Establishment and application of a PCR detection method for Tupaia (tree shrew)paramyxovirus(TPMV) / 中国比较医学杂志

Objective To establish and preliminarily apply an effective PCR assay for detection of Tupaia(tree shrew)paramyxovirus(TPMV). Methods Using TPMV genomic DNA from NCBI GenBank, bases 8231 to 8720 were synthesized and inserted into a plasmid as a positive standard. One primer pair was designed based on this sequence. In total,60 respiratory swabs and 12 lung tissues from the tree shrews were tested in this PCR assay. Results A PCR method for detection of TPMV was successfully established,with high specificity and sensitivity of 11.5 × 10 -5μg/mL. PCR result testing 60 respiratory swabs and 12 lung tissues were negative. Conclusions PCR for detecting TPMV has good specificity and high sensitivity and can be used for conventional tree shrew paramyxovirus detection.

Exploration of establishing a tree shrew model of chronic gastrointestinal mucosal injury / 中国比较医学杂志

Objective To explore the feasibility of establishing a tree shrew model of chronic gastrointestinal mucosal injury. Methods A total of 12 adult male tree shrews were randomly divided into 3 groups. The experimental groups 1 and 2 were administered with intraperitoneal injection of 2 mg/(kg·d)and 1 mg/(kg·d)of 1-methyl-4-phenyl-1,2, 3,6-tetrahydropyridine(MPTP)once every day for 56 days, respectively. The control group was given the same volume of sterile saline at the corresponding time points. Changes in the body weight of the tree shrews were observed. The contents of dopamine in the cerebrospinal fluid were detected. Gastrointestinal morphology was observed by stereoscope and histopathological changes of the gastrointestinal mucosa were examined by HE staining. Results The body weight and the contents of dopamine in the cerebrospinal fluid of the tree shrews in the model group were significantly decreased(P< 0.05 for both). Pathological changes to some extent of the gastric antrum, the gastric body and the duodenum were observed, without obvious differences between the 2 mg/kg group and the 1 mg/kg group. No obvious changes were found in the control group. Conclusions Long-term intraperitoneal injection with a low dose of MPTP is a feasible method for the establishment of a tree shrew model of chronic gastrointestinal mucosal injury. The optimal dose is 2 mg/(kg·d)every day for 56 days.

Research progress in culture and in vivo and in vitro HBV infection of primary hepatocytes of tree shrews / 临床肝胆病杂志

HBV is a major health problem faced by human beings. The study of the mechanism of HBV infection has been a key point in this field. To carry out the related research on HBV, establishment of simple and available in vivo and in vitro infection models is the trend. Due to the close relationship between tree shrews and human/primates, the tree shrew model of HBV infection has attracted more and more attention. This review summarizes recent research, both at home and abroad, about in vivo and in vitro HBV infection in tree shrews, especially the research progress in in vitro culture of primary hepatocytes of tree shrews, and looks to the future research directions as well.

Research progress in culture and in vivo and in vitro HBV infection of primary hepatocytes of tree shrews / 临床肝胆病杂志

HBV is a major health problem faced by human beings. The study of the mechanism of HBV infection has been a key point in this field. To carry out the related research on HBV, establishment of simple and available in vivo and in vitro infection models is the trend. Due to the close relationship between tree shrews and human/primates, the tree shrew model of HBV infection has attracted more and more attention. This review summarizes recent research, both at home and abroad, about in vivo and in vitro HBV infection in tree shrews, especially the research progress in in vitro culture of primary hepatocytes of tree shrews, and looks to the future research directions as well.

New advances in tree shrew model in experimental studies of hepatitis B virus / 临床肝胆病杂志

Hepatitis B virus (HBV) infection is the main cause of liver fibrosis, cirrhosis, and hepatocellular carcinoma, and it is also a major health problem around the world. How to establish an efficient, reliable, and standardized animal model of chronic HBV infection is essential to the study of the pathogenesis and prevention strategies for HBV infection. This review summarizes the general research and new advances in using tree shrews as the model of HBV infection. We believe that tree shrews, as lower primates, will provide a vital platform and have a huge potential for building a proper animal model in the future, and could become the essential animal model for simulating the process of HBV infection in humans.

Isolation and chatracterization of microsatellite markers in Tupaia belangeri chinensis / 中国比较医学杂志

Objective To screen out specific microsatellite markers for use in Tupaia belangeri chinensis genetic testing. Methods Firstly to screen about 700 microsatellite loci from whole genome.Secondly to choose about 100 better loci without defect factors.Lastly 46 primers were designed by 33 tree shrew’ s microsatellite loci obtained from whole genome and other references.Agarose gel electrophoresis and polyacrylamide gel electrophoresis were used for PCR products, and better loci based on electrophoresis results were chosen.Then STR scan was used to select the microsatellite loci combination for genetic testing.Results Twenty-two microsatellite loci were selected with a significant Stutter peak on STR scanning.Comparing the alternative loci and ultimately selected loci, there were two loci available in the five alternative loci of T.glis.The coincidence rate between T.glis and T.b.chinensis was 40%.There were two loci available in the five alternative loci of T.minor, and the coincidence rate between T.minor and T.b.chinensis was 40%.There were two loci available in the three alternative loci of T.belangeri, and the coincidence rate between T.belangeri and T.b. chinensis was about 70%.Conclusions The 22 microsatellite loci screened in this study are well applied for genetic testing of Tupaia belangeri chinensis, therefore, provide a scientific basis for the genetic quality monitoring of tree shrews.

Isolation and identification of two different genotypes of Tupaia orthoreoviruses / 中国实验动物学报

Objective To isolate and identify viruses from fecal samples of tree shrew with diarrhea.Methods Fecal sample supernatant of tree shrew with diarrhea was inoculated to three cell lines ( Vero, LLC-MK2 and KMB17 ) , and the cytopathic effects on the cells were observed.The infectious particles in the culture supernatant were further ana-lyzed by transmission electron microscopy ( TEM) , genomic RNA-PAGE, rotavirus detection kit, amplification of S1 com-plete segment and bioinformatics analysis.Results Constant cytopathic effects were induced in Vero, LLC-MK2 and KMB17 cell lines after three passages of culture.The results from TEM, RNA-PAGE and rotavirus analysis indicated that they belong to reoviruses.Analysis of the S1 segments revealed that the S1 sequence from KMB17 cell culture had the high-est homology with that of prototype isolate T1L (85%nucleotide homology and 90%amino acid homology), therefore this isolate was named as type I reovirus.The other two S1 sequences from LLC-MK2 and Vero cell culture were identical to have 85%nucleotide homology and 92%amino acid homology with the prototype isolate T3D, named as type III reovirus. Phylogenetic analysis indicated that the isolates in this study are evolutionally adapted to tree shrews.Conclusions It is the first report here that 2 genotypes of Tupaia orthoreovirus are isolated and identified from one fecal sample via three cell lines and viral S1-specific primers, which provides useful guidelines for the isolation and identification of other reoviruses from tree shrew or other hosts.

Adaptive thermogenesis of the brown adipose tissue in tree shrews (Tupaia belangeri) during cold acclimation / 中国实验动物学报

Objective To investigate the effect of ambient temperature on body mass, thermogenic activity and un-coupling protein-1 ( UCP1) content of brown adipose tissue ( BAT) in tree shrews ( Tupaia belangeri) , and to provide the-oretical basis for establishing tree shrews model of obesity.Methods Forty healthy adult tree shrews with similar body mass were uesd in our experiment.The tree shrews were divided into five groups (n=8):control group (0 d), the ani-mals were maintained under 25 ±1℃ and 12L:12D ( light : dark, lights on 08:00) photoperiod; and the animals were maintained under 5 ±1℃and 12L:12D photoperiod for 7 d, 14 d, 21 d and 28 d groups, respectively.At the end of ex-periment, the changes of body mass, nonshivering thermogenesis (NST), BAT mass and uncoupling protein 1 (UCP1) con-tent were determined.Results Compared with the control group (0 d), the body mass, NST, BAT mass and UCP1 con-tent of the cold acclimation groups were improved significantly, the BAT color also obviously deepened, and after cold accli-mation for 28 d, the body mass, NST, BAT mass and UCP1 content were increased by 26.32%, 20.65, 53.85%and 43%, respectively.Apparently, the UCP1 content was significantly positively correlated with BAT mass and NST.Conclusions BAT proliferation may be induced and UCP1 expression upregulated by cold acclimation in Tupaia belangeri, therefore, en-hancing the thermogenic activity of brown adipose tissue to increase energy expenditure.We would speculate that BAT might be used as a target organ for treatment of obesity by energetic approach in the future.

Isolation,culture, adipocgenic and osteogenic induction of Tupaia bone marrow mesenchymal stem cells / 中国比较医学杂志

Objective To study the isolation,culture, adipogenic and osteogenic induction Tupaia bone marrow mesenchymal stem cells(BM-MSCs).Method The BM-MSCs from tupaia were isolated and expended by combination of gradient centrifugation and adherence culture , then subcultured and observed for morphology under inverted phase contrast microscope.BM-MSCs were induced to adipocytes .and osteoblasts in vitro Result Cells were spindle or triangle-shaped, and clone proliferation .Cells were successfully induced into adipocytes .and osteoblasts Conclusions The method of isolation BM-MSCs from tupaia by combination of gradient centrifugation and adherence culture is simple and feasible , BM-MSCs have differentiation potential into adipocytes and osteoblasts .

Establishment and application of a PCR method for detection of the Tupaia (tree shrew) adenovirus (TAV) / 中国比较医学杂志

Objective To establish and apply an effective PCR assay for detection of the Tupaia ( tree shrew) adenovirus ( TAV) .Methods According to NCBI Genbank, TAV genome DNA from 19418 to 19917 were synthetized and inserted into a plasmid as positive standards.One pair of primers was designed based on this sequence.Sixty blood samples and fifty-six stool samples from tree shrew were detected with this PCR assay.Results A PCR method for detection of TAV was successfully established, with a high specificity and the sensitivity was 13.5 ×10 -7μg/mL.The PCR results of testing sixty tree shrew blood DNA samples were negative.24 positive cases were tested among 56 stool DNA samples.Sequencing of the samples confirmed a 42.9%infection rate of TAV in tree shrew stool samples, well consistent with the PCR results.Conclusions The PCR method for detecting TAV established in this study has good specificity and high sensitivity, therefore, can be used in conventional detection of tree shrew adenovirus.

Measurement and Analysis of Myocardial Enzymes and Some Renal Function Indice in Tupaia Belangeri Chinensis / 昆明医科大学学报

Objective To lay the physiological and biochemical basis for establishing and evaluating tree shrews model of human disease. Methods There were 92 Tupaia belangeri chinensis, in which half of them were male,they were allowed to eat nothing for 12 hours, then we sampled heart blood 0.8~1.0 mL without anesthesia and put blood samples into sterilized centrifuge tube for separation and preparation of serum. Olympus AU5400 automatic biochemistry analyzer was used to measure biochemical indexes. Then 1.5~2.0 mL of urine of each tree shrew was collected and put into sterilized centrifuge tube for measuring renal function by using Combi-scan500 urine analyzer. Fianally SPSS statistics software was used to analyse the measured values, and comapared the measured values with the human reference values. Results There were significant differences in myocardial enzymes and some renal function indexes such as lactic dehydrogenase,α- hydroxybutyric, acid dehydrogenase, creatinine, uric acid, urine specific gravity and pH value between male and female Tupaia belangeri chinensis ( 0.05) . Then determination value of Tupaia belangeri chinensis, male’s and female’s, myocardial enzyme and part of the renal function indexes were compared with the human reference values. Some indexes including urea, urine specific gravity, urine, urobilinogen were in the range of human reference value, while the values of urine bilirubin, urine nitrite. lactate dehydrogenase, creatine kinase, α-hydroxybutyrate dehydrogenase, creatine kinase isoenzyme CKMB were higher than the human reference value. White blood cell, urine protein, ketone, occult blood most of them were negative as the same to human reference value, but sometimes were positive, and the positive rates respectively were 3.95%and 46.1%,39.5%,2.63%. The measured value of Vitamin C was positive that is completely opposite to human reference value, but sometimes is negative, the negative rate was 6.6%. Conclusion Urea, urine specific gravity, urine, urobilinogen, urine bilirubin, urine nitrite can be directly used as the indexes for evaluating tree shrews models of human disease, other indexes can be used as indexes for judgment of the normal physiological and biochemical basis of tree shrews.