非酒精性脂肪肝与脂肪性肝炎动物模型研究进展

非酒精性脂肪肝是无酒精滥用的包括单纯性脂肪肝、脂肪性肝炎、脂肪性肝纤维化和肝硬化的肝病综合征,目前已成为广受关注的肝病医学难题。随着抗脂肪肝药物的深入研究,动物模型制作得到很好发展。近年来,在大鼠、沙鼠、小鼠、兔和小猪等动物种属成功地建立了食物、胃肠外营养与蛋氨酸胆碱缺乏等诱导的单纯性脂肪肝和脂肪性肝炎动物模型,这些模型为研究脂肪肝和脂肪性肝炎的发病机理与治疗提供了机会。每种动物模型各有优缺点,合理应用动物模型能更好地开展脂肪肝病的实验和临床研究。本文综述了非酒精性脂肪肝及脂肪性肝炎动物模型制作方法的若干研究进展。     

Animal models of NAFLD from a hepatologist's point of view

Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disorder closely linked to obesity, hyperlipidemia and type 2 diabetes and is increasingly recognized as a major health problem in many parts of the world. While early stages of NAFLD are characterized by a bland accumulation of fat (steatosis) in hepatocytes, the disease can progress to non-alcoholic steatohepatitis (NASH) which involves chronic liver inflammation, tissue damage and fibrosis and can ultimately lead to end-stage liver disease including cirrhosis and cancer. As no approved pharmacological treatment for NAFLD exists today, there is an urgent need to identify promising pharmacological targets and develop future therapies. For this purpose, basic and translational research in NAFLD animal models is indispensable. While a large number of diverse animal models are currently used in the field, there is an ongoing challenge to identify those models that mirror human pathology the closest to allow good translation of obtained results into further clinical development. This review is meant to provide a concise overview of the most relevant NAFLD animal models currently available and will discuss the strengths and weaknesses of these models with regard to their comparability to human disease conditions.     

病毒性肝炎树鼩动物模型研究与建模策略

病毒性肝炎是由肝炎病毒引起的肝脏疾病。在我国,病毒性肝炎高度流行,其中又以乙型肝炎病毒(Hepatitis B virus,HBV)和丙型肝炎病毒(Hepatitis C virus,HCV)危害较大。动物模型是研究疾病感染与发病机制,进行药物与疫苗研究的必要工具。目前病毒性肝炎实验动物模型的研究已取得长足的发展,主要集中于病毒在动物体内的感染特性及发病规律方面。本文仅就病毒性肝炎动物模型,尤其乙型、丙型肝炎树鼩动物模型的研究及建模策略进行综述。     

常用实验动物肝癌模型研究进展

肝癌动物模型是研究肝癌发病病因和机理的重要平台和手段,在肝癌研究过程中,肝癌动物模型的建立起着非常重要的作用,建立和提供良好的肝癌动物模型为今后进一步研究肝癌的致病机理,指导临床肝癌的诊断和治疗提供理论参考。     

Comparison of 3 Methods to Induce Acute Pulmonary Hypertension in Pigs

Large animal models for acute pulmonary hypertension (PHT) show distinct differences between species and underlying mechanisms. Two embolic procedures and continuous infusion of a stable thromboxane A₂ analogue (U46619) were explored for their ability to induce PHT and their effects on right ventricular function and pulmonary and systemic circulation in 9 pigs. Injection of small (100 to 200 µm) or large (355 to 425 µm) polystyrene beads and incremental dosage (0.2 to 0.8 µg kg⁻¹ min⁻¹) of U46619 all induced PHT. However, infusion of U46619 resulted in stable PHT, whereas that after bead injection demonstrated a gradual continuous decline in pressure. This instability was most pronounced with small beads, due to right ventricular failure and consecutive circulatory collapse. Furthermore, cardiac output decreased during U46619 infusion but increased after embolization with no relevant differences in systemic pressure. This result was likely due to the more pronounced effect of U46619 on pulmonary resistance and impedance in combination with limited effects on pulmonary gas exchange. Coronary autoregulation and adaption of contractility to afterload increase was not impaired by U46619. All parameters returned to baseline values after infusion was discontinued. Continuous infusion of a thromboxane A2 analogue is an excellent method for induction of stable, acute PHT in large animal hemodynamic studies.Abbreviations: PHT, pulmonary hypertensionSystematic investigation of the pathophysiology of acute pulmonary hypertension (PHT), especially adaption of the right ventricular function in response to increased afterload, requires valid animal models with conclusions that are transferable to humans. In addition, the availability of such models would promote the evaluation of treatment options for pulmonary vasodilatation and inotropic support of the right ventricle. The various models reported in the literature can be classified by animal size, developmental period, and techniques. Due to cardiac dimensions and basic regulatory principles, sophisticated and transferable hemodynamic measurements require large animals such as dogs, pigs, and goats, and differences in vasoconstrictory responsiveness and adaption to hypoxia between these species and humans must be taken into account.^9,16,21 Chronic models of PHT in large animals are used less frequently than acute models and typically are induced through injection of monocrotaline pyrrole,⁶ surgical creation of an aortopulmonary shunt,²² or pulmonary banding.⁵ Techniques for the induction of acute PHT can be weighed in light of their underlying mechanisms, side effects, stability, and reversibility. Exposure to hypoxia ⁶ and repeated embolism^{9,17, 23} are used more frequently than are constriction of the pulmonary artery or infusion of the stable thromboxane A₂ analogue U46619.⁵ Whereas hypoxia mediates vasoconstriction by means of endothelin 1, serotonin, and the inhibition of voltage-gated potassium channels in smooth muscle cells,⁷ embolic procedures reduce the vascular cross-sectional area and increase concentrations of thromboxane A2.¹⁹ The size of injected particles positively correlates with the degree of hypoxia⁹ and inversely correlates with induction of thromboxane A2 production, thereby resulting in PHT and circulatory collapse.¹⁹ These mechanisms influence the stability of PHT, cardiac function, and sympathetic tone as a consequence of hypoxia. A leading advantage of transient occlusion, constriction of the pulmonary artery, and infusion of U46619 is that the resulting PHT is reversible. Compared with embolic procedures, proximal occlusion of the pulmonary artery induced different grade of afterload increase for the right ventricle, whereas U46619 may have systemic and coronary vasoconstrictory effects, thus causing negative inotropy.¹¹ The design of a study involving a PHT model therefore is influenced not only by the animal and technique selected but also by the underlying mechanisms of the technique and the sensitivity of the resulting PHT to drug intervention.To study the effects of volatile anesthetics on right ventricular function during acute PHT, we aimed to develop a large animal model with stable increased afterload over several hours and minimal direct effects on cardiac function. We tested embolization techniques with different sizes of microbeads and the infusion of U46619. We favored pigs over dogs and goats because of the thickness of the arteriolar vascular muscle layer and the degree of collateral ventilation, which thus make the sensitivity of the pulmonary vasculature of swine more representative of that in humans.     

Animal models for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) represents ~90% of all cases of primary liver cancer and occurs predominantly in patients with underlying chronic liver disease and cirrhosis. Establishing appropriate animal models for HCC is required for basic and translational studies, especially the models that can recapitulate one of the human disease settings. Current animal models can be categorized as chemically-induced, genetically-engineered, xenograft, or a combination of these with each other or with a metabolic insult. A single approach to resemble human HCC in animals is not sufficient. Combining pathogenic insults in animal models may more realistically recapitulate the multiple etiologic agents occurring in humans. Combining chemical injury with metabolic disorder or alcohol consumption in mice reduces the time taken to hepatocarcinogenesis. Genetically-engineering weak activation of HCC-promoting pathways combined with disease-specific injury models will possibly mimic the pathophysiology of human HCC in distinct clinical settings.     

Wilson disease: Current status and the future

The focus of this minireview is on the current status and new advances in diagnosis and treatment of Wilson disease, an autosomal recessive disorder of copper metabolism. Molecular diagnostics have improved and complements current biochemical and clinical methods for screening for Wilson disease. Screening for Wilson disease in newborns is feasible and has been tested in limited populations, but is not yet widely performed. Identification of patients with Wilson disease as the cause of acute liver failure is possible using standard biochemical tests. Treatments for Wilson disease include chelating agents and zinc salts and liver transplantation. Future therapies may include hepatocyte transplantation and gene therapy, both of which have been tested and shown to work in animal models of Wilson disease. Future human studies await advances in these areas.     

Animal models for cystic fibrosis liver disease (CFLD)

Liver disease is a severe complication in patients with Cystic Fibrosis (CF), a genetic disease caused by mutations in the gene encoding for cystic fibrosis transmembrane conductance regulator (CFTR) channel. The sequence of events leading to CFLD is still unclear and has limited the development of more specific treatments other than the bile acid UDCA. However, in the last twenty years, several gaps have been filled, which have mainly been possible due to the availability of different animal models that mimic CF. CF mice, although they lack a spontaneous liver manifestation, have been essential to better understand the multiple functions of CFTR expression on the apical membrane of cholangiocytes, from chloride channel to regulator of epithelial innate immunity. Additionally, we have learned that the gut microbiota might be a pathogenetic factor for the development of liver disease. The recent creation of novel CF animal models (i.e. pig and ferret) that better reproduce the human disease, will allow for comparative studies with species that spontaneously develop the liver disease and will hopefully lead to novel therapeutic treatments. In this review, we have compared and summarized the main features of the current available CF animal models and their applicability for the study of the liver phenotype.     

Mixed cryoglobulinemia

Primary biliary cirrhosis (PBC) is a chronic and slowly progressive cholestatic liver disease of autoimmune etiology characterized by injury of the intrahepatic bile ducts that may eventually lead to liver failure. Affected individuals are usually in their fifth to seventh decades of life at time of diagnosis, and 90% are women. Annual incidence is estimated between 0.7 and 49 cases per million-population and prevalence between 6.7 and 940 cases per million-population (depending on age and sex). The majority of patients are asymptomatic at diagnosis, however, some patients present with symptoms of fatigue and/or pruritus. Patients may even present with ascites, hepatic encephalopathy and/or esophageal variceal hemorrhage. PBC is associated with other autoimmune diseases such as Sjogren's syndrome, scleroderma, Raynaud's phenomenon and CREST syndrome and is regarded as an organ specific autoimmune disease. Genetic susceptibility as a predisposing factor for PBC has been suggested. Environmental factors may have potential causative role (infection, chemicals, smoking). Diagnosis is based on a combination of clinical features, abnormal liver biochemical pattern in a cholestatic picture persisting for more than six months and presence of detectable antimitochondrial antibodies (AMA) in serum. All AMA negative patients with cholestatic liver disease should be carefully evaluated with cholangiography and liver biopsy. Ursodeoxycholic acid (UDCA) is the only currently known medication that can slow the disease progression. Patients, particularly those who start UDCA treatment at early-stage disease and who respond in terms of improvement of the liver biochemistry, have a good prognosis. Liver transplantation is usually an option for patients with liver failure and the outcome is 70% survival at 7 years. Recently, animal models have been discovered that may provide a new insight into the pathogenesis of this disease and facilitate appreciation for novel treatment in PBC.     

Role of cyclooxygenase isoforms in prostacyclin biosynthesis and murine prehepatic portal hypertension

Portal hypertension (PHT) is a common complication of liver cirrhosis and significantly increases morbidity and mortality. Abrogation of PHT using NSAIDs has demonstrated that prostacyclin (PGI(2)), a direct downstream metabolic product of cyclooxygenase (COX) activity, is an important mediator in the development of experimental and clinical PHT. However, the role of COX isoforms in PGI(2) biosynthesis and PHT is not fully understood. Prehepatic PHT was induced by portal vein ligation (PVL) in wild-type, COX-1(-/-), and COX-2(-/-) mice treated with and without COX-2 (NS398) or COX-1 (SC560) inhibitors. Hemodynamic measurements and PGI(2) biosynthesis were determined 1-7 days after PVL or sham surgery. Gene deletion or pharmacological inhibition of COX-1 or COX-2 attenuated but did not ameliorate PGI(2) biosynthesis after PVL or prevent PHT. In contrast, treatment of COX-1(-/-) mice with NS398 or COX-2(-/-) mice with SC560 restricted PGI(2) biosynthesis and abrogated the development of PHT following PVL. In conclusion, either COX-1 or COX-2 can mediate elevated PGI(2) biosynthesis and the development of experimental prehepatic PHT. Consequently, PGI(2) rather then COX-selective drugs are indicated in the treatment of PHT. Identification of additional target sites downstream of COX may benefit the >27,000 patients whom die annually from cirrhosis in the United States alone.     

Differential Emergence of Cortical Granule Breakdown and Electrophysiological Responses during Meiotic Maturation of Toad Oocytes

Oocytes of the toad, Bufo bufo japonicus , at various stages of progesterone-induced maturation were stimulated by pricking or treatment with Ca-ionophore A23187. Upon pricking, oocytes 14 hr after hormone treatment (PHT) underwent sequential activation responses, such as development of an activation potential, cortical granule breakdown (CGBD), and formation of a perivitelline space (PVS), like those of mature oocytes (18 hr PHT). When oocytes were pricked at 14 hr PHT, it took about 10 min for the wave of CGBD to spread over all the oocyte surface, in contrast to the case with mature oocytes in which it took about 150 sec. The rate of spread of CGBD was significantly less in the vegetal hemisphere than in the animal hemisphere in both mature and immature oocytes. Treatment with A23187 (1 μM) for 5 min induced an activation potential, and PVS formation by the oocytes from 10 hr PHT, which was 3–4 hr earlier than the time when these responses could be induced by pricking. Oocytes at 8–9 hr PHT also showed CGBD in response to A23187, but without formation of an activation potential. Several patches of local PVS caused by the non-propagating CGBD were observed in oocytes treated with the ionophore 5–7 hr PHT. When a high concentration (10 μM) of A23187 was employed, CGBD without PVS formation was induced even in oocytes at 0 hr PHT. These results indicate that the responsiveness to a Ca²⁺ surge that is a prerequisite for both CGBD and genesis of an activation potential is acquired for the repective responses at different stages of oocyte maturation.     

Functional analysis of the Arabidopsis PHT4 family of intracellular phosphate transporters

The transport of phosphate (Pi) between subcellular compartments is central to metabolic regulation. Although some of the transporters involved in controlling the intracellular distribution of Pi have been identified in plants, others are predicted from genetic, biochemical and bioinformatics studies. Heterologous expression in yeast, and gene expression and localization in plants were used to characterize all six members of an Arabidopsis thaliana membrane transporter family designated here as PHT4. PHT4 proteins share similarity with SLC17/type I Pi transporters, a diverse group of animal proteins involved in the transport of Pi, organic anions and chloride. All of the PHT4 proteins mediate Pi transport in yeast with high specificity. Bioinformatic analysis and localization of PHT4-GFP fusion proteins indicate that five of the proteins are targeted to the plastid envelope, and the sixth resides in the Golgi apparatus. PHT4 genes are expressed in both roots and leaves, although two of the genes are expressed predominantly in leaves and one mostly in roots. These expression patterns, together with Pi transport activities and subcellular locations, suggest roles for PHT4 proteins in the transport of Pi between the cytosol and chloroplasts, heterotrophic plastids and the Golgi apparatus.     

人源化鼠嵌合肝动物模型研究进展

动物模型是人类疾病研究、发病机制、药物研发的重要工具,对于困扰人类健康的肝脏疾病还没有理想的动物模型能有效地反映出人类疾病发病的机制。建立人源化鼠嵌合肝动物模型,对于研究人类肝脏疾病的发病机制、疫苗和药物的研发及疾病的诊治等方面都具有十分广阔的应用前景。     

乙型肝炎病毒DNA疫苗的研究进展

预防与控制乙型肝炎发病的乙型肝炎病毒(HBV)疫苗,是有重大的社会和经济意义。HBV的持续感染可引起慢性肝脏疾患,并逐步发展为肝硬化和肝细胞癌(HCC)。目前的乙肝重组亚单位疫苗可以使90％的接种产生保护性抗体;但是对慢性HBV携带,由于其机体对HBsAg蛋白产生耐受,不能产生体液和细胞免疫,因此它只能作为一种预防性的疫苗。DNA疫苗(基因疫苗)是一种新的疫苗技术,通过向体内递送编码抗原的细菌质粒,刺激产生特异的体液和细胞免疫反应。在小鼠和其他的肝炎病毒感染动物模型中,HBV DNA疫苗可以特异性地引起体液和细胞免疫,清除HBV转基因动物血循环中的HBsAg颗粒和HBV DNA。如果加入各种免疫调节细胞因子的基因,可以进一步提高HBV DNA疫苗的免疫效果,因此它不仅可作为预防性疫苗,也可作为治疗型疫苗。     

Systems biology approach to Wilson’s disease

Wilson’s disease (WD) is a severe disorder of copper misbalance, which manifests with a wide spectrum of liver pathology and/or neurologic and psychiatric symptoms. WD is caused by mutations in a gene encoding a copper-transporting ATPase ATP7B and is accompanied by accumulation of copper in tissues, especially in the liver. Copper-chelation therapy is available for treatment of WD symptoms and is often successful, however, significant challenges remain with respect to timely diagnostics and treatment of the disease. The lack of genotype-phenotype correlation remains unexplained, the causes of fulminant liver failure are not known, and the treatment of neurologic symptoms is only partially successful, underscoring the need for better understanding of WD mechanisms and factors that influence disease manifestations. Recent gene and protein profiling studies in animal models of WD began to uncover cellular processes that are primarily affected by copper accumulation in the liver. The results of such studies, summarized in this review, revealed new molecular players and pathways (cell cycle and cholesterol metabolism, mRNA splicing and nuclear receptor signaling) linked to copper misbalance. A systems biology approach promises to generate a comprehensive view of WD onset and progression, thus helping with a more fine-tune treatment and monitoring of the disorder.     

Animal models for the study of liver fibrosis: new insights from knockout mouse models

Fibrosis arises as part of a would-healing response that maintains organ structure and integrity following tissue damage but also contributes to a variety of human pathologies such as liver fibrosis. Liver fibrosis is an abnormal response of the liver to persistent injury with the excessive accumulation of collagenous extracellular matrices. Currently there is no effective treatment, and many patients end up with a progressive form of the disease, eventually requiring a liver transplant. The clarification of mechanisms underlying pathogenesis of liver fibrosis and the development of effective therapy are of clinical importance. Experimental animal models, in particular targeted gene knockouts (loss of function) in mice, have become a powerful resource to address the molecular mechanisms or significance of the targeted gene in hepatic functions and diseases. This review will focus on the recent advances in knowledge obtained from genetically engineered mice that provide novel insights into the pathophysiology of liver fibrosis.     

Liraglutide modulates gut microbiota and reduces NAFLD in obese mice

Metabolic disorders such as insulin resistance and diabetes are associated with obesity and nonalcoholic fatty liver disease (NAFLD). The aggressive form of a fatty liver disease may progress to cirrhosis and hepatocellular carcinoma. Furthermore, recent studies demonstrated that there is a dysbiosis in the gut microbiota associated with early stages of metabolic disease. Therefore, the identification and repurposing of drugs already used to treat insulin resistance may be an excellent option for other disorders. We evaluated the effect of liraglutide on obesity, NAFLD and gut microbiota modulation in two different animal models of obesity: the ob/ob mice and the high-fat diet (HFD)-fed mice. Liraglutide treatment induced significant weight loss in both obesity models, showed improvements in glycemic parameters and reduced inflammatory cell infiltration in the cecum and the liver. In ob/ob mice, the liraglutide treatment was able to reduce the accumulation of liver fat by 78% and reversed steatosis in the HFD mice. The gut microbiota analysis showed that liraglutide changed the overall composition as well as the relative abundance of weight-relevant phylotypes such as a reduction of Proteobacteria and an increase of Akkermansia muciniphila in the treated HFD group. We show that liraglutide can lead to weight loss and gut microbiota modulations, and is associated with an improvement of NAFLD. Furthermore, by generating a profile of the intestinal microbiota, we compiled a list of potential bacterial targets that may modulate metabolism and induce a metabolic profile that is considered normal or clinically controlled.     

Lessons from experimental Mycobacterium tuberculosis infections

Mycobacterium tuberculosis is the cause of enormous human morbidity and mortality each year. Although this bacterium can infect and cause disease in many animals, humans are the natural host. For the purposes of studying the pathogenesis of M. tuberculosis, as well as the protective and immunopathologic host responses against this pathogen, suitable animal models must be used. However, modeling the human infection and disease in animals can be difficult, and interpreting the data from animal models must be done carefully. In this paper, the animal models of tuberculosis are discussed, as well as the limitations and advantages of various models. In particular, the lessons we have learned about tuberculosis from the mouse models are highlighted. The careful and thoughtful use of animal models is essential to furthering our understanding of M. tuberculosis, and this knowledge will enhance the discovery of improved treatment and prevention strategies.     

肝靶向动物模型研究与展望

肝癌动物模型是抗肝癌药物实验及肝靶向给药系统验证的重要方法和手段。本文对用于研究肝靶向制剂的动物模型的种类、特征、不足及应用进行了研究论述,提出了目前较适于应用的模型,应用肝癌动物模型可以提供与肝癌病人相似的肝癌生物学特性,也为肝靶向给药制剂药代动力学指标的可靠性提供了保障。     

A comparison of experimental visceral leishmaniasis in the opossum, armadillo and ferret

Visceral leishmaniasis is a severe, chronic protozoal disease of humans and animals. Although chemotherapeutic agents are available for the treatment of this disease, problems such as drug toxicity, drug ineffectiveness and drug resistance of the parasite are responsible for treatment failures. To determine whether a drug is a potential antileishmanial agent, screening tests are performed using in vitro and in vivo models. Subsequently, a study using an appropriate animal model is performed to clearly determine the efficacy of a drug against Leishmania. Due to current public concerns regarding the use of companion animals in addition to the high costs of obtaining and maintaining these animals for research use, conventional animal models used in these chemotherapy studies, notably the dog and monkey, are becoming less acceptable. Therefore, new, less expensive and more accessible animal models are needed for the study of antileishmanial compounds. In this study, the armadillo, ferret and opossum were evaluated as possible new animal models for visceral leishmaniasis. The marked body weight loss, hepatomegaly, splenomegaly, large amastigote densities and the microscopic lesions observed in the infected opossums indicated that the opossum was more susceptible to visceral leishmaniasis than the armadillo or ferret.