Abstract:Objective To establish a stable and real-time monitorable nude mouse model of orthotopic glioma xenograft. Methods U251 glioma cell line was infected by a lentiviral vector containing green fluorescent protein (GFP) and luciferase (Luc) gene. Cells stably expressing fluorescence of GFP and Luc were sorted by flow cytometry. CCK-8 test and Transwell tumor invasion and migration assay were used to compare the biological features between the cells stably expressing GFP-Luc fluorescence and cells without fluorescence. Then the cells were implanted intracranially in the right caudate nucleus of athymic Balb/c nude mice to establish the tumor model. The growth of intracerebral tumor was monitored over time by a bioluminescence imaging (BLI) system. Hematoxylin-eosin (HE) staining was used to evaluate the histopathological features and tumorigenicity of the transplanted glioma cells in the brain of nude mice. Results U251 glioma cell line with stably expressing GFP-Luc fluorescence and the corresponding orthotopic xenograft model were successfully established. There was no statistically significant difference in the proliferation, invasion and migration abilities between the cells with stably expressing GFP-Luc fluorescence and the control cells. This model showed a high tumor formation rate and stable tumor growth, and takes a moderate time to establish this model. Conclusions Compared with the traditional glioma cells, GFP-Luc-transfected human glioma cells are more feasible for the studies of glioma in vivo. The tumor growth and pathological characteristics in this U251-GFP-Luc glioma model are similar to human glioma, and the growth of this tumor can be real-time monitored. It can be used as an ideal animal model for experimental studies of glioma.

Abstract:Objective To compare the organ coefficients and expressions of hypoxia-related genes in Bama and Juema pigs. Method Real-time quantitative PCR was used to detect the changes of hypoxia gene expressions in the heart, liver, spleen, lung, and kidney of Juema and Bama miniature pigs. Results The organ coefficients of kidney and spleen of Juema pigs were significantly lower than Bama miniature pigs (P<0.05 for both). The heart and lung coefficients of Juema pigs were significantly higher than that of Bama miniature pigs (P<0.05 for both).The VEGF and HIF-1α expressions in the lung and kidney in Juema pigs were significantly higher than Bama pigs (P<0.05 or P<0.01). Only the EPO expression in in the lung of Juema pigs was significantly higher than that of the Bama miniature pigs (P<0.05). Conclusions These results indicate that the variation in organ coefficients may be resulted from evolutionary factors such as adaptiveness to environmental physical and energy conditions, pathogens, and energy metabolism demands, etc. in combination. Juema miniature pigs showing a significantly higher expression of hypoxia-related genes than that in Bama minipigs indicate that it has a strong plateau adaptability by higher gene expressions.

Abstract:Objective To explore the protective effect of nimodipine on PC12 cell apoptosis induced by hydrogen peroxide (H₂O₂). Methods The cells were randomized into five groups: normal group, model group (200 μmol/L H₂O₂), nimodipine low-, medium- and high-dose groups (1, 10, 100 μmol/L nimodipne + 200 μmol/L H₂O₂). Cell viability was measured by MTT assay. Cell apoptosis was assessed by Hoechst staining. The activity of cysteinyl aspartate specific proteinase 3 (caspase 3), caspase 9 and superoxide dismutase (SOD), the level of malonic aldehyde (MDA) were measured by colorimetry. The expression of B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax) and p53 were detected by western blot. Results Compared with the normal group, the cell apoptosis rate, the activity of caspase 3 and caspase 9, the MDA level, and the expression of Bax and p53 were significantly increased, the cell viability, SOD activity, and expression of Bcl-2 were decreased in the model group (P<0.01). Compared with the model group, the cell apoptosis rate, the activity of caspase 3 and caspase 9, the level of MDA, and the expression of Bax were decreased; and the cell viability, SOD activity, and the expression of Bcl-2 were increased in the nimodipine low-, medium- and high-dose groups (P<0.01). Conclusions Nimodipine suppresses cell apoptosis induced by H₂O₂ in PC12 cells, which may be related to regulation of expression of cell apoptosis-related proteins.

Abstract:Objective To observe the changes of bone mass in reloaded rats after tail-suspension, and the effect and mechanism of simvastatin on this process. Methods Twenty-four 5-month old rats were divided into 4 groups of 6 animals in each group: Control (CL) group without tail-suspension, unloaded (UL) group with tail-suspension for 6 weeks, other 12 rats received tail-suspension for 3 weeks, then reloaded for subsequent 3 weeks (UL+RL) or combined with simvastatin treatment (UL+RL+SIM) at a dose of 10 mg/kg/d. All rats were sacrificed 6 weeks later, and the left femur was used for examination of bone mineral density, left tibia was used for bone histomorphometry analysis, the right femur and tibia were harvested for biomechanical test, and expression levels of type I collagen by real-time PCR and Western blot, respectively. Results 1. BMD of the CL group was significantly higher than those of the other three groups (P<0.05), and was markedly lower than those in the UL+RL and UL+RL+SIM groups (P<0.05). 2. The bone histomorphometry showed that BV/TV in the CL group was significantly higher than those in the other 3 groups, and the UL+RL and UL+RL+SIM groups showed a significantly higher BV/TV than that of UL group (P<0.05). The Tb.Th was significantly higher in the CL group than in the UL group. The Tb.Sp in the CL group was significantly lower than those in the other 3 groups (P<0.05). The UL+RL and UL+RL+SIM groups showed significantly lower Tb.Sp than that of the UL group (P<0.05). 3. Biomechanical test showed that the maximal load and elastic modulus in the CL groups were significantly higher than those of the other three groups (P<0.05). 4. Real-time PCR showed that no significant difference in the mRNA expression level of Col I was found between any two groups. 5. Western blot showed that the IOD of Col I is significantly lower than that in the CL group. Conclusions Bone loss, destruction of trabecular bone micro-architecture and biomechanical properties and reduction of type 1 collagen are present in tail-suspension treated rats, which are partially restored after reloading, and this recovery process is not enhanced by simvastatin treatment.

Abstract:Objective To study the changes of behavior and depression-like classic indicators after hippocampal microinjection of K252a, and to establish a new animal model of depression. Methods SD rats were randomly divided into five groups, namely the control group, sham group, chronic stress depression model group, hippocampal of K252a microinjection group, and hippocampal microinjection K252a plus chronic stress group. Open field experiments, sucrose consumption test, and Morris water maze behavioral assay were used to assess the behavioral changes in the rats. ELISA was used to detect the plasma monoamine neurotransmitter, radioimmunoassay was used to determine the plasma CRH, ACTH, CORT contents, and western-blotting was performed to observe the protein expression of BDNF, CREB, ERK1/2, and BCL-2 in the hippocampus. Results Compared with the control group, the amount of activity, sugar consumption, learning and memory abilities were decreased(P<0.05 or P<0.01), also the serum monoamine neurotransmitters were decreased (P<0.01), HPA axis function was improved (P<0.01), and the expression of BDNF, CREB, ERK1/2, BCL-2 decreased in the CUMS group(P<0.05 or P<0.01), but there was no significant difference in the DMSO group.Compared with the DMSO group, the activity, consumption of sucrose, learning and memory ability were significantly decreased(P<0.05 or P<0.01),while the HPA axis function was increased (P<0.05 or P<0.01),the serum monoamine neurotransmitters decreased(P<0.05 or P<0.01), and the BDNF, CREB, ERK1/2, BCL-2 expressions in the hypocampus were significantly decreased(P<0.05 or P<0.01) in the K252a group and K252a + CUMS group. Compared with the CUMS group,the K252a group and K252a + CUMS group did not show significant changes in these parameters. Compared with the K252a group, these indicators were not significantly changed in the K252a + CUMS group. Conclusions The results of behavior, hematology, and molecular biology analysis show that this model has a great similarity to the classical model of CUMS in surface validity, construct validity, and functional validity. It may provide an alternative investigative technology platform for basic research and antidepressant drug screening.

Abstract:Objective To explore the preventive and therapeutic effects of diminazene (DIZE) on pulmonary arterial hypertension (PAH) in rats. Methods Left pulmonary lobectomy combined with injection of monocrotaline was used to establish a rat model of pulmonary arterial hypertension. One hundred adult male Wistar rats were randomly divided into blank control group (group A, n=20), DIZE control group (group B, n=20), PAH model group (group C, n=20), PAH model plus DIZE group (group D, n=20) and PAH model plus DIZE and C-16 group (group E, n=20). Angiotensin-converting enzyme 2 (ACE2), IL-6 and IL-8 levels were determined by fluorescence resonance energy transfer (FRET) and enzyme linked immunosorbent assay (ELISA), and the mean pulmonary arterial pressure (mPAP) and right ventricular hypertrophy index (RVHI) were measured. The wall thickness (WT) and intimal hyperplasia score were calculated, and the pulmonary vascular lesions were analyzed using elastic fiber staining. Results RVHI, ACE2 enzyme activity and WT in the group A were significantly different from those of the groups C, D and E (P<0.05). Those of the group D and E were significantly different (P<0.05). The five groups showed significant differences in the overall analysis of each index (P<0.05). Conclusions Diminazene can increase the ACE2 enzyme activity, and decrease the mPAP, RVHI and WT, while reducing the pulmonary arterial medial hypertrophy, and inhibit intimal hyperplasia of pulmonary arterioles. The results of this study provide an experimental basis for the use of diminazene in the treatment of human pulmonary hypertension.

Abstract:Objective We compared the effect of three anesthetic procedures on the establishment of and recovery effect on the young minipig models of skull defect, and explore an optimal anesthetic procedure for long-lasting surgical experiment in minipigs. Methods Thirty 3-month old Guizhou minipigs (male:female=1:1) were randomly divided into three groups, 10 in each group. The group A was given with midazolam and ketamine i.p., the group B received lumianning II i.p., and the group C received midazolam combined with ketamine and lumianning II i.p. The induction time of anesthesia, the first anesthesia maintenance time, the first anesthesia maintenance period after additional use of anesthetics, the second time anesthesia maintenance period after additional use of anesthetics, the recovery period, the number of times of additional intraoperative use of anesthetics, cumulative amount of anesthetics used, and the adverse reaction and mortality rates of the animals after anesthesia were observed and analyzed. Results The anesthesia induction time in the group B was significantly longer than that in the groups A and C (P<0.05 for both). The anesthesia maintenance time and the anesthesia maintenance after first and second additional use of anesthetics in the groups A were significantly longer than those of the groups A and B (P<0.05 for both). The recovery periods in groups A and C were shorter than that of the group B (P<0.05 for both). The number of times of additional intraoperative use of anesthetics, the total dose of anesthetics, the adverse reaction and mortality rates in the group C were significantly lower than those of the groups A and B (P<0.05 for both). Conclusions The combination of midazolam with ketamine and lumianning II is a simple, easy to control the anesthesia depth, and a safe method to anesthetize young minipigs in long-lasting surgical experiment.

Abstract:Objective To establish lentiviral expression vectors for Smurf1 silencing and assess the effects of Smurf1 silencing on cell migration. Methods HeLa and A549 cells were infected with lentiviral expression vectors for Smurf1 silencing respectively. After 7 days, the stable cell lines with Smurf1 silencing were obtained after puromycin-resistance screening, enrichment and expansion. The intracellular gene and protein levels of Smurf1 were detected by qPCR and western blot. Transwell assay was used to assess the effect of Smurf1 silencing on cell migration. Results The stable cell lines with Smurf1 silencing are constructed successfully. Silencing of Smurf1 down-regulated cell migration rate detected by Transwell assay. Conclusion Smurf1 promotes cell migration.

Abstract:Objective To knockout the MATP gene of mouse melanoma cell line B16F10 using CRISPR/Cas9 system, and to lay foundation for the functional study of MATP gene.Methods Specific primers of MATP were designed according to the report in http://crispr.mit.edu/ website. The primers were linked to pCAS9/gRNA1 vector. Then the positive vector was transfected into mouse melanoma B16F10 cells, and monoclonal cell lines were obtained by the infinite dilution method. After the genomes of different monoclonal cell lines were extracted and sequenced, the cell lines with MATP gene cleavage were screened, and the expression of MATP in these cell lines was verified by Western-blot analysis. Results Three MATP gene knockout cell lines were successfully obtained. The western-blot results showed that the cell lines did not express MATP protein. Conclusions The knockout of MATP gene in B16F10 cell line can be successfully achieved using the pCAS9/gRNA1 vector.

Abstract:Objective Shkbp is also called Shkbp1, can competitively inhibit binding CIN85 and c-Cbl, thereby blocking the epidermal growth factor receptor (EGFR) endocytosis and degradation, to play a role in tumor promotion. This study aims to explore the changes in blood cell classification and T cell subsets in blood, bone marrow, and spleen in Shkbp1-deletion (Shkbp-1^-/-) mice. Methods Shkbp-1^-/- transgenic mice were identified by PCR genotyping. Blood cell classification was performed using an automatic classification system. Flow cytometry was used to detect the T lymphocyte subsets in the blood, bone marrow, and spleen of Shkbp-1^-/- and control mice. Results Routine blood examination showed that neutrophils and eosinophils tended to increase and showing significant differences, and there was no significant difference in lymphocytes. The flow cytometry results showed that there was a decrease of CD4⁺CD8⁺double positive cells and increase of bone marrow CD3⁺ and CD4⁺ cells in the control group. However, there was a decreasing trend of CD3⁺, CD4⁺, CD8⁺, and CD4⁺CD8⁺cells in the spleen tissues. Conclusions Shkbp1 is involved in the maturation and differentiation of blood cells, and affects the number of immune cells. This study lays a foundation for the study of how Shkbp1 is involved in the differentiation of blood cells.

Abstract:Objective This study aims to understand the characteristics of the time sequence of ICR mouse first mandibular molar tooth germ development through dynamic observation. Methods Tooth germ of Embryos (E11.5, E12.5, E13.5, E14.5, E15.5, E16.5, E17.5 and E18.5) and postnatal (PN1, PN2) mice were obtained. The heads (E11.5-E15.5) and mandibles (E16.5-PN2) of mice were dissected, fixed and embedded for serial sections and HE staining. All the results were assessed under light microscopy. Results The tooth germ underwent various development stages including the bud, cap and bell stages. Mouse odontogenesis was initiated at E11.5. Proliferation of oral epithelium formed the bud stage at E13.5. Then the cap stage was observed at E14.5- E15.5 and the bell stage was appeared beginning from E16.5. The pre-dentin was observed at PN1, as well as the dentin at PN2. Conclusions Establishing the regular development pattern of the first mandibular molar of ICR mice will provide a reliable basis for the future use in the specific tooth germ developmental research.

Abstract:Objective To investigate the changes of average body weight gain and serum biochemical indexes of C57BL/6J mice (B6 mouse) and their offspings after frozen-thawed embryo transfer of B6 mice. Methods The mice were divided into three groups in this study. In the experimental group I (E-I, 30 males and 20 females), 2-cell embryos after in-vitro fertilization were collected, and cryopreserved by EFS method, then obtained the offsprings after transplantation of the recovered embryos to oviduct of recipient mice (ICR mouse). In the experimental group II (E-II, 26 males and 17 females), when the mice from E-I grew to maturity, the offsprings were obtained from natural mating of mice from E-I. In the control group (20 males and 20 females), the offsprings came from conventional feeding and natural mating. The three groups of mice were raised to 16 weeks old, weighing the body weight at a regular time intervals, and the serum biochemical indexes were obtained from 16-week-old mice. Then the changes of average body weight and serum biochemical indexes of the mice were analyzed. Results The average body weight of E-I mice was significantly higher than that of control group at each week-age (P<0.01). The average body weight of E-II female mice was significantly higher than that of the control group in 12-16-week old mice (P<0.01), but the average body weight of E-II male mice showed no significant differences compared with the control group except for few weeks. The serum biochemical indexes of E-I and E-II mice were changed in all items except for AST, TP and Ca. Conclusions There are some effects on the average body weight gain and serum biochemical indexes of C57BL/6J mice and their offspings after frozen-thawed embryo transfer.

Abstract:Objective To investigate the natural infection of Theiler's murine encephalomyelitis virus (TMEV) in mice, and to survey the distribution of virus in tissues and the changes of serum antibody in the experimentally TMEV-infected mice. Methods Enzyme linked immunosorbent assay (ELISA) and fluorescence quantitative RT-PCR (qRT-PCR) assay were used to detect the antibody and nucleic acid of TMEV in clinical samples. These samples included SPF mice collected from Guangdong area in 2010-2015, mice obtained from a non-barrier laboratory rodent colony, and wild Rattus norvegicus live-trapped around the non-barrier laboratory rodent colony. 36 ICR mice were intracerebrally inoculated with TMEV BeAn strain. The clinical signs of the animals were observed daily post-inoculation. Three mice were euthanatized at day 0, 3, 7, 10, 17, 21, 31, 39 and 46 post-inoculation (dpi), respectively. Tissue and serum samples were collected for TMEV detection. Results The TMEV antibody and nucleic acid positive rates of SPF mice collected from Guangdong area in 2010-2015 were 5.29% (n=2834) and 27.27% (n=457), respectively. The TMEV antibody and nucleic acid positive rates of the mice obtained from a non-barrier laboratory rodent colony were 71.95% (n=82) and 53.66% (n=82), respectively. The TMEV nucleic acid positive rate of wild Rattus norvegicus was 25.93% (n=27). In the TMEV positive mice, only two mice showed obvious clinical symptoms. The cecal contents, feces and brain samples were the best candidates for qRT-PCR assay. The viral nucleic acid could be detected in the brain, heart, liver, lung and stomach of ICR mice at 3 dpi, but no viral nucleic acid was detected in the spleen, kidney, and cecum. The viruses in liver, heart, lungs and stomach were completely cleared at 10 dpi, and the viruses persisted in the brain throughout the experiment. The TMEV antibody could be detected at 7 dpi, and then the antibody positive rate reached 100% at 17 dpi. The antibody level increased gradually and maintained up to 46 days. ICR mice showed latent infection after TMEV inoculation, with no obvious symptoms and eye pathological changes. Conclusions The experimental mice and wild Rattus norvegicus in Guangdong area are both infected with TMEV, and the infection rate is high. The mice inoculated with TMEV BeAn strain show latent infection. The TMEV antibody produced in mice can be detected at 7 dpi and persisted until the end of the experiment. The viruses are found in the liver, heart, lung and stomach for a short time, but are persisted in the brain for a long time. There is a good consistency of TMEV detection between qRT-PCR and ELISA. The qRT-PCR assay can be used as a powerful complement method for the national standard of laboratory animals.

Abstract:Objective To compare the effects of single and complex emotional stimuli on animal model of liver depression syndrome, in order to find out an animal model of liver depression syndrome with more similar clinical manifestations. Methods Based on the previous screening work, different methods, i.e. tail clamping, limb binding, putting a cangue on the neck, were used respectively, or using the three methods in combination to establish rat models of liver depression syndrome. A comparison table of human and rat liver depression syndrome was prepared, a mark sheet of evaluation criteria was made, general condition was observed and body weight was measured, and positive response to drugs and changes of hormonal levels were evaluated to identify whether the models were established successfully. Results Rats in the control group grew well and showed no abnormalities. There were lots of abnormal phenomena in the model group especially in the complex emotional stimulus modeling group. They were maniac with acute stress or depressive with chronic stress, had dry hair, small and less feces, many of the rats had hair loss, red eyes and shortness of breath symptom. They showed a significantly lower body weight gain and higher levels of CRH in hypothalamus and plasma ACTH and CORT. The manifestations were much more severe in the complex-emotional stimuli-induced model group than that in the single-stimulus prepared model groups. The drug-treated group showed considerable alleviation of the symptoms and improvement of the parameters closing to normal levels. Conclusions Establishing the rat model of stagnation of liver depression syndrome by complex emotional stimuli modeling method has good stability, with a short preparation period and high success rate, and presenting a better consistence with clinical liver stagnation of depression syndrome, so as to provide a suitable tool for TCM research.

Abstract:Objective To evaluate the effect of maternal separation stress on the behavior of neonatal rd mice. Methods Neonatal rd mice were divided into maternal separation (MS) group (n=9) and control group (n=9). MS-stress was induced in the MS group by 4-hour-separation per day for 28 days. Open field test, elevated plus maze test, forced swim test and tail suspension test were used to evaluate the anxiety-like and depression-like behavior of the neonatal rd mice.Results The stay time and distance travelled of MS group in the central zone were 0.88% and 28.17±5.65 cm, respectively, significantly shorter than that of the control group (2.61%,109.9±9.79 cm. P=0.04, P=0.001). Compared with the control group, the stay time in open arms of the MS group was significantly decreased (P<0.01), while the immobility time in forced swim test and tail suspension test of the MS group were 126.5±10.22 s and 21.56±6.83 s, significantly longer than that of the control group (77.75±16.83 s, P=0.02, 7.37±3.22 s, P=0.03). Conclusions The 28-day maternal separation stress can significantly increase the anxiety-like and depression-like behavior in neonatal rd mice.

Abstract:Objective To establish an in vitro skin sensitization test, human cell line activation test ( h-CLAT), based on THP-1 cell line (a human acute monocytic leukemia cell line), and to assess the sensitizing potency of plant raw materials of chemical and cosmetic products by this in vitro skin sensitization test. Method THP-1 cells were cultured in vitro and exposed to 11 reference skin sensitization chemicals and 9 samples, by monitoring the cell viability, cell surface marker CD54/CD86 and relative fluorescence intensity of cells surface after the cells was exposures to the substances, and to discover whether there is a positive reaction. At the same time, Buehler test was used to validate the results of samples tested by h-CLAT. Results 11 reference chemicals were distinguished correctly by h-CLAT. Among the 9 samples tested, 7 samples were recognized as negative sensitizer and 2 plant extracted substances were identified as suspicious skin sensitizer. The qualitative classification of the 9 samples by h-CLAT test was consistent with the results obtained by animal test. Conclusions The h-CLAT-in vitro test can be used to replace some animal tests for the prediction of soluble skin sensitizing substances.