Abstract:Objective To generate transgenic mice expressing human APOC3, and establish a mouse model of hyperlipidemia. MethodsThe transgenic plasmid was constructed by inserting human APOC3 gene into the downstream of the human ubiquitin C (Ubc) promoter. The transgenic mice were produced by microinjection and the genotyping was detected by PCR. The expression level of the gene was determined by Western blotting. The levels of blood lipids of the mice at different ages were detected using a biochemical analyzer. Fat in the liver tissue was observed by histology with fat staining. ResultsThe transgenic mouse model with overexpression of human APOC3 gene was established, showing overexpression of human APOC3 gene in the blood, liver, intestine, muscle, heart, kidney and spleen tissues, compared with those of the wild type mice. The plasma triglyceride phosphate (TG) level and liver fat content of the transgenic mice were significantly higher than those in the wild type mice. ConclusionsA transgenic mouse model overexpressing human APOC3 gene and phenotypes of hyperlipidemia has been developed, providing a useful animal model for hyperlipidemia and related angiocardiopathy studies.

Abstract:ObjectiveTo observe the effects of rituximab injection, an anti-human B-lymphocyte monoclonal antibody, on eliminating the B lymphocytes from Chinese origin rhesus macaques, and to provide some experimental data for establishing B-cell-depleted animals models and better define the role of B cells in the control of pathogenic simian immunodeficiency virus (SIV) replication in future studies. MethodsTwo healthy Chinese origin rhesus monkeys were selected and infused with rituximab via the tail vein by intravenous drip. Peripheral blood, lymph nodes and duodenum biopsy samples were collected at different time points after injection. Lymphocytes were isolated from all the samples, stained and analyzed by FACS. ResultsThe B lymphocytes in peripheral blood of rhesus monkeys were completely depleted 24 hours after the rituximab infusion and lasted for 14 days, while 100% depletion in the inguinal lymph nodes and 90% depletion in the duodenal mucosa at 7 days after infusion. At the same time, CD4⁺ T and CD8⁺ T-cells in peripheral blood maintained at a stable level without significant fluctuation. ConclusionsB lymphocytes can be effectively eliminated from various tissues in Chinese origin rhesus macaques by rituximab injection in vivo.

Abstract:ObjectiveTo analyze the effects of ultrasonic homogenization on the stability of Sendai virus (SeV) in ELISA. MethodsThe SeV amplified in BHK-21 cells was purified by differential centrifugation, and then homogenized by ultrasonication. The ultrasonic-treated SeV antigen and the untreated antigen were coated in the wells of ELISA plates. The coefficients of variation (CV) between the two kinds of plates were compared by testing SPF mouse serum and SeV-immunized serum. Resultsthe CV of gradient diluted Sendai virus-immunized serum was between 1.97% to 6.02% in untreated antigen coated plates and 0.53% to 2.26% in ultrasonic-treated antigen coated plates. The CV of SPF sera was higher than that of SeV-immunized serum. The CV of all the tested samples in ultrasonic-treated antigen coated plates was lower than that in the untreated antigen coated plates. ConclusionUltrasonic treatment can homogenize the SeV antigen and improve the SeV antigen stability in ELISA.

Abstract:ObjectiveTo study the expression of death receptor (CD95) on the surface of peripheral blood CD4⁺T cells and the CD4⁺ T cell numbers during the acute phase in SIVmac239 virus-infected rhesus monkeys challenged by rectal (rectal infection:IR) and intravenous (intravenous infection:IV) routes, respectively.MethodsFourteen rhesus monkeys were challenged with SIVmac239 intrarectally or intravenously. Infection was determined by monitoring the viral load, CD4⁺T lymphocyte number and CD4⁺/CD8⁺ ratio. Flow cytometry analysis was used to assess the CD95 expression on the surface of CD4⁺ T cells at nine time points during the acute-phase infection. ResultsIn the intravenous infection group, CD95 expression on the CD4⁺T cells began to increase on the next day after infection, and reached the peak value on the 7th day, meanwhile the CD4⁺T lymphocyte number was declined to the lowest point. In the rectal infection group it also began to rise on the following day after infection and the CD95 expression reached the peak value at 10 days after infection, but the number of CD4⁺T cells was reduced to the lowest point until the 17th day. Compared with the intravenous infection, the peak value of CD95 expression and the lowest point of CD4⁺T cell number appeared later in the IR group, and the peak value of CD95 expression occurred a week earlier to the lowest point of CD4⁺T cells. Conclusions CD95 expression of the CD4⁺T cells is increased and the amount of CD4⁺T cells is decreased in SIVmac 239-infected rhesus monkeys challenged intrarectally or intravenously. Our results show that CD95 expressed on the surface of CD4⁺T cells vary in the SIVmac 239-infected rhesus monkeys. It may be due to the different effects of CD95 during the acute phase infection induced by different routes.

Abstract:ObjectiveTo determine the genetic characteristics of PLCε knockout mice by polymorphic microsatellite DNA loci analysis．MethodsGenome DNA of 28 PLCε gene knockout mice were amplified by PCR using screening 15 microsatellite DNA loci,and population genetic diversity was identified by gene fragments．ResultsAmong 13 microsatellite DNA loci (D1Mit365,D3Mit51,D4Mit235,D6Mit102,D7Mit281,D8Mit113,D9Mit23,D10Mit180,D13Mit88,D16Mit145,D17Mit36,D18Mit94,D19Mit97),each locus of electrophoresis distance of DNA fragments in the 28 PLCε gene knockout mice kept consistent and presented monomorphism,indicating the genetic stability. However the two loci Dq (knock genotype) and Dy (wild-type) were used to discriminate 28 PLCε gene knockout mice by PCR amplification．Among them, 6 mice were of gene knock mice,7 mice were of wild-type mice,and 15 mice were of heterozygous type．ConclusionsThe method of microsatellite marker analysis can be used to monitor population genetic quality and accurately distinguish different genotypes of mice,providing a feasible method for the detection of genetic quality in mice.

Abstract:ObjectiveTo compare three rat models of hyperuricemia induced by different agents.Methods Twenty healthy male adult (SPF) Sprague-Dowley rats were randomly distributed into three model groups and control group. Rats in the model groups received 5% oxonate diet (5% OA), 10% yeast extract diet (10% YE), 10% yeast extract + 2% oxonate diet (10%YE +2%OA), respectively, for three weeks, and then normal diet for one week. The control group received normal diet. Blood and urine samples were assayed to determine the levels of uric acid, creatinine, nitrogen, albumin, triglyceride and cholesterol. ResultsCompared with the control group, serum uric acid levels in the 5% OA group were significantly higher and consistently (P<0.05). The serum uric acid levels in the 10% YE group increased only in the second week (P <0.05). In the 10% YE + 2% OA group, serum uric acid levels increased in the first and second weeks (P<0.05), then decreased. There were no significant differences in the serum creatinine, nitrogen, albumin and body weight among the four groups (P>0.05). ConclusionsThe rat model of hyperuricemia induced by 5% OA is stable and enduring. The 10% YE models hardly reach hyperuricemia status. The 10% YE + 2% OA models have unstable high level of serum uric acid.

Abstract:ObjectiveTo comparatively analyze the impact of intraperitoneal injection of acidic phosphate buffer solution at a late stage of gestation on the reproductive physiology in two inbred strains of C57BL/6J (B6) and BALB/c (B/c) mice. MethodsOne or two sisters were randomly mated with one brother in the same strain overnight, starting at the age of 10 weeks. The correlation of mating mode (1∶1 or 2∶1) and vaginal plug with pregnancy rate was observed. The pregnant dams were intraperitoneally injected with acidic phosphate buffer solution at 17.5^th day of their gestation (day 0=day post mating). The observation was carried out to detect the impact of the injection of acidic buffer on pregnant dams and their fetuses, and also the growth of the offspring from weaning to 8 weeks. ResultsThe pregnancy rate was higher in B6 than in B/c females regardless of mating mode (29.4% vs. 21.1% ［2∶1］, 33.2% vs. 29.7% ［1∶1］). The better way to accurately determine pregnancy was to examine the expanded belly combined with increased body weight from day 10 to 14 post-mating. Compared with the dams of B/c strain, the impact of the peritoneal injection on those of B6 strain was stronger. It manifested a reduction in number of weaned offspring (4.7±3.1 vs. 6.1±2.1), a significant decrease (P<0.05 in both genders) of the offspring''s body weight at the time of weaning in both genders (g,female:11.7±1.1 vs. 12.7±1.5; male:12.8±1.3 vs. 13.6±1.5), and a significantly different growth pattern of the offsprings (P=0.000). ConclusionsThere is a strain-dependent difference in some aspects of reproductive physiology in inbred mice. The tolerance to the intraperitoneal injection of acidic phosphate buffer solution at a late stage of gestation varied in the two strains. The procedure may produce some effect on dams'' breeding after baby-delivery, which may continue to influence the early developmental growth of the offspring.

Abstract:ObjectiveTo observe the effect of caloric restriction on oxidative damage in human neuroblastoma cell line SH-SY5Y cells in vitro. MethodsTo establish an in vitro model of H₂O₂-induced oxidative stress damage of SH-SY5Y cells. SH-SY5Y cells were cultured in vitro. The cells were divided into four groups:control group, H₂O₂ (250 μmol/L) group, low glucose (2g/L) group, and low glucose + H₂O₂ group. Cell morphology, thiazolyl blue (MTT) metabolism rate, and lactate dehydrogenase (LDH) leakage rate were measured to observe the cell growth status in different groups. ResultsCompared with the control group, the MTT metabolism rate in the cells treated with 50 μmol/L H₂O2 for one hour was not significantly changed (P>0.05), but it was significantly decreased in the 0,0, 0,0 μmol/L H₂O₂-treated groups than that of control group (P<0.01), and there was a tendency that along with the increase of H₂O2 concentration, the cell activity was increasingly decreased. So, the H₂O2 concentration of 250 μmol/L was selected to generate oxidative stress. The MTT metabolism rate in the cells pretreated with low glucose for 24 hours and treated with 250 μmol/L H₂O2 for one hour was significantly decreased than that of control group (P<0.01), but significantly increased than that of the H₂O2 group (P<0.01). When the cells were further cultured for 7 hours, the metabolism rate of the low glucose group was significantly higher than that of the control group (P<0.01). Compared with the H₂O2 group, the metabolism rate of the low glucose + H₂O2 group was significantly increased (P<0.01). The LDH leakage rate in the cells treated with 250 μmol/L H₂O2 for 1 hour was significantly increased than that of the control group (P<0.05). The LDH leakage rate in the low glucose treated group was slightly decreased, and the rate of the low glucose + H₂O2 group was significantly lower than that of the H₂O₂-treated group (P<0.01). When the cells were further culture for 7 hours, the LDH leakage rate of the low glucose group treated for 7 hours was slightly higher than that of cells cultured for 1 hour (P＞0.05). The LDH leakage rate of the low glucose + H₂O2 group cultured for 7 hours was slightly higher than that of cells cultured for 1 hour (P＞0.05). The histological observation revealed that the morphology of cells treated with low glucose was similar to that of the control group, and it was similar at one hour after H₂O₂ added. At 7 hours after addition of H₂O₂, the cells of the low glucose group and control group had well streching cytoplasmic projections, but in the H₂O₂ group, the cell number was significantly reduced, with a lot of dead cells, and the cells became rounded in shape and with poor adherence and transparence. ConclusionCaloric restriction can improve the viability and anti-oxidative stress ability of neurons, and reduce the cell mortality.

Abstract:Objective To establish a single genome amplification assay for obtaining complete rt gene sequence from RT-SHIV chimeric virus. MethodSpecific primers were designed with Oligo and screened through serial dilution, then PCR was optimized to amplify complete rt gene by selecting the best annealing temperature and cycles. Serial dilutions of RT-SHIV chimeric plasmid was used as template standards to establish this system. Complete rt gene of infected monkey in vivo were amplified using this method,and the obtained sequences were analyzed with BioEdit software. ResultsA group of nested PCR primers was got and a single genome amplification assay was established to obtain complete rt gene. Single genome sequences were obtained using dilution of 100 copies/μL plasmid. The results show that the RT-SHIV-infected monkeys had one and six amino acid variations at d266 and d294. ConclusionsThe single genome amplification assay established in this study is an highly sensitive, specific and reproducible method. It can be used to analyze complete rt gene from various types of RT-SHIV.

Abstract:ObjectiveTo establish a stable and reversible rat model of aplastic anemia.MethodsForty-four healthy adult female Wistar rats were randomly divided into 3 groups:control group (n=8), modeling groups A and B (n=18, each). The rats of modeling group A received a total body irradiation by a linear accelerator at a dose of 240 cGy·min-1, SSD=100 cm and for 1.2 min on the first day of modeling, and intraperitoneal injection of cyclophosphamide 35 mg/kg and chloromycetin 43.75 mg/kg once a day on the 4th, 6th and 8th days of modeling. The rats of modeling group B received a total body irradiation at a dose of 300 cGy·min-1, SSD=100 cm and for 1.2 min on the first day of modeling, and the same doses of intraperitoneal injection of cyclophosphamide and chloromycetin but on the 4th, 5th and 6th days of modeling. The rats of control group received sham operation of irradiation only. On the 9th, 12th, 15th days of modeling, reticulocyte count, peripheral blood examination and bone marrow biopsy were performed. ResultsOn the 9th day of modeling, compared with the control group, the WBC, RBC, PLT, HGB, RET in the experimental groups A and B were significantly decreased (all P<0.05 ). On the 15th day of modeling, compared with that in the control group, the RBC and HGB were significantly and continuously decreased, but WBC, PLT and RET significantly increased in the experimental group A (P<0.05). In the experimental group B, the WBC, RBC, HGB, PLTand RET were significantly decreased, and the RET was significantly increased (all P<0.05). ConclusionsStable and reversible rat models of aplastic anemia can be prepared by the protocol of the modeling group A in this study, and they have advantages such as short experiment time, high successful rate, good reproducibility, and low mortality. This animal model is suitable for experimental studies on therapeutic drugs for the treatment of aplastic anemia.

Abstract:ObjectiveTo find the best feed and feed supplier for 24-month rodent carcinogenicity studies by assessing the quality, supply risks and price of three rodent feeds. MethodsSamples of the MF18 feed (Japan), LabDiet feed (USA) and SPF domestic rodent feed (Beijing) were sent to a professional laboratory for testing the conventional nutrients, metals and pesticides, and microbiological indicators, and to evaluate the quality of the feeds. Risks on feed transport and non-controllable factors which might affect feed quality and supply were evaluated. The price of each feed as a reference factor was also considered. ResultsThe quality of the three feeds met the requirements of standard GB14924-2001. According to the evaluation criteria for general toxicological indicators concerned in toxicity studies, the quality sort of the three feed was ranked as:MF18 feed (the best) -> LabDiet feed (better) -> local feed produced in Beijing (good). There were significant risks in transportation and supply for the imported feeds. The price of the domestic feed was relatively cheap but was not considered as a critical factor. ConclusionsAfter a comprehensive comparison, the special customized domestic feed is selected for the 24-month carcinogenicity study to be conducted in the National Centre for Safety Evaluation of Drugs.

Abstract:Objective To investigate the respiratory tract flora and mycoplasma status in Mongolian gerbils from a conventional facility, and provide basic information for the developing microbiologic testing standards of gerbils. Methods Sixty-five gerbils from conventional facility of Capital Medical University were dissected to obtain the tracheal secretion, and then the samples were seeded in blood agar base, cultured in common or 5% CO₂ incubator for 48 h, separately. In order to accurately identify the genus to which the isolated strain belongs, biochemical analysis and PCR sequencing were performed. On the other hand, DNA was isolated from tracheal lixivium, and mycoplasma DNA was amplified by specific primer. The detection rate was calculated. ResultsIn this study, 21 kinds of bacteria and mycoplasma were detected in the tracheas of these mongolian gerbils. The positive rates of different bacterial and mycoplasma infection were varying greatly, between 1.5% (1/65) to 64.6% (42/65), among which, some bacteria were pathogenic to mice and rats. Conclusion This study provides reference information for the development of microbiological standards of Mongolian gerbils.

Abstract:Objective To prepare neutralizing monoclonal antibody of tetrodotoxin (TTX) and establishment of a detection method of the antibody based on competitive ELISA. MethodsBlab/c mice were immunized with TTX-KLH and its monoclonal antibody was detected by TTX-BSA coated indirect ELISA. A hybrydoma cell line was established and its ascites was injected into peritoneal cavity of the mice. The monoclonal antibody was purified with protein A sepharose CL4B affinity column and identified with SDS-PAGE electrophoresis and indirect ELISA. LD50 of tetrodotoxin for Kunming mice was determined by routine method. A mixture of monoclonal antibody and TTX was injected into the peritoneal cavity of mice, and the neutralizing capacity of monoclonal antibody was detected. A detection method of TTX was also established by competitive ELISA. ResultsTwo kinds of neutralizing monoclonal antibodies were obtained, and their purity was above 95% obtained by Protein A sepharose CL 4B affinity column. Monoclonal antibody 5E7 had a higher binding capacity with TTX than that of monoclonal antibody 5E4. The neutralizing antibody showed a protection rate of 50% for Kunming mice against 2 LD50 TTX attack. A detection method of TTX with the monoclonal antibody based on competitive ELISA was established and the detectable minimal concentration of TTX was 1.56 μg/mL. Conclusion Two kinds of neutralizing monoclonal antibody have been obtained. The protection rate was 50% for Kunming mice against 2 LD50 TTX attack. A detection method of neutralizing monoclonal antibody established based on competitive ELISA, with a minimum detection limit of 1.56 μg/mL.

Abstract:With the rapid development of fish used as an important material for scientific research, fish welfare aroused the attention of researchers. This paper aimed to elaborate the fish welfare development history and current situation, to analyze the implementation difficulties of fish welfare, and to provide the reference countermeasure for improving welfare. It would help scientists further understand the laboratory fish welfare, and promote the implementation of fish welfare during experimental studies.

Abstract:Lung cancer is one of the common malignant tumors in the world. Animal models of lung cancer have played very important role in the research on etiology, diagnosis and treatment of human lung cancers. Transgenic animal models of lung cancer are more similar to human lung cancer and are better for studies on the etiology and pathogenesis of lung cancer. This article is focused on the research progress of transgenic animal models of lung cancer in recent years.

Abstract:Glutamate is an essential excitatory neurotransmitter regulating brain functions. Excitatory amino acid transporter (EAAT)-2 is one of the major glutamate transporters expressed predominantly in astroglial cells and is responsible for 90% of total glutamate uptake. Glutamate transporters tightly regulate glutamate concentration in the synaptic cleft. Dysfunction of EAAT2 and accumulation of excessive extracellular glutamate has been implicated in the development of several neurodegenerative diseases including Alzheimer’s disease, Huntington’s disease, and amyotrophic lateral sclerosis. Analysis of the human EAAT2 promoter showed that NF-kB is an important regulator of EAAT2 expression in astrocytes. Screening of approximately 1,0 FDA-approved compounds led to the discovery that many b-lactam antibiotics such as ceftriaxone etc. are transcriptional activators of EAAT2 resulting in increased EAAT2 protein levels and produce neuroprotective effects.

Abstract:Sentinel animals are rarely used in microorganism detection as a ‘tool’ in our country, but along with the increasing application of various species of laboratory animals and special animals, the currently used laboratory animals are more and more not enough to satisfy the requirement of various detection tests. It is very important to explore and learn from developed countries how to use sentinel animals. In this paper, we will introduce the concept and application methods of sentinel animals.

Abstract:Objective To establish commonly used experimental methods of Guizhou miniature pigs under the guidance of animal welfare and “3R” principle. MethodsUsing minimal irritative and less painful way to make intramuscular injection and intragastric gavage, blood collection, anesthesia, intravenous injection and euthanasia to Guizhou miniature pig. Results and ConclusionA set of experimental methods of Guizhou miniature pigs have been established in accordance with requirements of animal welfare and "3R" principle.