重组8型腺相关病毒介导HBV急性感染树鼩模型建立

目的利用重组8型腺相关病毒介导1.3拷贝HBV基因组（1.3HBV,ayw亚型）在树鼩肝脏表达,建立HBV急性感染树鼩模型。方法通过大腿内侧静脉注射将携带有1.3 HBV的重组8型腺相关病毒（recombi-nant adeno-associated virus 8,rAAV8-1.3HBV）导入树鼩肝脏,通过ELISA检测树鼩血清中HBsAg、HBeAg、HBsAb、HBeAb、HBcAb,荧光定量PCR检测树鼩肝脏和血清中HBV DNA,全自动生化分析仪检测血清中ALT水平,并观察感染后肝脏的病变情况。结果 HBV感染主要血清标志物1～2周内均检测阳性;30 d后肝组织仍可检测到病毒抗原阳性细胞;55 d时肝组织HBV DNA拷贝数仍可达到104～105;树鼩血清中HBV DNA拷贝数持续一个月高于正常组;肝组织炎细胞略增多,血清ALT水平持续升高。结论 rAAV8所携带的HBV基因组高效专一导入树鼩肝细胞并复制表达,成功建立HBV急性感染树鼩模型,为进一步探索rAAV8树鼩慢性感染模型打下一定的基础。     

乙型肝炎病毒进入肝细胞机制研究进展

乙型肝炎病毒(hepatitis B virus,HBV)感染早期进入肝细胞机制研究一直是HBV研究领域的热点和难点．简单易得的HBV体外感染细胞模型是HBV感染进入机制研究无法逾越的主要障碍．近年来,随着新型HBV体外感染细胞模型的建立和应用(HepRG细胞和树鼩原代肝细胞),HBV的进入机制研究取得了一系列重大发现．综述了近几年HBV进入肝细胞机制的最新研究进展,主要包括HBV表面蛋白进入相关结构域的鉴定,已发现的候选HBV进入相关分子和尚待解决的问题．     

乙型肝炎病毒假病毒体外感染树鼩原代肝细胞模型的建立

目的：建立乙型肝炎病毒假病毒（HBVpp）体外感染树鼩原代肝细胞（PTH）模型。方法：通过肝脏原位两步灌注法分离PTH并对其冻存方法进行优化,通过共转染293T细胞生产基于慢病毒包装系统的HBVpp并考察其感染的种属和组织特异性。结果：通过肝脏原位两步灌注法分离了PTH,并优化了PTH冻存液的配方;包装的HBVpp具有与HBV真病毒类似的感染种属和组织特异性。结论：该模型的建立对深化HBV进入肝细胞机制的研究具有重要意义。     

EV71病毒对树鼩原代肾上皮细胞感染模型的建立

目的建立EV71对树鼩原代肾细胞的感染模型。方法胰蛋白酶消化法获得树鼩的原代肾细胞,用EV71感染树鼩肾细胞,测定1、2、4、6和8 d培养上清病毒滴度,分别用Western blot和间接免疫荧光法检测细胞中EV71病毒VP1蛋白的表达,以确定EV71病毒对树鼩原代肾细胞的感染性。结果对分离得到的树鼩原代肾细胞进行传代纯化和形态鉴别,建立以树鼩原代肾细胞为主的细胞培养。用EV71病毒感染树鼩原代肾细胞,感染后48~96 h病毒滴度可达到1.3×10~6TCID_(50)/m L,说明EV71病毒可有效感染树鼩原代肾细胞并有效增殖。Western blot检测发现,EV71病毒VP1蛋白可在感染后2~8 d的树鼩原代肾细胞中有效检出,间接免疫荧光法则在感染后2~6 d细胞的细胞质中检测到病毒VP1蛋白的分布。结论在成功建立树鼩原代肾细胞培养的基础上,确定了EV71病毒对树鼩原代肾细胞的感染性和病毒增殖特性,初步建立了EV71树鼩原代肾细胞感染模型。     

动物模型与人类疾病机理重点实验室成功破译树鼩基因组

由中科院昆明动物所动物模型与人类疾病机理重点实验室、深圳华大基因研究院等单位合作完成的破译树鼩基因组研究工作于2013年2月在《自然·通讯》(Nature Communications)杂志上在线发表。研究人员通过完成高质量的树鼩(Tupaia belangeri chinensis)全基因组测序及比较基因组分析,阐明了其系统分类地位和相关生物学特征的遗传基础,尤其是树鼩用于若干重要疾病如HBV、HCV感染以及抑郁症模     

树鼩驯养繁殖实验动物标准及丙型肝炎动物模型技术平台建设的探讨

目前用于生物医学研究的树鼩大多数来源于野生捕获,由于个体间的差异较大,其实验结果的均匀性和可重复性较差,亟待解决树鼩人工饲养设施条件、繁殖关键技术、动物质量标准等一系列基础科学问题。许多文献报道了树鼩与其他实验动物比较,在肝病研究中具有巨大的开发应用潜力,在丙型肝炎动物模型研究中,亟待解决丙型肝炎病毒(HCV)细胞培养体系、HCV病毒感染剂量与感染途径、HCV病毒对树鼩体外、体内感染后病毒核酸检测、抗体检测和病理检测等技术方法的建立及动物模型评价标准问题。本文就上述问题系统性地进行初步报道,旨在为树鼩早日实现实验动物化和丙型肝炎疾病研究中应用提供科学依据。     

人轮状病毒G1P[8]型感染树鼩原代小肠上皮细胞模型的建立

目的探究树鼩原代小肠上皮细胞的增殖特性,建立人轮状G1P[8]型病毒体外感染树鼩原代小肠上皮的细胞模型。方法采用胶原酶XI和中性蛋白酶I联合消化法获取树鼩原代小肠上皮细胞,经纯化和鉴定,用人轮状G1P[8]型病毒感染细胞,测定培养上清的病毒滴度和载量,并用Western blot和间接免疫荧光检测法检测人轮状病毒G1P[8]型VP6蛋白的表达情况,评价人轮状G1P[8]型病毒体外对树鼩原代小肠上皮细胞的感染性。结果分离的树鼩原代小肠上皮细胞经传代培养纯化,获得纯度达90%树鼩原代小肠上皮细胞。对树鼩原代小肠上皮细胞、原代肾细胞、HCT116细胞和MA104细胞进行轮状病毒易感性比较,确定树鼩原代小肠上皮细胞可以被人轮状病毒G1P[8]感染,培养72 h时病毒滴度可达到2.0×105TCID_(50)/m L。经Western blot和间接免疫荧光发现在人轮状G1P[8]型病毒感染树鼩原代小肠上皮细胞1~5 d均能检测到人轮状病毒VP6蛋白的表达和分布。结论确立了树鼩原代小肠上皮细胞的分离、纯化与培养方法,并建立了人轮状G1P[8]型病毒感染树鼩原代小肠上皮细胞的体外模型。     

树鼩在眼科学的基础研究进展

树鼩具有发达的视觉系统,视锥细胞数量占感光细胞的96%,具有较好的色觉及立体视觉。我国的树鼩资源丰富,成本较低,比较医学和基因组学研究证实树鼩是理想的眼科实验动物模型。利用树鼩开展眼科研究主要集中在近视模型的建立、近视对巩膜和脉络膜造成的变化,以及树鼩视网膜、视神经、角膜及视皮质的基础研究。本文对树鼩在眼科学方面的基础研究结果进行综述。     

树免疫细胞体外感染Ⅰ型人免疫缺陷病毒的实验研究

至今可以感染Ⅰ型人免疫缺陷病毒(HIV-1)的动物只有黑猩猩和长臂猿,这严重阻碍了HIV-1的疫苗研究和治疗研究。因此,寻找新的可以感染HIV-1的动物模型成为十分迫切的课题。已知树鼩对许多重要的医学病毒易感,为了探讨树鼩是否可以感染HIV-1,利用不同辅助受体的5种HIV-1病毒株,体外感染云南野生成年树鼩的淋巴细胞和单核/巨噬细胞;同时还用这些病毒感染人外周血淋巴细胞或单核细胞。然后用RT-PCR、PCR和流式细胞术分别进行检测。用RT-PCR方法未检测到感染上清中有病毒粒子的存在,用PCR法未能发现树鼩的这些免疫细胞中有前病毒DNA,用流式细胞术也未能在这些感染HIV-1的树鼩细胞的表面检测到特异抗原;而感染HIV-1的人免疫细胞均为阳性结果。实验结果表明树鼩的这些免疫细胞在体外未能感染上HIV-1,可能的原因是树鼩的这些免疫细胞的HIV-1受体(CD4)和辅助受体(CCR5或CXCR4)与人的免疫细胞差别较大。     

The tree shrews: adjuncts and alternatives to primates as models for biomedical research

The tree shrews are non-rodent, primate-like, small animals. There is increasing interest in using them to establish animal models for medical and biological research. This review focuses on the use of the tree shrews in in vivo studies on viral hepatitis, hepatocellular carcinoma (HCC), myopia, and psychosocial stress. Because of the susceptibility of the tree shrews (Tupaia belangeri) and their hepatocytes to infection with human hepatitis B virus (HBV) in vivo and in vitro, these animals have been used to establish human hepatitis virus-induced hepatitis and human HBV- and aflatoxin B1-associated HCC models. As these animals are phylogenetically close to primates in evolution and have a well-developed visual system and color vision in some species, they have been utilized to establish myopia models. Because dramatic behavioral, physiological, and neuroendocrine changes in subordinate male tree shrews are similar to those observed in depressed human patients, the tree shrews have been successfully employed to experimentally study psychosocial stress. However, the tree shrews holds significant promise as research models and great use could be made of these animals in biomedical research.     

野生中缅鼩病毒携带情况的初步调查

病毒学检测和监测是树鼩实验动物化和质量控制的重要标准和依据,而野生中缅树鼩是否携带人兽共患病毒鲜见报道。本研究采用酶联免疫吸附(ELISA)方法,对来源于云南昆明市城郊青龙峡地区的野生树鼩是否携带单纯疱疹病毒、轮状病毒、流感病毒、柯萨奇病毒、甲肝病毒、乙肝病毒、丙肝病毒、丁肝病毒、登革热病毒、出血热病毒和麻疹病毒等11种常见病毒进行筛查。结果表明,在已筛查的60只野生中缅树鼩中,可检测到单纯疱疹病毒和柯萨奇病毒,其血清抗体阳性比例分别为36.7%(22/60)和1.67%(1/60),而在粪便中仅检测到轮状病毒,其抗原阳性为6.7%(4/60),未检测到其他病毒,初步显示了野生树鼩自然状态下携带病毒的状况。为此,建议将单纯疱疹病毒、柯萨奇病毒和轮状病毒列为普通级树鼩病毒质量控制的首检项目,进一步大样本筛查将显示是否将其他病毒列为必检项目。     

饲料营养对饲血蚊传播HBV动物实验的影响

以含乙型肝炎病毒的人血饲喂三属蚊叮咬树，复制人类乙型肝炎病毒感染动物模型１～２个月后，由于饲料营养配比不当，树的出现消瘦、被毛蓬松、死亡率较高。经调整饲料有效营养配比，制成均衡营养颗粒饲料。提高了树乙型肝炎病毒感染模型的存活率，取得了较好的效果。     

Mutations in the p53 tumor suppressor gene in tree shrew hepatocellular carcinoma associated with hepatitis B virus infection and intake of aflatoxin B1

Infection with hepadnaviruses and exposure to aflatoxin B1 (AFB1) are considered to be major risk factors in the development of hepatocellular carcinoma (HCC) in humans. A high rate of p53 mutations at codon 249 has been reported in these tumors. The tree shrew (Tupaia belangeri chinensis) is a useful animal model for the development of HCC after human hepatitis B virus (HBV) infection or AFB1 treatment. Therefore, it was of particular interest to determine whether the p53 gene in tree shrew HCCs associated with HBV infection and/or with exposure to AFB1 is affected in the same manner as in human HCCs. We determined the tree shrew p53 wild-type nucleotide sequences by RT-PCR and automatic DNA-sequencing. Tree shrew wild-type p53 sequence showed 91.7 and 93.4% homologies with human p53 nucleotide and amino acids sequences, respectively, while it showed 77.2 and 73.7% homologies in mice. One HCC and normal liver tissue from AFB1 treated and one HCC from AFB1- and HBV-treated tree shrew showed no change in p53 sequences, while three HCCs from AFB1- and HBV-treated tree shrews showed point mutations in p53 sequences. One HCC showed point mutations at codon 275, which is on the DNA-binding domain of p53 gene, which might be a cause of gain-of-function during the development of HCC. As a result, our finding indicates that tree shrews exposed to AFB1 and/or HBV had neither codon 249 mutations nor significant levels of other mutations in the p53 gene, as is the case with humans.     

Experimental chronic hepatitis B infection of neonatal tree shrews (Tupaia belangeri chinensis): A model to study molecular causes for susceptibility and disease progression to chronic hepatitis in humans

ABSTRACT: BACKGROUND: Hepatitis B virus (HBV) infection continues to be an escalating global health problem. Feasible and effective animal models for HBV infection are the prerequisite for developing novel therapies for this disease. The tree shrew (Tupaia) is a small animal species evolutionary closely related to humans, and thus is permissive to certain human viral pathogens. Whether tree shrews could be chronically infected with HBV in vivo has been controversial for decades. Most published research has been reported on adult tree shrews, and only small numbers of HBV infected newborn tree shrews had been observed over short time periods. We investigated susceptibility of newborn tree shrews to experimental HBV infection as well as viral clearance over a protracted time period. RESULTS: Forty-six newborn tree shrews were inoculated with the sera from HBV-infected patients or tree shrews. Serum and liver samples of the inoculated animals were periodically collected and analyzed using fluorescence quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, Southern blot, and immunohistochemistry. Six tree shrews were confirmed and four were suspected as chronically HBV-infected for more than 48 (up to 228) weeks after inoculation, including three that had been inoculated with serum from a confirmed HBV-infected tree shrew. CONCLUSIONS: Outbred neonatal tree shrews can be long-term chronically infected with HBV at a frequency comparable to humans. The model resembles human disease where also a smaller proportion of infected individuals develop chronic HBV related disease. This model might enable genetic and immunologic investigations which would allow determination of underlying molecular causes favoring susceptibility for chronic HBV infection and disease establishment vs. viral clearance.     

Diabetes Mellitus Is Associated with Hepatocellular Carcinoma: A Retrospective Case-Control Study in Hepatitis Endemic Area

Background

A number of case-control patient studies have been conducted to investigate the association between diabetes mellitus (DM) and hepatocellular carcinoma (HCC). Despite some controversial reports, it has been suggested that DM is associated with HCC. The previous studies on this subject vary in the selection of populations, sample sizes, methodology, and analysis results. Therefore, it is necessary to further delineate the involvement of DM, together with other related risk factors, in HCC with large sample size and strict analysis methodology.

Methods

We conducted a hospital-based retrospective case-control study at Perking Union Medical College Hospital, China. A total of 1,568 patients with liver diseases were enrolled in the statistical study to evaluate the association of DM and other risk factors with HCC. Among these patients, 716 of them were diagnosed with benign liver diseases, and 852 patients were diagnosed as HCC. We utilized binary logistic regression and stepwise logistic regression to investigate the associations among DM, hypertension, fatty liver, cirrhosis, gallstone, HBV infection, HCV infection, and HCC.

Results

Statistical analysis through the stepwise regression model indicated that the prevalence of DM, male gender, cirrhosis, HCV infection, or HBV infection is higher in the HCC patient group compared to the control group. However, the prevalence of gallstone is negatively associated with HCC cases. DM co-exists with HBV infection, male gender, and age in the HCC cases. Binary logistic regression analysis suggested that DM may synergize with HBV infection in HCC development.

Conclusion

DM is strongly associated with the increased risk of HCC regardless of the prevalence of HBV infection, HCV infection, cirrhosis, male gender, and age. However, the synergistic interaction between DM and HBV in HCC occurrence is significant. Therefore, DM patients with HBV infection represent a very high HCC risk population and should be considered for HCC close surveillance program.     

IL-23 production of liver inflammatory macrophages to damaged hepatocytes promotes hepatocellular carcinoma development after chronic hepatitis B virus infection

Liver inflammation after chronic hepatitis B virus (HBV) infection is essential for hepatocellular carcinoma (HCC) development. We did a nested case-control study based on QBC chronic HBV infection cohort to identify HCC-related inflammatory cytokines. Serum levels of distinct Th-cell representative cytokines at varied periods before HCC diagnosis were determined in 50 HCC cases and 150 age- and gender-matched controls who did not develop HCC in 8–10?years. The individuals with HCC outcome had statistically higher serum levels of IL-23 than controls (P?<?0.01). Further analysis in HCC tissues showed that CD14⁺ inflammatory macrophages were the major IL-23 producers. Monocytes-derived macrophages generated more amount of IL-23 after being stimulated with cell-associated HBV core antigen from damaged HBV-infected hepatocytes than the cells being stimulated with HBV-S and HBV e antigen, which are secreted from infected hepatocytes. IL-23 upregulated IL-23 receptor expressions on macrophages, enhanced macrophage-mediated angiogenesis. In HBV-transgenic (Alb1HBV) mice, administration of diethylnitrosamine induced more liver tumors than in wild-type mice. The livers of Alb1HBV mice had higher concentrations of IL-23 and vascular endothelial growth factor (VEGF) than the wild-type mice. Neutralizing IL-23 activity, diethylnitrosamine-treated Alb1HBV mice developed significantly less tumors and produced less VEGF, tumor angiogenesis was inhibited with dramatically decreased CD31⁺ cells within tumor mass (all P?<?0.01).

Interaction of aflatoxin and hepatitis B virus in the pathogenesis of hepatocellular carcinoma

Aflatoxin-B1 (AFB) and chronic hepatitis B virus (HBV) infection epidemiologically correlate with the geographic distribution of hepatocellular carcinoma (HCC). Integration of HBV DNA into the cellular genome of HCCs and the in vivo formation of adducts between AFB and nucleic acids lead us to suggest that hepatocytes with integrated HBV DNA preferentially accumulate AFB; the AFB-adducts formed may then initiate cell transformation by modifying the expression of critical host genes. The altered molecular biology of liver cells in HCC is evidenced by the fact that HBV does not replicate in HCC tissues or cell lines. The effect of AFB on the expression of cellular genes such as endogenous retrovirus(es) and possibly cellular oncogene(s) can be analyzed in HCC cell lines with and without integrated HBV DNA. In addition, human HCC tissues can be probed for HBV sequences and AFB-DNA adducts at the single-cell level. The presence of HBV and AFB can be correlated with the expression of putative transforming genes, providing a new insight into the interaction between liver cells, HBV and AFB in the pathogenesis of HCC.     

Association of interleukin-18 genotypes (−607C > A) and (−137 G > C) with the hepatitis B virus disease progression to hepatocellular carcinoma

Chronic infection with HBV has been reported to be associated with the development of HCC. The inflammation mounted by cytokine-mediated immune system plays an important role in the pathogenesis of HBV-associated HCC. IL-18 is a pro-inflammatory cytokine whose role in the development of HBV-associated chronic to malignant disease state has not been much studied. The present study was conceived to determine the role of genetic polymorphisms in IL-18, serum levels of IL-18, and expression level of its signal transducers in the HBV disease progression. A total of 403 subjects were enrolled for this study including 102 healthy subjects and 301 patients with HBV infection in different diseased categories. Polymorphism was determined using PCR–RFLP. Genotypic distributions between the groups were compared using odd’s ratio and 95% CI were calculated to express the relative risk. Circulating IL-18 levels were determined by ELISA. Expression levels of pSTAT-1 and pNF?B was determined by western blotting. In case of IL-18(??607C?>?A), the heterozygous genotype (CA) was found to be a protective factor while in case of IL-18(??137G?>?C) the heterozygous genotype (GC) acted as a risk factor for disease progression from HBV to HCC. Moreover, serum IL-18 levels were significantly increased during HBV disease progression to HCC as compared to controls. Also the levels of activated signal transducers (pSTAT-1 and pNF-κB) of IL-18 in stimulated PBMCs were significantly increased during HBV to HCC disease progression. These findings suggest that IL-18 has the potential to act as a biomarker of HBV-related disease progression to HCC.

     

An intronic polymorphism in the corticotropin-releasing hormone receptor 2 gene increases susceptibility to HBV-related hepatocellular carcinoma in Chinese population

Corticotropin-releasing hormone receptor 2 (CRHR2) plays a role in both the central nervous system (CNS) and the peripheral nervous system. CRHR2 together with its ligands, urocortins (Ucns) and corticotropin-releasing hormone (CRH), functions as a mediator of inflammatory response and inhibitor of angiogenesis. Recently, it has been reported to be expressed in many human cancers. An association between rs2267716 polymorphism in the CRHR2 gene and susceptibility to hepatocellular carcinoma (HCC) was found in patients with chronic hepatitis C virus (HCV) infection. In the present study we analyzed, using a polymerase chain reaction–ligation detection reaction (PCR–LDR), the rs2267716 polymorphism in 364 hepatitis B virus (HBV)-related HCC patients, 196 non-HCC patients with HBV infection, and 404 healthy controls. The aim was to detect the possible association of this single-nucleotide polymorphism (SNP) with susceptibility to HBV-related HCC. Significant differences of rs2267716 allele were detected between HBV-related HCC patients and healthy controls (OR = 1.55, 95% CI 1.13–2.15, P = 0.007) or non-HCC patients with HBV infection (OR = 1.61, 95% CI 1.13–2.31, P = 0.009). These results suggest that the rs2267716 polymorphism in the CRHR2 gene might influence the risk of developing HCC in patients with HBV infection in Chinese population.