Abstract:Objective To analyze and compare the pathological changes of lung tissue in mice infected with the novel H7N9 influenza virus and 2009 pandemic H1N1 influenza virus, respectively, and to preliminarily study the mechanisms of acute lung injury induced by those virus infection. Methods SPF 6-week old BALB/c mice (body weight 18-20 g, male :female=1:1) (n=3 in each subgroup) were intranasally infected with H7N9 virus and H1N1 virus, respectively. The behavior and survival time of mice after virus infection were observed and the survival rates were analyzed. The heart, liver, spleen, lung, kidney, intestines, and brain were collected at indicated time points for histopathological examination using H&E staining. The distribution of virus antigen was detected by immunohistochemistry. The neutrophil infiltration was also observed. The correlation of lung injury with virus replication and host immune responses was analyzed. Results The lung and spleen injury of mice infected with H7N9 virus was slighter and their survival rate (100%) was higher than those of mice infected with H1N1 virus. The damages of the lung and spleen in H1N1virus-infected mice were more severe than that in H7N9 virus-infected mice, and all the 10 mice in this group died within 9 days after virus inoculation. The distributions of both the virus antigens were mainly in the bronchial epithelial cells, a few stromal cells and alveolar epithelial cells. The levels of virus replication in the two groups were not significantly different. There were more intense neutrophil infiltration in the lung and inflammatory response in the H1N1 virus-infected mice than those in the H7N9 virus-infected mice. Conclusions There are some differences of the pathological characteristics and extent of lung injury in the mice infected with H7N9 virus and H1N1 virus, respectively. The virus replication is a precipitating factor but not the decisive factor of the lung injury, and there is a close relationship between the host immune responses and acute lung injury.

Abstract:Purpose Polymorphisms of candidate gene Agouti was analyzed in order to reveal the molecular mechanisms of coat color difference in chromosome engineering mice. Methods Firstly, differences of mouse coat color was detected by color measurement spectrophotometer. Then, candidate gene Agouti was found by whole genome scanning based on DNA chip. Finally, cDNA and amino acid sequence polymorphisms were analyzed, as well as the influence of protein properties and function after mutation was predicted by bioinformatics software. Results There are five SNPs in the Agouti cDNA sequences, resulting in three missense mutations in the amino acid sequence of Agouti signaling protein. Bioinformatics analysis revealed that one β sheet deletion in the secondary structure of the mutant protein, as well as tertiary structure changed, leading to decrease of binding ability. Conclusion A novel missense mutation is found in candidate Agouti gene. It plays critical role in receptor binding activity, and may reflect on mice coat color changing from light gray to dark gray eventually.

Abstract:Objective To establish a mouse model of acute liver failure induced by lipopolysaccharide/D-galactosamine (LPS/D-GalN). Methods The optimum dose of LPS/D-GalN was determined by i.p. injection of eight different doses of LPS and D-GalN into 40 female C57BL/6 mice and observation of their survival time. Then, 32 female C57BL/6 mice were i.p. injected with the optimal dose of LPS/D-GalN and sacrificed at 0, 1, 4, 8 hours after the injection, 8 mice in each group. The control mice received saline injection. Hepatic changes were observed by pathology and serum ALT, IL-6, MCP-1 and TNF-α were measured by biochemistry or flow cytometry. Results LPS (2.5 mg/kg) and D-GalN (0.3 g/kg) were determined as the optimal dose for the establishment of mouse model of acute liver injury. Compared with the control group, the hepatocellular damages were progressing in a positive correlation with the time course after LPS/D-GalN administration. The level of serum ALT was significantly increased after LPS/D-GalN administration(P<0.001). The levels of inflammatory cytokines IL-6, MCP-1 and TNF-α were increased and reached a peak at one hour after LPS/D-GalN administration and then decreased almost to that of the control group 8 hours later(P<0.001). Conclusions The mouse model of acute liver injury is successfully established by LPS/D-GalN administration, and provide an effective animal model for the study of pathogenic mechanisms of acute liver failure and evaluation of therapeutic drugs.

Abstract:Objective To establish a diarrhea rat model using multiple-stimulating factors and choosing the best indexes to assess whether the model is consistent with the disease characteristics of liver-QI stagnation with spleen deficiency in traditional Chinese medicine. Methods Newborn SD rats were randomly divided into model group (n=20) and control group (n=10). The rats of model group were stimulated by maternal separation, restraint stress and rectum acetic acid irritation, while the rats in control group were fed as normal. Weight changes, rectal sensitivity, Bristol scores and water content of feces and histology of the colon tissues were used as evaluation indexes to assess whether the model meets the demands for further studies. Results The rats in the model group showed loss of appetite, increase of water intake and urine reduction. Some rats showed increased activity, and even mania. Bristol scores and water content of feces were significantly higher than that of the control group, and the rectal sensitivity was significantly increased. The colon mucosa showed slightly thickened submucosal layer and mild inflammatory cell infiltration in the model rats. Conclusions The rat model established in this study is better to simulate the clinical manifestation of liver-QI stagnation with spleen deficiency in Chinese medicine, and may meet the demands of related researches of this disease.

Abstract:Objective To explore whether the cAMP-PKA-pCREB signal pathway plays a role in promoting the recovery of motor function after rehabilitation training in cerebral ischemia-reperfusion rats. Methods The middle cerebral artery occlusion model (MCAO) was established by modified Longa nylon occlusion method in adult male Sprague-Dawley rats. The 84 MCAO rats were selected and randomly assigned to four groups: the natural recovery group without any special training (group B, n=24),natural recovery group with Rp-cAMP (group C, n=24), rehabilitation training group (group D, n=18) and rehabilitation training with Rp-cAMP (group E, n=18), and in addition a control group (group A, n = 12). To establish rat MCAO models immediately after injection of Rp-cAMP into the lateral ventricle of the brain. The rats in the groups D and E were trained by balance beam, bar rotating and rolling exercises started at 48 h after MCAO. The expression of PKA was determined by enzyme-linked immunosorbent assay (ELISA) and the pCREB protein expression was detected by Western blot assay. Motor function was assessed by balance beam test. Results (1) The motor function score in the group C was significantly higher than that of group B, suggesting that Rp-cAMP inhibited the recovery of motor function in the cerebral ischemia-reperfusion rats. The score of group D was significantly lower than that of groups B and E, indicating that Rp-cAMP inhibited the promoting effect of rehabilitation training on motor function in the cerebral ischemia-reperfusion rats. (2) The expressions of PKA and pCREB proteins detected at 2nd, 7th, 14th, and 21th days after surgery showed that their expressions in the group D were significantly higher than those of the groups B and E, indicating that rehabilitation training promoted the expression of PKA and pCREB, and Rp-cAMP significantly inhibited the promoting effect of rehabilitation training on the expressions of PKA and pCREB proteins. Conclusion cAMP/PKA-pCREB signal transduction pathway may mediate a promoting effect of rehabilitation training on the recovery of motor function after ischemic stroke in rats.

Abstract:Objective To study the impact of establishment of lipopolysaccharide (LPS)-induced rat model of chronic obstructive pulmonary disease(COPD)on the function of multidrug resistance-associated protein 1(MRP1)in the rat bronchial epithelium. Methods Using intratracheal instillation of LPS to establish COPD rat model. 8-week old healthy male Wistar rats were divided into 3 groups (10 rats in each group): (1) Normal control;(2) Modeling for 14 days after LPS instillation; (3) Modeling for 28 days after LPS instillation. Pulmonary function and the concentration of phenol red in bronchoalveolar lavage fluid (BALF) and plasma were measured. The ratio of phenol red concentration in BALF/plasma was used as an index of the MRP1 function in the rat bronchial epithelium and the expression of MRP1 in the bronchial epithelium was also observed by immunohistochemistry. Results Compared with the normal group, the pulmonary functions of the rats in the model groups were significantly reduced along with the modeling progress. After intravenous administration of phenol red, the ratio of phenol red concentration in BALF/plasma was gradually reduced, and the expression of MRP1 in the bronchial epithelium was significantly decreased. Conclusions COPD rat model can be established by intratracheal LPS instillation, and the function of MRP1 in bronchial epithelium was gradually reduced along with the modeling progress.

Abstract:Objective In order to provide the basis for establishment of food allergy models, we compared the differences of sensitivity and alterations of intestinal flora of food allergy models in two strains of mice. Methods Forty 4-5-week old female BALB/c and Kunmimg mice were divided into control group (n=10) and food allergy goup (n=30), respectively. Ovalbumin (OVA) was injected to the mice to establish food allergy models. Serum OVA-specific IgE of the mice was assayed by ELISA. The jejunum tissue was examined by pathology with HE staining. The changes of fecal flora were detected by denaturing gradient gel electrophoresis (DGGE). Results (1)Among the sensitized 60 mice, OVA-sIgE levels were significantly increased in 27/30 BALB/c mice and 21/30 KM mice compared with those of control groups(P<0.001). Moreover, there were more evident inflammatory cell infiltration, epithelial cell shedding and cytolysis in the jejunal villi of BALB/c mice than those of KM mice. (2) After food allergy modeling, there were significant changes of intestinal flora in the BALB/c mice (P<0.001), while only significant change of evenness was found in the KM mice (P<0.05). (3)There were changes of abundance, Shannon index and evenness of intestinal flora in the model groups of BALB/c and KM mice. Conclusions BALB/c mice are more sensitive to OVA allergy than KM mice. The composition of intestinal flora is different among different strains of mice. The changes of intestinal flora after OVA challenge in BALB/c mice are more obvious than those in KM mice.

Abstract:Objective To define the functional mechanisms of two NSAIDs, paracetamol and ibuprofen, in specific brain regions in headache control by observing the distribution of Fos-immunoreactive neurons in trigeminal ganglia and trigeminal nucleus caudalis in conscious rat models of vasculogenic headache. Methods Thirty male Sprague-Dawley rats were randomly divided into three groups: control group (saline group), acetaminophen group and ibuprofen group. Each rat was given electrical stimulation (frequency 20 Hz, current 3-5 mA, pulse width 0.25 ms) at 50 minutes after injection. The rats were killed and perfusion fixed after electrical stimulation. Trigeminal ganglia and trigeminal nucleus caudalis of the brains were taken out for paraffin sections and immunohistochemical staining, and Fos-immunoreactive neurons were counted under the Image J system. Results After electrical stimulation, there were significant differences of Fos protein expression in bilateral trigeminal ganglia and spinal trigeminal nucleus caudalis between the saline group and groups of non-steroidal anti-inflammatory drugs, but no significant difference of Fos protein expression in bilateral trigeminal ganglia and spinal trigeminal nucleus caudalis between the acetaminophen group and ibuprofen group. Conclusions The changes of Fos expression in bilateral trigeminal ganglia and spinal trigeminal nucleus caudalis after treatment with NSAIDs suggest that such structures participate in the pain transmission and expression and the pharmacology course of analgesic drugs.

Abstract:Objective To evaluate the application value of PCR-sequencing in clinical detection of hantavirus in rodents. Methods Based on 7 subtypes and 24 strains of representative hantavirus strains downloaded from Genbank, the virus S gene fragments were used for primer design and neighbor joining method was applied for phylogenetic analysis. Thereafter, we identified hantavirus strains isolated from wild rodents in recent years in Zhejiang Province by this method. Results The 24 analyzed strains were divided into 5 regions in the phylogenetic tree. Four of them with topology structure were more stable. Eleven strains of the virus were amplified by PCR and sequenced, and the results showed that the primers were with high sensitivity and specificity. Three HTN strains and 1 strain of serotype SEO were distinguished from 9 strains of unknown strains isolated in Zhejiang Province. We also found that 5 strains of hantavirus belonging to two unknown serotypes. Discussion Our results suggest that the PCR-sequencing method proposed in this study can be used for clinical detection of hantavirus.

Abstract:Objective To establish a new rat model of biliary atresia by pure ethanol injection into the common bile duct. Methods A catheter was inserted and fixed in the common bile duct in male SD rats. Saline (8 rats) or pure ethanol (16 rats) was injected through the catheter,respectively, and the biochemical and pathological changes in the rats were examined. Results SD rats in the experimental group were divided into a persistent injury and a restoration of liver dysfunction groups according to pathological and biochemical detection. In the persistent injury group, biochemical impairments were significantly higher at 8 weeks after ethanol injection than those in the control group and restoration group. Distinct pathological changes in the liver were observed using HE, SMA, and Masson staining. Conclusions It is a reliable animal model of biliary atresia induced by injection of pure ethanol into the common bile duct in the rat. It will provide a useful tool in future studies of biliary atresia.

Abstract:Objective The aim of this study was to investigate the differences of the cell ultrastucture of normal mouse hatched blastocysts and their dormant ones cultured in vitro after freezing-thawing, and to explore whether the dormant embryos have a better anti-freezing shock property than the normal hatched mouse embryos. Methods By transmission electron microscopy, the ultrastructure of these two types of mouse embryos was observed and analyzed. Results By comparative analysis of their ultrastructure, the results showed that the dormant embryos before freezing are being austerity and with lower energy metabolism at a‘ground state'. After freezing-thawing and culture, their cellular structure seemed to be similar to that of the normal embryos cultured in vitro before freezing. However, after freezing-thawing and culture, the number of mitochondria decreased, the nuclei were loose, and their heterochromatin also increased. Conclusions From the ultrastructural observation, compared with the normal mouse hatched embryos, the cellular state of dormant mouse embryos after freezing-thawing is more favorable for material storage and energy metabolism, thus, indicating that they have a better anti-freezing property than normal hatched embryos.

Abstract:Objective Uterine epithelial cells were isolated from pregnant mice and cultured in vitro, and examined the effect of CD82 on the expression of integrin αV, β3, E-cadherin and β-catenin in the cells. Methods The uterine epithelial cells were primarily isolated from pregnant mouse uterus. The recombinant adenovirus containing mouse CD82 gene which had been constructed in our lab infected the uterine epithelial cells. Immunocytochemistry was used to detect the protein expressions of integrin αV, β3, E-cadherin and β-catenin in the uterine epithelial cells, which were infected with CD82 adenovirus or not. Results 1. The purity of primary cultured cells was (93.2 ± 0.6)%. 2. The transfection efficiency of CD82 recombinant adenovirus was (92±4.5)%. The adenoviral particles carrying CD82 gene indeed expressed CD82 gene and protein in the primary uterine epithelial cells after 24 hours or 48 hours. 3. The uterine epithelial cells of pregnant mice on d4 expressed integrin αV, β3, E-cadherin and β-catenin. 4. In contrast to the control group, when CD82 adenovirus infected cells, the uterine epithelial cells of pregnant mice on d4 increased the expression of integrin αV, β3 and β-catenin protein, had no significant changes of E-cadherin. Conclusions CD82 may have effect on the expression of integrin αV, β3 and β-catenin in mouse uterine epithelial cells before implantation.

Abstract:Objective To study the effects of living environment conditions and animal-animal interaction on the blood hormone levels and psychological behavior in Chinese tree shrews. Methods Chinese tree shrews were raised in cages of different space sizes or were administered reserpine for 15, 30, 60, 120, 180 days, respectively. Then the animals were anesthetized by ether inhalation, and blood samples were taken from the heart to detect the levels of blood testosterone (T), estradiol (E2), endothelin (ET), adrenaline (A) and noradrenaline (NA) by radioimmunoassay (RIA). Results 1. Chinese tree shrews were bred in large cage (D1group) or small cage (X1 group) for 15, 30, 60, 120, 180 days, respectively. Compared with the animals bred in the large cage (D1 group), the level of blood testosterone (T) was significantly reduced (P< 0.01), and the levels of adrenaline, noradrenaline and endothelin were significantly increased in the small cage group (P< 0.01 for all). 2. The animals raised in small cages in close neighborhood with large cages for 15, 30, 60, 120, 180 days, respectively. The levels of testosterone, adrenaline and noradrenaline in the large cage group (X2 group) were significantly higher than those of the small cage group (X1 group) (P< 0.01 for all). 3. The adrenaline and noradrenaline levels were significantly lower in all the reserpine groups (P<0.01 for all). 4. The animals bred in small cages (X1 group) and in small cages in close neighborhood with large cages showed sudden loss, reduced appetite, testicular atrophy, penile prolapse and stress symptoms. The animals of the reserpine groups appeared gentle temperament, significantly reduced activity and reduced appetite. However, after stopping the reserpine administration and feeding them in large cages, the animals gradually returned to normal behavior. Conclusion Both animals living environment conditions and animal-animal interaction may cause changes of blood hormone levels and psychological behavior in Chinese tree shrews.

Abstract:Objective To compare the therapeutic effect of three treatment regimens on H22 tumor-bearing mice. Methods H22 tumor-bearing mice were treated by local injection of compound chemotherapeutic agents (5-FU, mitomycin and cisplatin), oral administration of sorafeni, or the both combined, respectively. Standardized and quantitative syndrome differentiation methods were used to assess the syndromes and tumor-inhibition rate in the mice. Results All these three therapeutic regimens were effective in suppressing the tumor growth in mice. Among them, the combined therapeutic regimen of local injection of compound chemotherapeutic drugs plus oral administration of sorafeni was the best. Conclusion The effect of sorafenib combined with local injection of compound chemotherapeutic agents is better than the other two regimens used separately.

Abstract:Objective To establish a simple, stable, specific and sensitive method for detection of Tyzzer's organism by reverse dot blotting (RDB). Methods Primers and specific probes were designed according to the conservative sequence of Tyzzer 16S rDNA. The forward primer was labeled with biotin. The reverse dot blotting method was established followed by PCR amplification. The specificity and sensitivity of this method were determined. Next, 41 mice and 38 rats were examined by RDB, ELISA and IFA. Results The RDB method showed a high specificity, and in the testing of the 79 laboratory animals, its limit of detection was 4.5 ng/μL. Compared the results of ELISA and IFA, its consistence with ELISA was 100% and the positive rate was 7.59% (6/79), the consistence with IFA was 92.4% (73/79), and the positive rate was 0%.Conclusions An accurate, sensitive and specific method in combination with PCR and RDB in detection of Tyzzer's organism is established in this study.

Abstract:Objective To screen microsatellite DNA markers from genome of guinea pigs for further genetic quality control and gene-mapping of this species. Methods Microsatellite sequences were obtained by magnetic bead enrichment and genome database screening, and candidate loci were chosen to design primers. Thereafter, genomic DNA of 5 different guinea pig strains were employed to select polymorphic microsatellite DNA markers based on PCR amplification results. Results A total of 304 microsatellite sequences were analyzed by magnetic bead enrichment and 125 primers were designed. One polymorphic microsatellite DNA marker and 17 specific sites (no polymorphic was found) were determined. By gene-mapping, 292 microsatellite sequences were obtained and 178 primers were analyzed, totally 25 polymorphic microsatellite DNA markers and 28 specific sites (without polymorphics) were discovered. Conclusions We obtained 26 polymorphic microsatellite DNA markers and 45 potential markers in guinea pigs, and these may lay a foundation for application of microsatellite DNA markers in genetic quality control and gene-mapping of guinea pigs.

Abstract:Objective The aim of this study was to observe the growth difference and expression of cytochrome C of skin hair follicles in neonatal mice. Methods The morphology of different skin hair follicles of neonatal mice(postnatal day 1-9)were observed by HE staining histology and cytochrome C was detected by immunohistochemistry. Results The skin hair follicles in different parts of neonatal mice showed differences not only in morphology but also in developmental periods. Hair follicle growth in the back and tail skin had a nonlinear and growing period. After the nonlinear and growing period they began to grow rapidly. The tail development was slightly slower than that on the back. The hair follicles of vibrissae were very special, and started to develop without a stable period. Conclusions The results of morphological observation and cytochrome C immunohistochemistry demonstrate that differences exist in the hair follicle morphology and developmental times in the skin of different parts of the body in neonatal mice.

Abstract:Objective To reduce the animal component contamination for human embryonic stem cells (hESCs) and to simplify hESCs culture process, we develop a new coating substrate which can support the hESCs growth without differentiation, and is easy to store and use. Methods Mouse embryonic fibroblasts(MEF)were fixed on the surface of plate by methanol. hESCs were cultured on this new substrate and were passaged every 5 to 6 days. After 10 passages, we checked the cell morphology, alkaline phosphatase expression, embryonic specific markers and the differentiation ability in vitro. Results After 10 passages, the hESCs grew well on this new substrate and maintained the typical hESCs morphology. Alkaline phosphatase staining was positive. Immunofluorescence staining showed that the expressions of Oct4, SSEA4, Tra-1-60 were positive. The cells formed embryoid body in vitro. Conclusions This methanol-fixed MEF substrate can support the growth of undifferentiated hESCs. The coating material can be produced in large scale and stored for a long time. It provides a new and relatively easy way to amplify hESCs.

Abstract:Objective To study the effects of bone marrow mesenchymal stem cells modified by brain-derived neurotrophic factor (BDNF) gene on the repair of spinal cord injury by electrophysiological assay. Methods Thirty healthy Sprague-Dawley rats (male and female) were randomly divided into 3 groups: Blank group, 10 rats (removal of the lamina only and exposed spinal dura mater);spinal cord injury (SCI) group,10 rats;and cell transplantation after SCI group, 10 rats. Eight rats of them were selected randomly and detected their SEP and MEP, and evaluated the degree of recovery of motor scores in the rats at 1 d, 7 d, 14 d, 21 d, 30 d, and 60 d. Result Since 4 days after cell transplantation, the process of hind limbs changes was as follows: at the 1-4 days after injury, the injury side hind limb had flaccid paralysis, mopping the floor walk, the movement of contralateral hind limb was gradually recovered from the initial injury, the injury side hind limb had spastic paralysis in 5-9 days after SCI; during 10-14 days, the injury side had a few activities; the contralateral side recovered to a less normal state; At 15-21 days, activities of the injury side improved obviously, until the 30th day. The activity and muscle tension degree of the injury side recovered most obviously. After 30 days no more obvious improvement was observed. Immunohistochemistry showed that the transplanted mesenchymal stem cells, which were induced and labeled firstly, survived at the damage spinal cord, and behavioral observation found that the cell transplantation improved exercise capacity of the rats injured before. Conclusion Bone marrow mesenchymal stem cells modified by BDNF gene can partially promote the recovery of nerve transmission function and nerve regeneration.

Abstract:Objective To explore whether the Chinese medicine Guben Yiliu Ⅲ can improve the effect of gemcitabine on human pancreatic cancer xenograft in nude mice. Methods Nude mice with transplanted human pancreatic cancer were divided randomly into 4 groups: control group, gemcitabine treatment group, combined (Guben Yiliu Ⅲ + gemcitabine) group, and Guben Yiliu Ⅲ group, 10 mice in each group. The gemcitabine group and combined group were treated with gemcitabine from the 8^th day after transplantation in a dose of 100 mg/kg by i.p. injection, twice a week. Guben Yiliu Ⅲ and combined groups were given the aqueous solution of Guben Yiliu Ⅲ granules p.o. since the 8^th day after transplantation. Result The inhibition rate of transplanted tumor in the three treatment groups were 48.9% in the gemcitabine group, 68.9% in the combined group, and 28.0% in the Guben Yiliu Ⅲ group. The combined group showed a significantly higher inhibition rate than the gemcitabine group (P<0.05). The gemcitabine group, combined group and Guben Yiliu Ⅲ group showed a significantly slower growth rate than the control group. However, the combined treatment group showed a pronounced side effect and body weight loss than the other 3 groups. Conclusions The Chinese medicine Guben Yiliu Ⅲ can improve the inhibitory effect of gemcitabine on nude mice with human pancreatic cancer xenograft in the auxilla of nude mice.