Abstract:In this study, a Specific Pathogen-Free (SPF) Bama miniature pig herd was established. The standards for breeding experimental pigs, genetic test and microbiological quality control had been drafted. The local standards of Heilongjiang province “SPF” technical specifications for microbiological monitoring of pigs had been formulated. The genetic and microbiological quality criterion had been used to control of the SPF pig herd. Classical swine fever virus (CSFV) and highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) infection models of Bama miniature pigs were established, especially the infection miniature pig model of PRRSV was used to evaluated commercially vaccine. The comparison of the cytokine homology between Bama miniature pigs and domestic pigs showed that the Bama miniature pigs can be used to instead of domestic pigs as an ideal experiental animal. At present the SPF Bama miniature pig colonies have been widely used to study the mechanism of prevention and pathogenic in the classical swine fever virus, porcine reproductive and respiratory syndrome virus and porcine transmissible gastroenteritis virus and porcine circovirus and so on. The completion of the project solved the bottleneck problem of the experimental pig usage which provided a new ideal experimental animal for animal disease and life science research.

Abstract:Chinese hamster is an important laboratory animal in medical and biological researches,but the molecular genetic markers research was rarely reported.In our study the base composition,gene structure,genetic evolution and other characteristics of mitochondrial genome of Chinese hamster were analyzed using the methods of bioinformatics and comparative genomics,genetic quality detection system of Chinese hamster were also established.These results would supply genome data for animal models of human diseases,and lay the foundation for scientific evaluation and reasonable utilization.

Abstract:There are some shortcomings of animal experiments applied in chemical toxicity testing, such as long period, large cost, species differences and dose differences, which limit the use of animal experiments' results in predicting human toxicity. Accordingly, we established a toxicity testing alternative screening system in line with the toxicity endpoints which required in chemical safety evaluation and risk assessment (genotoxicity, carcinogenicity, reproductive toxicity, acute toxicity and general toxicity) based on “3R” principles (replacement, reduction, refinement) for animal experiments. This system covers most of the endpoints of toxicity assessment, and molecular biology technology was also applied to integrate the toxicity test, as well as some operation was optimized in order to shorten the experimental period, reduce experimental costs, improve animal welfare. Furthermore, the results from the screening system have higher clinical relevance because it is based on the toxicity mechanisms.

Abstract:Non-alcoholic fatty liver disease (NAFLD) has become one of the major metabolic diseases. In view of the defects of traditional animal models, this study was the first to establish the NAFLD model of Mongolian gerbil (Meriones unguiculatus) with simple feed formula which is similar to human (from simple fatty liver to steatohepatitis, fibrosis,Liver cirrhosis). This study discussed the mechanism of rapid fatty liver deposition in Mongolian gerbil, revealed its molecular mechanism,main regulatory target and network function of fatty liver susceptibility. We provide a new animal model of NAFLD with relatively clear background and less time-consuming for clinical treatment and new drug development. The theoretical and practical basis for the breeding of inbred strain NAFLD gerbil was established.

Abstract:The adverse reactions caused by traditional Chinese medicine have occurred frequently, but there is a lack of scientific, objective and standardized methods for safety evaluation of traditional Chinese medicine. In the process of preclinical evaluation of traditional Chinese medicine, it is imperative to form a set of scientific, standardized and feasible evaluation system of modem Chinese herbal drug. We established the preclinical safety evaluation system of modem Chinese herbal drug including the quality control system of samples for the preclinical safety evaluation, the toxicity evaluation system of modem Chinese herbal drug and its preparation and the evaluation management system, and standardized each research link of preclinical evaluation of traditional Chinese medicine. Whether from protecting patients' health and increasing the safety of clinical medication, or from enriching and improving traditional Chinese medicine science, developing traditional Chinese medicine and promoting mutual connection of traditional Chinese medicine and international medicine, it has important instructional significance and application value.

Abstract:In order to establish the minipig model of chronic myocardial ischemia and apply non-invasive telemetry technique, the minipig model of chronic myocardial ischemia was made induced by Vitamin D3, isoproterenol and combined with high fat diet, and the non-invasive telemetry technique was used to detect and evaluate the symptoms of minipig model of chronic myocardial ischemia. Moreover, the effects of transport stress and the risk factors of atherosclerosis (AS) induced by high fat diet among Wuzhishan minipigs, Bama minipigs and Tibetan minipigs were also evaluated. Our study has successful established the Bama minipig model of chronic myocardial ischemia and the technical specification for evaluation,. The non-invasive telemetry technique can be used to detect and evaluate the symptoms of chronic myocardial ischemia model, and defines minipigs at least need to keep for more than 4 weeks after transport stress to adaptive recovery period. In addition, the different characteristics of AS risk factors such as hyperlipidemia, hypertension and hyperinsulinemia were observed in Wuzhishan minipigs, Bama minipigs and Tibetan minipigs in high fat environment, and this provides a reference for the selection and application of minipigs in the research of cardiovascular diseases.

Abstract:In this paper, the present situation and development trend of the laboratory animal industry in China are expounded comprehensively and objectively. Through comparative analysis, relative to Europe and the United States one hundred years of accumulated huge resources and strong financial support, the overall laboratory animal industry in China is far behind. The whole nation system should make great efforts to realize leaping development and to catch up. Laboratory animal resources are still the foundation and weak link of the development of laboratory animal industry. This study can provide a scientific basis for the competent authorities at all levels to develop fast and efficient laboratory animal industry development strategy, and also provide reference for the development of the laboratory animal industry itself.

Abstract:Objective To investigate the role and mechanism of platelet in the development of salt-sensitive hypertension.Methods 25 Dahl salt-sensitive rats (Dahl SS) were divided into three groups:low-salt diet (0.12% NaCl, LS), high-salt diet (8%NaCl, HS) and high-salt diet + platelet inhibitor (8%NaCl+busulfan, HS+bus). Blood pressures were measured by tail-cuff method. After six weeks, animals were sacrificed. Platelet p-selectin expression, platelet cytosolic Ca²⁺ concentration, platelet-leukocyte aggregation (PLA) in peripheral blood, and immune cells infiltrated on aortic walls were assessed by flow cytometry, and serum IL-6 level was tested by ELISA in vivo. Platelets purified from SD rats were treated with normal salt (0.9%NaCl) and high salt (1.3%NaCl), then the cytosolic Ca²⁺ concentration and p-selectin expression of platelet were detected. Results We found that Dahl SS rats with high-salt diet, relative to low-salt diet, presented with high blood pressure and increased the ratio of platelet p-selectin expression, Ca²⁺ concentration. IL-6 level and PLA in peripheral blood, and the number of infiltrated immune cells on aortic walls were also significantly elevated in high-salt diet group. The whole events were ameliorated by the platelet inhibitor busulfan. Cytosolic Ca²⁺ concentration and p-selectin expression were also increased in purified platelets treated with high salt than those treated with low salt (P< 0.05). Conclusions Our findings suggest that high salt induced platelet activation with increased Ca²⁺ concentration may play an important role in the development of salt-sensitive hypertension via vascular inflammation. However, the detailed mechanisms of platelet activation and development of high blood pressure via inflammation induced by high salt intake remain to be determined.

Abstract:Objective The purpose of this study is to understand the influence of Fkbp51 gene knockout on alternative splicing of mRNA in liver tissues. Methods mRNAs of liver from both Fkbp51 knockout(KO) and wild type(WT) mice were isolated. mRNA expression profiling was performed using RNA-seq reads. The mRNA reads produced from RNA-seq was analysed by TopHat for alternative splicing. Exon skipping and intron retetion were identified according to alignment analyses. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were analyzed using online tools DAVID. All genes which had differently expressed alternative splicing between WT and KO were annotated using NCBI database. Results (1) Fkbp51 KO can introduce new mRNA alternative splicing; (2) Fkbp51 KO also cause mRNA alternative splicing expression change; (3) According to GO and KEGG analysis, it was found that the mRNA alternative splicing changes mainly enriched in fat metabolism, immune, bile acid secretion, and PPAR signaling pathway etc. (4) The genes which have different intron retetion events are mainly involved in regulation of actin cytoskeleton organization and amino acids metabolism. Conclusions Fkbp51 gene knockout can cause the change of mRNA alternative splicing, and thereby affect the metabolism of liver in mice.

Abstract:Objective To screen and determine the effective silencing targets of β2-microglobulin(B2m)gene at the cellular level in marmoset.Methods By homology comparison of the b2m gene in human and the B2m gene in marmoset, choose homology small hairpin RNA(shRNA)sequences targeting marmoset B2m gene were designed, We choose homology small hairpin RNA(shRNA)sequences targeting designed B2m gene to make homology analysis, and insert into lentivirus-based gene silencing constructs FUGW-TDT. The vectors were transfected into HEK293T cells induced by polyethylenimine(PEI). The suppression of B2m mRNA was detected by real-time PCR. Results Two gene-silencing sequences were screened that lied in 290~310 bp and 665~685 bp of the marmoset B2m mRNA, and have statistical significance in the silencing rate:(46.54±7.91)% (P< 0.05) and(83.22±4.37)%(P< 0.0001). Conclusions Two effective silencing target sequences are screened at cellular level, which can be further used in studies on gene silencing in marmoset.

Abstract:Objective To investigate the effects of Kruppel-like factor 6 (KLF6) on the apoptotic and migration ability of HepG2 cell, and the developmental role of KLF6 on zebrafish liver. Methods Constructed plasmid with shRNA-KLF6 was transfected in HepG2 and L-02. The impacts of loss of KLF6 on HepG2 was investigated by Western bolt, apoptosis analyses, cell cycle detection and scratch experiment; KLF6 morpholino oligonucleotides was microinjected into the Tg(lfabp:eGFP) transgenic zebrafish embryos. The morphant phenotype of the liver was imaged and the protein expression of KLF6 after knockdown of KLF6 was analyzed by Immunofluorescence staining. Results The expression of KLF6 in L-02 was significantly higher than in HepG2. After knockout of KLF6, KLF6 protein expression and apoptosis were significantly reduced. In addition, the cell cycle mainly stated in S phase and the migration ability of HepG2 was enhanced. After klf6 knockdown in transgenic zebrafish larvae, the development of zebrafish liver was delayed and KLF6 expression was obviously decreased in the liver. Conclusions The reduction of KLF6 expression increased the proliferation and migration ability, and reduced the apoptosis of HepG2.Loss of KLF6 affects the development of zebrafish liver, which may open a possibility to use zebrafish as a liver cancer model and for anti-liver cancer drug screening.

Abstract:Objective To investigate the effect of uterine cavity injection of absolute ethanol on estrous cycle in mice.Methods Twenty mice with regular estrous cycle were selected by vaginal exfoliated cells staining, and then were injected absolute ethanol into their uterine cavity. The estrous cycle was observed and recorded every day. Ten mice were selected randomly to observe the changes of second estrous cycle every two hours.Results After the injection of absolute ethanol into the uterine cavity of the experimental mice, the estrous cycle was greatly prolonged, and the various degree of disorder was observed in estrus and pre-estrus. Conclusions The disorder was observed in estrous cycle of mice after the injection of absolute ethanol, which maybe provides some references for clinical use of absolute ethanol.

Abstract:Objective To study the effect of Interleukin-22 (IL-22) on murine asthmatic airway inflammation and airway remodeling, observe the effect of budesonide on IL-22 of asthmatic mouse model, explore the mechanism of budesonide in the treatment of asthma. Methods Ovalbumin(OVA) was used as an allergen to sensitize and challenge the mice. 24 female specific-free (SPF) BALB/c mice aged four weeks were randomly divided into 3 groups:control group, asthma group and budesonide treatment group (BUD group). For Histopathological Examination, HE staining was used to measure the inflammation scores, AB-PAS staining was used to measure the hyperplasia of goblet cells and mucin. Enzyme-linked immunosorbent assay(ELISA) was used to analyze IL-22 levels in bronchoalveolar lavage fluid (BALF). Quantitative Real-time PCR was performed to analyze the effects of budesonide on IL-22 mRNA levels in lung tissue. Results The inflammation scores of asthma group were elevated compared with the control group. An overall change towards less severe asthmatic airway inflammation by the end of the trial was observed in the BUD group. IL-22 levels in BALF were significant decreased after the treatment of budesonide, the mRNA levels of IL-22 were obviously decreased in BUD group, too. A significant positive correlation was observed between the mRNA levels of IL-22 and airway inflammation. Conclusions The increasing IL-22 secretion can lead to the occurrence of airway inflammation of asthma. Budesonide can inhibit the expression of IL-22, thereby Budesonide could inhibit the development of airway inflammation of asthma.

Abstract:Objective To investigate the synergism and attenuation effects of Pini Pollen on cyclophosphamide chemotherapy in model mice.Methods The models of mouse were induced with S180 tumor and cyclophosphamide. The tumor inhibiting rates, hematology, Liver function, giant cell phagocytosis, hemolysin level and indexes of thymus and spleen were determined to evaluate the synergism and attenuation effects of Pini Pollen(150,300,600 mg/kg) on cyclophosphamide chemotherapy.Results Compared with cyclophosphamide chemotherapy group,the tumor inhibiting rates,white blood cell number,giant cell phagocytosis, hemolysin level and indexes of thymus and spleen were increased, at the same time liver function damage were alleviated significantly in cyclophosphamide plus Pini Pollen groups. Conclusions Pini Pollen has a remarkable synergism and attenuation effects on cyclophosphamide chemotherapy through its immune adjustment functions.

Abstract:Objective To study the expression and significance of EGFR, PDCD4, TGF-β1, Smad3, Smad7 in oral normal mucosa, oral simple hyperplasia, oral epithelial dysplasia and oral squamous cell carcinomas(OSCC). Methods Serum levels of EGFR, PDCD4 were measeured with ELISA in Chinese hamster during the oral mucosa carcinogenesis; The expressions of TGF-β1, Smad3, Smad7 in oral normal mucosa, oral simple hyperplasia, oral epithelial dysplasia and OSCC tissues were examined by immunohistochemistry. Results In the process of oral carcinogenesis,the expression level of EGFR increased significantly,while the expression of PDCD4 was decreased, the negatively correlation was evident between expression of these two proteins in ED and SCC. The expression of TGF-β1, Smad7 was higher in OSCC than in oral normal mucosa, oral simple hyperplasia, oral epithelial dysplasia(P < 0.05), while the expression of Smad3 was decreased. Further analysis showed the expression of TGF-β1 was correlated with the expression of Smad7. Conclusions The expressions of EGFR, PDCD4, TGF-β1, Smad3, Smad7 are closely related with the biological behaviors of oral squamous cell cancinoma.This experiment provides a theoretical basis for the study of oral squamous cell carcinomas mechanism research. So it has important significance for the development and prognosis of oral squamous carcinomaare.

Abstract:Objective To discuss the effects of Paclitaxel(PTX) on levels of CD28 and CTLA-4,B lymphocyte stimulator(BAFF) in experimental autoimmune encephalomyelitis(EAE).Methods The 50 rats were divided into 5 groups by the random number table, 10 rats in each group,the doses of small group,Middle group, High group were 1 mg/kg,2 mg/kg,4 mg/kg by intraperitoneal injection for 10 consecutive days, the normal group and model group were injected 0.9% NS 2 mL,Using brain tissue score to estimate the neurological dysfunctions of rats.Using flow cytometry to detect the levels of CD28 and CTLA-4,using enzyme-linked immunosorbent (ELISA) to detect the levels of BAFF.Results The brain tissue score in PTX experimental groups were lower than model group,the comparative differences between groups were statistically significant(P< 0.01);The levels of CD28 in PTX groups were lower than EAE group,the comparative differences between groups were statistically significant(P< 0.01). The levels of CTLA-4 in PTX groups were higher than EAE group,the comparative differences between groups were statistically significant(P< 0.01);the content of BAFF in all PTX groups were lower than EAE control group. Conclusions PTX could decrease the brain tissue score,the mechanism may adjust the express of CD28、CTLA-4 in brain and the expression of BAFF.PTX may have preventive and therapeutic effects on EAE rats.

Abstract:Objective To observe the effect of Guanxinning Tablet (GXNT) on myocardial infarction and cardiac autonomic nervous function in rats with myocardial ischemia reperfusion injury (MI/RI). Methods Seventy SD rats were divided into 7 groups randomly (n=10); the sham group, the MI/RI group, 75 mg/kg, 150 mg/kg, 300 mg/kg and 600 mg/kg GXNT groups and 300 mg/kg Compound Danshen Tablets (DST) group. All rats were administered orally for 7 days, and then the MI/RI model was made by ligating the left anterior descending branch of coronary artery in rats. The changes of electrocardiogram were recorded and the electrocardiogram of J points and heart rate variability (HRV) parameters were analyzed. At the end of reperfusion, the myocardial infarct size was measured by using Evans blue and tetrazolium chloride (TTC) double staining, and pathological changes of myocardium were observed by HE staining. The changes of serum lactate dehydrogenase (LDH), creatine kinase (CK), malondialdehyde (MDA) and nitric oxide (NO) levels were also detected. Results Compared with MI/RI group, GXNT and DST groups were significantly reduced myocardial infarct size and inhibited the rising of serum LDH and CK activities (P< 0.05, P< 0.01), and also reduced the total or average value of J point during reperfusion (P< 0.05, P< 0.01). Meanwhile, GXNT and DST groups were markedly increased HRV and serum NO level as well as decreased serum MDA content (P< 0.05, P< 0.01), and improved myocardial tissue pathology. Conclusions GXNT can reduce the myocardial infarction in rats with MI/RI, and also improve the cardiac autonomic nervous function.

Abstract:Objective To explore the role of miR-221 in the injury induced by hydrogen peroxide (H₂O₂) in rat myocardial cells (H9c2). Methods The viability of H9c2 cell induced by cell different concentrations of H₂O₂ was determined by MTT. The expression of miR-221 was detected by RT-PCR method. The miR-221 inhibitor and negative control were transferred into H9c2 cells by Lipofectamine 2000, then the cells were divided into normal control group, model control group (H₂O₂ group), negative control group (H₂O₂+ negative control group), inhibition group (H₂O₂+miR-221 inhibitor group). The cell viability was measured by MTT assay. Cell apoptosis was detected by acridine orange staining method. The expression of Bcl-2, Bax, phosphatase and tensin homolog deleted on chromosome ten (PTEN, p-protein kinase B (AKT) were assayed by Western Blot. Results 0,25,50,100,200,400 μmol/L H₂O₂ inhibited H9c2 cell activity gradually, of which 200 mol/L inhibition of cell viability moderate, so as a subsequent induction dose. Compared with normal control group, cell viability was decreased (P< 0.01), cell apoptotic rat was increased (P< 0.01), the expression of Bax and PTEN was upregulated (P< 0.01), the expression of Bcl-2 and p-AKT was downregulated (P< 0.01) in model control group and negative control group. Compared with model control group and negative control group, inhibition group proves the contrary. Conclusions Down-expression of miR-221 could significantly inhibit oxidative stress damage in H9c2 cells, which related to regulation of PTEN/AKT signal pathway.

Abstract:Objective To evaluate the effect of deoxyviolacein on the apoptosis in HT29 cells and its possible mechanisms of anticancer effects. Methods In vitro,human colon cancer HT29 cells were treated by deoxyviolacein in a series of concertrations.The absorbance value was detected by means of MTT assay and growth inhibition rate was calculate.Cell cycle and apoptosis was assessed by flow cytometry.The expression of Bax,Bcl-2 and Caspase9 were analyzed by Western blotting.Results Deoxyviolacein could inhibit the growth of human colon cancer HT29 cells.The cells at period G₀/G₁ and the apoptosis rates were significantly increased,and the cells at period S were significantly decreased.The expression of Bax,Caspase9 were upregulated and Bcl-2 was downregulated by deoxyviolacein at 48 hours. Conclusions Deoxyviolacein could inhibit the growth and induce apoptpsis of human colon cancer HT29 cells.The mechanism may be associated with up-regulation of Bax and Caspase9 and down-regulation of Bcl-2 expression.

Abstract:Objective Aiming at detecting Staphylococcus aureus、Pseudomonas aeruginosa and Klebsiella pneumoniae in laboratory animals,the paper provides a rapid,sensitive and simple test method.Methods According to Staphylococcus aureus nuc gene,Pseudomonas aeruginosa LasI gene,Klebsiella pneumonia PhoE geneand general 16S rRNA gene,designed specific primers; Through the optimization of multiplex PCR primer concentrations and annealing temperature, the specificity and sensitivity of detection, establishing multiplex PCR system.Application of the PCR system test specimens of artificial infections and experiment animal feces is compared with traditional test method.Results Multiplex PCR amplification of Staphylococcus aureus (153 bp), Pseudomonas aeruginosa (600 bp) with Klebsiella pneumoniae (368 bp) and general (520 bp).The multiplex sensitivity for the purpose of 10pg, specificity of detection was not detected from other pathogens.Application of establishing multiplex PCR system to detect the artificial positive samples, and detect 1 Pseudomonas aeruginosa positive case in 76 fecals. Conclusions This paper established the multiplex PCR method which has the advantages of specific,sensitive,simple and rapid, and provides a reliable way for rapid test in laboratory animals microbiology.

Abstract:Objective Allograft Vasculopathy(AV), which limits the function and long-term survival of transplanted organs, it is the most urgent problem need to be solved in the field of organ transplantation. Traditional vascular anastomosis requires excellent microsurgical techniques, it is difficult to carry out. To explore a simple and feasible AV model can provide ideas for clinical research. Methods We make vessel cannulas by using vein detained needles, pulling the thoracic aorta of donor rat in vessel cannula, both ends turned over and fixed,and then transplant it in recipient rat abdominal aorta. 1, 4 and 8 weeks after transplantation, we take out of the transplanted arteries, HE stain, to observe the histomorphology and measurement the vascular intimal thickness. Results 38 of the 40 recipient rats healthy survival to the detection time points, the legs and tail can move freely, defecating and urinating normally. It is found that transplanted arteries concentric circles thickening, its histomorphology just like the AV by using HE staining. Conclusions The method of rats artery transplantation is simple and feasible, significantly shorten the time of abdominal aortic blocking, reduce surgical trauma, can improve the success rate and the reproducibility of operation.

Abstract:In this paper, we mainly introduced the construction of the National Primate Research Center in USA, and the development of these research departments, include the distribution of these research centers in the mainland of USA and their work mechanism. We want to get their experiences in non-human primates breeding and research, and want to find the work flow in serving bio-medical research. All these findings are focus on the discovery of new non-human primate resources in China mainland, and on research of primate genetics, medical models, systemic biology analysis, et al. Which will help us to develop the study of this laboratory animal, non-human primates, in China, and will improve our serving ability of non-human primate animals supplement for bio-medical research in the 13^th Five Year program in China.