜下可见小血管扩张瘀滞,红细胞粘集贴壁、纤维蛋白血小板构成之微血栓形成等严重微循环障碍的改变。同时可见肝细胞变性、小灶状坏死及GPT(血清谷丙转氨酶)等也明显升高。如先给予山莨菪碱,GPT等升高受到明显抑制。实验Ⅱ,麻疹活疫苗仅单独注入门静脉,24小时GPT并不升高,仅门脉血管对静注去甲肾上腺素的收缩反应性上升。实验Ⅲ,麻疹活疫苗仅注入耳缘静脉,24小时GPT也不升高,但血小板粘附功能增强。结果提示:(1)在病毒感染中肝血管收缩反应性上升及血小板粘附功能增强是肝微循环障碍发生的基础。(2)肝微循环障碍是病毒所致肝损伤的机理之一。(3)山茛菪碱是防治病毒性肝损伤的有效药物。     

Computer-assisted liver surgery: clinical applications and technological trends

Oncological liver surgery and interventions aim for removal of tumor tissue while preserving a sufficient amount of functional tissue to ensure organ regeneration. This requires detailed understanding of the patient-specific internal organ anatomy (blood vessel system, bile ducts, tumor location). The introduction of computer support in the surgical process enhances anatomical orientation through patient-specific 3D visualization and enables precise reproduction of planned surgical strategies though stereotactic navigation technology. This article provides clinical background information on indications and techniques for the treatment of liver tumors, reviews the technological contributions addressing the problem of organ motion during navigated surgery on a deforming organ, and finally presents an overview of the clinical experience in computer-assisted liver surgery and interventions. The review concludes that several clinically applicable solutions for computer aided liver surgery are available and small-scale clinical trials have been performed. Further developments will be required more accurate and faster handling of organ deformation and large clinical studies will be required for demonstrating the benefits of computer aided liver surgery.     

Three-dimensional observations on the alterations of lobular architecture in chronic hepatitis with special reference to its angioarchitecture for a better understanding of the formal pathogenesis of liver cirrhosis

For a better understanding of the formal pathogenesis of liver cirrhosis, the angioarchitecture of the liver lobule in chronic viral hepatitis was investigated three dimensionally. The histological reconstruction method, using serial histological sections, was adopted for the three-dimensional observation. Histology of the case showed chronic active hepatitis with occasional fibrous bridging of the portal to portal tract or hepatic vein. Graphic reconstructions revealed various degrees of altered angioarchitecture from place to place. While the conducting portion of the portal vein was almost preserved, the pathological changes mostly began at the parenchymal portion, especially second step or subsequent branches of the portal vein. In general, portal vein branches showed damage such as stenoses, disappearance, an increase and decrease in number and distorted spatial arrangements. Even in less damaged portal tracts, portal veins showed such changes to some extent. In severely damaged places with bridging fibrosis, a normal lobular angioarchitecture was completely lost; instead, portal veins, arteries and hepatic veins were tangled with each other. Parenchymal nutrition was suggested to be dependent on the remaining third-step portal branches or newly formed ones. However, the hepatic vein system had a tendency to be preserved and distributed fluently in the parenchyma. The distortion of these portal vessels indicated various degrees of loss of the lobular architecture. In conclusion, it is suggested that an early histological sign of cirrhosis develops in the course of chronic hepatitis.     

Stem Cells in Liver Regeneration and Their Potential Clinical Applications

Stem cells constitute a population of “primitive cells” with the ability to divide indefinitely and give rise to specialized cells under special conditions. Because of these two characteristics they have received particular attention in recent decades. These cells are the primarily responsible factors for the regeneration of tissues and organs and for the healing of lesions, a feature that makes them a central key in the development of cell-based medicine, called Regenerative Medicine. The idea of wound and organ repair and body regeneration is as old as the mankind, reflecting the human desire for inhibiting aging and immortality and it is first described in the ancient Greek myth of Prometheus. It is of interest that the myth refers to liver, an organ with remarkable regenerative ability after loss of mass and function caused by liver injury or surgical resection. Over the last decade there has been an important progress in understanding liver physiology and the mechanisms underlying hepatic development and regeneration. As liver transplantation, despite its difficulties, remains the only effective therapy for advanced liver disease so far, scientific interest has nowadays been orientated towards Regenerative Medicine and the use of stem cells to repair damaged liver. This review is focused on the available literature concerning the role of stem cells in liver regeneration. It summarizes the results of studies concerning endogenous liver regeneration and stem cell experimental protocols. Moreover, this review discusses the clinical studies that have been conducted in humans so far.     

Ang-2对肝再生作用机制的研究进展

对于肝脏的晚期病变,一般的治疗手段往往不能奏效,很多患者只能通过肝脏切除术或肝移植进行治疗,因此探究肝再生的影响因素就变得十分必要。近年来研究发现促血管生成素-2（Ang-2）在肝再生过程中发挥着极其重要的调控作用,本文对Ang-2及其受体做了简要概述,对近几年Ang-2在肝再生作用机制的研究情况进行综述。     

小鼠实验性肝内胆汁郁积的肝脏微循环活体观察

异硫氰酸－１－茶酸（ＡＮＩＴ）造成的小鼠肝内胆汁郁积的肝微循环活体观察表明：２４ｈ、４８ｈ主要是渗出和白细胞增加，血流减慢；７２ｈ后渗出增加，出现血栓、出血、红细胞集聚。胆汁郁积血液粘度升高，血管中摩擦力增加，使血栓Ａ＿２（ＴＸＡ＿２）和前列环素（ＰＧＩ＿２）平衡受到破坏，微血管壁与血小板相互粘附，毛细胆管内压增加而扩张，邻近静脉受压，肝血窦胆汁郁滞，造成肝微循环障碍，致使肝细胞变性、坏死。因此对长期高胆红素血症病例，采用活血化瘀，改善肝微循环，有利于肝脏病的恢复。     

Experimental models of hepatectomy and liver regeneration using newborn and weaning rats

OBJECTIVES: Liver regeneration is a complex process that has not been completely elucidated. The model most frequently used to study this phenomenon is 70% hepatectomy in adult rats; however, no papers have examined this effect in developing animals. The aims of the present study were: 1) to standardize two models of partial hepatectomy and liver regeneration in newborn suckling and weaning rats, and 2) to study the evolution of remnant liver weight and histological changes of hepatic parenchyma on the days that follow partial hepatectomy. METHODS: Fifty newborn and forty-four weaning rats underwent 70% hepatectomy. After a midline incision, compression on both sides of the upper abdomen was performed to exteriorize the right medial, left medial and left lateral hepatic lobes, which were tied inferiorly and resected en bloc. The animals were sacrificed on days 0 (just after hepatectomy), 1, 2, 3, 4 and 7 after the operation. Body and liver weight were determined, and hepatic parenchyma was submitted to histological analysis. RESULTS: Mortality rates of the newborn and weaning groups were 30% and 0%, respectively. There was a significant decrease in liver mass soon after partial hepatectomy, which completely recovered on the seventh day in both groups. Newborn rat regenerating liver showed marked steatosis on the second day. In the weaning rat liver, mitotic figures were observed earlier, and their amount was greater than in the newborn. CONCLUSIONS: Suckling and weaning rat models of partial hepatectomy are feasible and can be used for studies of liver regeneration. Although similar, the process of hepatic regeneration in developing animals is different from adults.     

兔肝脏及附属管道的应用解剖学研究

目的　对兔肝脏及其附属管道进行应用解剖学研究。 方法　对20只日本大耳兔分别进行活体和离体形态学观察,制作门静脉和肝静脉管道铸型标本观察其分支与走行,测定各肝叶质量及其所占肝脏百分比。 结果　兔肝肝裂明显,依据肝叶形态、肝裂走行和门静脉主干分支形式将兔肝脏分为五叶,分别为尾状叶、左外叶、左中叶、右中叶、右外叶,各肝叶质量分别为(g)：3.93±1.13、15.93±3.50、14.83±3.31、15.08±4.34、12.08±3.55。左中叶和右中叶根部肝组织融合,其余各肝叶相对独立,尾状叶包括相对独立的乳头突和尾状突两部分。各肝叶有相对独立的Glisson系统和肝静脉走行于肝蒂内。 结论　兔肝解剖学特点与多数哺乳类实验动物肝脏解剖相似,同时又具有其自身特点,适合于肝脏外科疾病动物模型的制作。     

Biliary cytokeratin expression but not CD56 (N-CAM) expression aids in the differential diagnosis of non-neoplastic bile duct diseases

Non-neoplastic bile duct diseases include several entities with a variety of clinical and histopathologic features. In needle biopsies, however, these may overlap. Here, auxiliary diagnostic markers would be helpful. CD56 (N-CAM) has been reported in bile duct development, liver regeneration, and different liver diseases. This study was performed to evaluate the diagnostic value of CD56 immunohistochemistry compared to biliary cytokeratins in the diagnosis of non-neoplastic biliary liver diseases in liver needle biopsies.     

胚胎来源肝干细胞肝内移植后在小鼠体内的分布及分化

BACKGROUND:As many factors can lead to liver injury, we attempt to use the “therapeutic liver regeneration” technology in clinical treatment of liver diseases by promoting liver regeneration. OBJECTIVE:To investigate distribution and differentiation of embryonic liver stem cells in mice after intrahepatic transplantation via a transplantation approach. METHODS:Liver injury models were prepared in 20 BALB/c mice, and then randomly equivalently assigned into two groups: 70% partial hepatectomy with intrahepatic transplantation with 1×10⁵ embryonic liver stem cells in control group; therapeutic liver regeneration model plus intrahepatic transplantation with 1x10⁵ embryonic liver stem cells in observation group. At 1 and 2 weeks after cell transplantation, the liver parenchyma of mice was observed. And at 2 weeks, both of the two groups underwent confocal immunofluorescence assay. Besides, blood samples of mouse tail vein were collected to detect levels of serum albumin. RESULTS AND CONCLUSION:At 1 week after cell transplantation, in the liver parenchyma, green fluorescence was sparsely distributed in the two groups, and the distribution density had no significant difference between the two groups; at 2 weeks after cell transplantation, hepatic cord-like structures appeared in the liver parenchyma of two groups, and the green fluorescence distribution in the control group was limited, but significantly expanded in the observation group. At 2 weeks after cell transplantation, positive albumin expression in the liver parenchyma was significantly higher in the observation group than in the control group, and there was no significant difference in levels of serum albumin between two groups (P > 0.05). To conclude, after transplantation of embryonic liver stem cells in the therapeutic liver regeneration model mice hepatocytes can be effectively integrated into the host hepatic plate, differentiate in the liver, and partially trigger the function of hepatocytes.     

#br# microRNAs在肝再生中的作用研究进展

目的 microRNAs是一类非编码蛋白的小RNA分子,通过与靶基因mRNA 3’端的非编码区（3’UTRS）结合而在转录后水平调控基因表达,调节细胞增殖、分化、代谢、凋亡、器官发育等生物学过程。肝脏有很强的再生能力,是研究再生的重要材料。因此,我们在文中总结了microRNAs对肝再生调节作用的研究进展。