Animals ; Behavior, Animal ; Reproduction ; physiology ; Social Behavior ; Tupaiidae ; physiology

The aim of this study is to establish a method to culture and purify cerebral astrocyte of tree shrew (Tupaia belangeri), a kind of new laboratorial animal which is a relative of primates. Newborn tree shrews were used in this experiment. The cortex of cerebrum was isolated and placed in 4°C for 20 min to injure neurons. The cortical tissue was disaggregated by trypsin digestion. Differential attachment method was used to remove fibroblasts. The mixed culture was rinsed by trypsin (0.005%) solution to remove neurons. Upon reaching 70% confluence, the culture was subjected to static trypsin digestion until a white slice film exfoliated from the bottom of culture bottle. This film, i.e. astrocyte layer, was taken out and cultured, and the third passage was identified by immunocytochemical staining and immunofluorescence with anti-glial fibrillary acidic protein (GFAP) antibody. The result showed the purity of tree shrew astrocytes was more than 98%. Thus the method to culture highly purified astrocyte of tree shrew was successfully established, which would contribute to further study in central nervous system physiology and diseases in this new laboratorial animal.
Animals ; Astrocytes ; cytology ; Brain ; cytology ; Cell Separation ; methods ; Primary Cell Culture ; methods ; Tupaiidae

Using tree shrew as an animal model, our previous studies have demonstrated synergistic effects of aflatoxin B-1 (AFB(1)) and human hepatitis B virus (HHBV) in the induction of hepatocellular carcinoma (HCC). In the present study, we have examined expression of p53 gene in HCCs induced by AFB(1) with or without HHBV infection in tree shrews. Avidin-biotin-peroxidase complex immunohistochemical method with human p53-CM1 polyclonal antibody has been used to detect p53 expression in serial sections of paraffin-embedded liver and HCC tissues. Five out of 9 animals with HCCs (55.6%) induced by AFB(1) with HHBV infection and 2/3 animals with HCCs (66.7%) induced by AFB(1) alone expressed the p53 protein. Out of 18 HCCs examined, expression of p53 protein was observed in 9/10 moderately and poorly differentiated HCCs (0/8). None of the well differentiated HCCs (0/8) expressed p53 (0%). Similarly, no p53 expression was observed in either non-tumorous or hyperplastic liver tissues or nodules. These results suggest that p53 expression associated with p53 mutation is a late event occurring probably during tumor progression in AFB(1) and HHBV induced hepatocarcinogenesis in the tree shrew. This report is the first example of an experimental animal model where combination of human HBV and AFB(1)-induced HCCs demonstrate p53 expression.
Aflatoxin B1* ; Aflatoxins* ; Animals ; Carcinoma, Hepatocellular* ; Genes, p53* ; Hepatitis B virus* ; Hepatitis B* ; Hepatitis* ; Humans* ; Liver ; Models, Animal ; Tupaiidae*

OBJECTIVETo understand the molecular mechanism and find out the responsible genes for liver cancer by exploring the regulation of gene expression during hepatocarcinogenesis in tree shrew induced by aflatoxin B1 (AFB1).

METHODSThe tissues from tree shrew of different stages during the pathogenesis and development of hepatocellular carcinoma (HCC), liver cancer tissue, para-cancerous tissues, pre-cancerous liver tissues, liver tissues of the same stage from normal controls and the liver tissues taken before AFB1-treatment were analyzed for gene expression by cDNA array.

RESULTSFour patterns of gene expression were observed during AFB1-induced hepatocarcinogenesis. They were: genes up-regulated in HCC tissue and para-cancerous tissue, especially in HCC tissues; genes with similar expressing level in both HCC tissue and para-cancerous tissue, but higher than that in pre-cancerous tissue; genes down-regulated in HCC tissue; genes up-regulated before HCC appeared but down-regulated after HCC appeared.

CONCLUSIONDynamic observation of gene expression will be beneficial to elucidate the mechanisms of AFB1- induced hepatocarcinogenesis and locate the responsible genes.

Aflatoxin B1 ; toxicity ; Animals ; Gene Expression Profiling ; Liver Neoplasms, Experimental ; chemically induced ; genetics ; Oligonucleotide Array Sequence Analysis ; methods ; Tupaiidae

OBJECTIVESTo study the differential expression of genes in signal transduction pathway (STP) during the hepatocarcinogenesis in tree shrews induced by AFB1 and/or HBV and to elucidate the molecular mechanism of hepatocellular carcinoma (HCC) development.

METHODSAdult tree shrews were divided into three groups: Group A was fed AFB1 only, Group B was infected firstly with HBV then fed AFB1 as in Group A, Group C served as the normal control. Liver biopsies were obtained at the 30th, 60th and 90th week of the experiment or until HCC occurred and the animals were sacrificed. Tree shrew-specific cDNA microarray was applied for detecting the differential expression of corresponding genes in each group at different time points during the experiment, and real time RT PCR was applied to verify the results of the cDNA microarray.

RESULTSGenes of IGF-II, C-rel, and NF-kappaB2 were differentially expressed between para-cancerous tissues and HCC tissues in both group A and group B, and the differential expression of bcl-2, cyclin A and CNTF was only seen in group B. Between the experimental groups A and B and the control group C, there were differential expressions of CNTF and cyclin A in the early 30th week and middle 60th week stage of hepatocarcinogenesis in tree shrews. Real time RT PCR results showed that the expression level of IGF-II and C-Rel in group A and of IGF-II in group B in HCC tissues were significantly lower than that in the adjacent non-cancerous tissues and in the biopsies taken at the 30th and 60th week of the experiment. Nevertheless, there were no significant differences between the para-cancerous tissues and the cancer tissues at the 30th and 60th week. These results were consistent with the cDNA microarray assay. The expression levels of C-Rel and CNTF in group B were not obviously altered in the para-cancerous tissues, HCC and at the 60th week, but they were significantly lower in these tissues than that in the tissues at the 30th week. In group A, the expression levels of CNTF in adjacent liver and HCC tissues were higher than that in para-cancerous lesions, but the difference did not reach a statistically significant level. In group C, the expression level of IGF-II, C-Rel and CNTF at different stages showed no significant differences, which was consistent with the cDNA microarray results.

CONCLUSIONSTo apply the tree shrew-specific cDNA microarray to detect the differential expression of genes related to signal transduction pathway during tree shrew hepatocarcinogenesis could be a valuable utility for further comprehending the mechanism of HCC. IGF-II, NF-kappaB2, C-rel, Bcl-2, and cyclin A. CNTF may be involved in the occurrence and progress of HCC in tree shrews.

Animals ; Carcinoma, Hepatocellular ; genetics ; Female ; Gene Expression Profiling ; Liver Neoplasms, Experimental ; genetics ; Male ; Oligonucleotide Array Sequence Analysis ; Signal Transduction ; Tupaiidae

OBJECTIVETo evaluate the mRNA and protein expressions of peroxiredoxin II (PrxII) in hepatocellular carcinoma (HCC) and their significance.

METHODSHCC was induced by aflatoxin B1 (AFB1) in 6 tree shrews (Tupaia belangeri chinensis). The expression levels of PrxII mRNA and protein were detected by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot on HCC tissues and on their surrounding liver tissues (para-HCC). Biopsied liver tissues were taken before the HCC induction (pre-HCC) from the same animals and from a group of blank controlled animals that served as controls. Liver biopsy specimens from 18 cases of human HCC and from 17 healthy human volunteers were studied using the same methods.

RESULTSThe mRNA and protein expressions of PrxII in tree shrew HCC tissues were significantly higher than those in para-HCC and pre-HCC tissues, and also higher than those in the liver tissues from the control animals (all P < 0.05). The expression levels of PrxII mRNA and protein in human HCC tissues were also significantly higher than those in their para-HCC tissues and in the human normal liver tissues (P < 0.05).

CONCLUSIONPrxII might play an important role in hepatocarcinogenesis and might be used as a molecular target for HCC prevention and treatment.

Adult ; Aged ; Animals ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Female ; Humans ; Liver ; metabolism ; pathology ; Liver Neoplasms ; metabolism ; pathology ; Liver Neoplasms, Experimental ; metabolism ; pathology ; Male ; Middle Aged ; Peroxiredoxins ; genetics ; Tupaiidae

OBJECTIVETo acquire cDNA sequence of lecithin-cholesterol acyltransferase (LCAT) from tree shrew and analyze the sequence structure.

METHODSThe first strand cDNA was acquired by reverse transcription using mRNA from tree shrew liver as template. By the method of SMART RACE PCR, tree shrew LCAT cDNA was acquired and deduced its amino acids sequence. The sequence and structure of tree shrew LCAT cDNA and amino acid were analyzed and predicted by the molecular software.

RESULTSTree shrew LCAT cDNA is composed of 1,340 bp, including 2 bp 5' untranslated region (5' UTR), 1,320 bp open reading frame (ORF) which encodes protein precursor of 440 amino acids (24 amino acids signal peptide and 416 amino acids mature peptide), and 18 bp 3' untranslated region (3'UTR). The stop codon is TAA and there is a poly (A) signal sequence AATAAA and a 25 bp poly (A) tail. Tree shrew LCAT cDNA sequence has been accepted by GenBank as a new gene, accession number AF272861 and its homology with human and baboon was 90% and 89%, respectively.

CONCLUSIONThe sequence of LCAT cDNA in tree shrew has high identity with that of human and other experimental animal species.

Amino Acid Sequence ; Animals ; Base Sequence ; DNA, Complementary ; genetics ; Liver ; enzymology ; Molecular Sequence Data ; Open Reading Frames ; genetics ; Phosphatidylcholine-Sterol O-Acyltransferase ; chemistry ; genetics ; Sequence Analysis, Protein ; Tupaiidae

Chronic hepatitis B affects 400 million people worldwide and is one of the leading causes of liver-related morbidity and mortality. All clinically available hepatitis B virus (HBV) drugs are nucleoside or nucleotide analogs that inhibit viral reverse transcriptase (RT) activity. Resistance to these HBV drugs has been widely reported, and is due to specific mutations in the viral RT domain. Therefore, the development of new, non-polymerase targeting anti-HBV agents is urgently needed. A potential drug target, the HBV receptor that mediates the viral entry process, has been recently identified using human primary hepatocytes, northern tree shrew (Tupaia belangeri) hepatocytes, and HepaRG cells. A transporter of bile acids, sodium taurocholate cotransporting polypeptide (NTCP), was identified as the receptor for HBV and hepatitis D virus, and the transport function of NTCP was correlated with HBV entry. Therefore, functional inhibitors of NTCP may inhibit HBV infection, and viral entry was blocked by several NTCP receptor-targeting compounds. The HBV receptor is an attractive target for development of entry inhibitors, and serves as a model for the mechanistic study of HBV entry and infection. This review will summarize the characteristics and clinical importance of NTCP, and will discuss the potential therapeutic use of NTCP inhibitors to prevent HBV entry.
Bile Acids and Salts ; Hepatitis B virus ; Hepatitis B* ; Hepatitis B, Chronic ; Hepatitis Delta Virus ; Hepatocytes ; Humans ; Mortality ; RNA-Directed DNA Polymerase ; Taurocholic Acid ; Tupaiidae

BACKGROUNDTo serially observe the pathologic changes in livers of tree shrews and macaca assamensises infected with HHBV.

METHODS10 adult tree shrews and 28 macaca assamensises were inoculated with HBV rich human sera. The liver of the animals were regularly biopsied. The liver samples were examined histopathologically by HE staining. Some samples were stained for HBsAg by immunohistochemistry (IH), and HBV DNA by in situ hybridization (ISH).

RESULTSHBsAg in 80% of tree shrews infected with HHBV can be detected by IH, HBV DNA in 50% of those can be found by ISH.The positive rates of HBsAg in macaca assamensises' livers were 25% by IH, none HBV DNA was detected.

CONCLUSIONThe tree shrew model seems to be applicable for the research of human hepatitis B.

Animals ; Antibodies, Viral ; immunology ; Disease Models, Animal ; Female ; Hepatitis B ; immunology ; pathology ; virology ; Hepatitis B virus ; immunology ; physiology ; Humans ; Liver ; pathology ; virology ; Macaca ; Male ; Tupaiidae

Diffusion-weighted magnetic resonance imaging (dMRI) is widely used to study white and gray matter (GM) micro-organization and structural connectivity in the brain. Super-resolution track-density imaging (TDI) is an image reconstruction method for dMRI data, which is capable of providing spatial resolution beyond the acquired data, as well as novel and meaningful anatomical contrast that cannot be obtained with conventional reconstruction methods. TDI has been used to reveal anatomical features in human and animal brains. In this study, we used short track TDI (stTDI), a variation of TDI with enhanced contrast for GM structures, to reconstruct direction-encoded color maps of fixed tree shrew brain. The results were compared with those obtained with the traditional diffusion tensor imaging (DTI) method. We demonstrated that fine microstructures in the tree shrew brain, such as Baillarger bands in the primary visual cortex and the longitudinal component of the mossy fibers within the hippocampal CA3 subfield, were observable with stTDI, but not with DTI reconstructions from the same dMRI data. The possible mechanisms underlying the enhanced GM contrast are discussed.
Animals ; Brain Mapping ; Diffusion Tensor Imaging ; methods ; Hippocampus ; diagnostic imaging ; Image Processing, Computer-Assisted ; methods ; Male ; Neural Pathways ; diagnostic imaging ; Tupaiidae ; anatomy & histology ; Visual Cortex ; diagnostic imaging