Abstract:Objective To establish uPA inducible expression system using recombinant retroviral system for the further construction of inducible uPA-SCID animal model. Methods The Inducible expression system need to construct two plasmids:pLNHXO1O2-Alb-GLUC-FMN2A -rtTA and pLNHXO5O6-TRE2-uPA-IRES-ZsGreen respectively. Both plasmids were based on retroviral vector pLNHX, Albumin promoter gene (Alb) and rtTA gene or uPA gene and ZsGreen were obtained by PCR reaction and inserted into pLNHX. The Gaussia enzyme fluorescent element (GLUC) was used to monitor rtTA expression in pLNHXO1O2-Alb-GLUC-FMN2A-rtTA,and the ZsGreen for uPA expression monitoring in pLNHXO5O6-TRE2-uPA-IRES-ZsGreen.The correct constructed plasmids were transfected into packaging cell line GP2-293 to gain recombinant viral particles.NIH/3T3 cells were infected with these viral particles and selected with G418.Gene expression in the surviving cells was confirmed by the PCR method. Results The recombinant retroviral vectors harbouring target genes were successfully cloned.The rtTA gene in pLNHXO1O2-Alb-GLUC-FMN2A-rtTA was expressed, and uPA can be induced to express in pLNHXO5O6-TRE2-uPA-IRES-ZsGreen by doxycycline (Dox) when the plasmid transfected into the HepG-Tet-on cell. The constructed recombinant two retroviral vectors were transfected into GP2-293 packaging cells respectively to gain infectious viral particles.Then,NIH/3T3 cells were infected with these viral particles and single-cell clones which stably expressed the transgenes were successfully established. Conclusion This study primarily established uPA inducible expression system, it laid a foundation for the murine model of inducible liver damage, and provided a novel technical platform for further building the liver humanised murine models for viral hepatitis studying.

Abstract:Objective To explore the role of HIF-1 and its downstream SDF-1α/CXCR4 and VEGF/VEGFR pathway in mediating MSC mobilization with DMOG. Methods Male SD rats were randomly divided into five groups: Normal saline control group, DMOG group, YC-1 group, AMD3100 group, SU5416 group. We used CFU-F assay and flow cytometry to determine the number of MSCs in rat bone marrow (BM) and peripheral blood (PB) in each group, respectively. The concentrations of SDF-1α and VEGF both in BM and PB serum in each group were detected by ELISA. Western blotting was used to test protein levels of HIF-1α, SDF-1α and VEGF in BM. Results Compared with NS group, the number of CFU-Fs as well as the percentage of CD45^-CD90⁺cells increased in DMOG group (P< 0.05); Compared with DMOG group, the number of CFU-Fs as well as the percentage of CD45^-CD90⁺cells decreased in YC-1 group, AMD3100 group and SU5416 group (P< 0.05). Compared with DMOG group, the concentration and protein expression of HIF-1α decreased significantly in YC-1 group (P< 0.05), the concentration and protein expression of SDF-1α decreased significantly in AMD3100 group (P< 0.05), the concentration and protein expression of VEGF decreased significantly in SU5416 group (P< 0.05). Conclusion DMOG can induce MSCs mobilization possibly via up-regulating the expression of HIF-1α and activating its downstream SDF-1α/CXCR4 and VEGF/VEGFR pathway.

Abstract:Objective To observation dynamic changes and gender difference of heart rate variability and blood pressure variability in unbonded spontaneously hypertensive rats using Telemetry technology. Method Taking sixteen spontaneously hypertensive rats of 3-month-old in SPF grade, there were implanted TL11M2-C50-PXT device intraperitoneally. Seven days later, respectively monitoring chainless blood pressure and electrocardiogram for 24 h continuously time using telemetry system when 3, 5, 7 months of age in spontaneously hypertensive rats. Analysing 24 h systolic pressure, mean blood pressure, diastolic blood pressure, heart rate variability and blood pressure variability. Result Systolic pressure, diastolic pressure and mean pressure, blood pressure variability increased with age of the moon increased in spontaneously hypertensive rats, moreover female SHR rats of blood pressure variability was significantly lower than that of the male rats. Correlated indexes of heart rate variability had no obvious change Between three to seven month-old in male spontaneously hypertensive rats. But RR interval, SDNN, TP, VLF and HF increased significantly with age of the moon increased in female spontaneously hypertensive rats, moreover the female SHR rats of TP, VLF and HF index of heart rate variability were lower than the male rats. Conclusion With the SHR rats age and blood pressure, blood pressure variability increased, and the female SHR rat variability in blood pressure and heart rate was significantly lower than that of the male.

Abstract:Objective To observe the different characteristics of spontaneous activitiy after simulated weightlessness 21 days in rats, aimed to provide a evaluation method for space weightlessness induced function change in human beings and to provide a reference for researches on the astronauts protective measures. Methods 30 Wistar male rats were randomly divided into three groups, the control group, the sham tail-suspended hindlimb unloading group (the sham group), the tail-suspended hindlimb unloading group (the suspending group), ten animals in each group. All animals were placed in the simulated space flight environmental equipment which has a real-time monitor system for 21 days. During the 21 days, the intake of water, food and the body weight were measured every week. Meanwhile, five independent activity data were collect every day, for example, morning(8:00am～ 12:00am), afternoon(2:00pm～6:00pm), daytime(8:00am～8:00pm),night(8:00pm～8:00am),and whole day (8:00am～8:00am). Results The spontaneous activity of normal rats in the control group between morning and afternoon had no significant difference, but it is significantly between night and daytime. The movement time and distance in night are 2-3 times than that of the daytime. After 10 days of tail suspending, the circadian rhythm was disordered, and the spontaneous activity in day and night become more similar in rats of the suspending group. Because of the individual difference, the spontaneous activity is not stable at the first 10 days in rats of the sham group, but after 10 days, it become close to the control group. Conclusion Rat is nocturnal animal and sleeps in the daytime, the spontaneous activity in night is 2-3 times as compared with the daytime. The sham tail-suspended hindlimb unloading 21 days can not influence the circadian rhythm in rats. Tail suspending 21 days will caused to the disappearance in the circadian rhythm in rats.

Abstract:Objective To investigate the change of mu opioid receptor (MOR) in dorsal root ganglion (DRG) in rat chronic inflammatory pain model and the effect of MOR agonist and antagonis tintraplantarly (i.pl.) injected on pain threshold, so as to determine the role of peripheral MOR in chron in inflammatory pain. Methods Chronicin flammatory pain model was established by i.pl. injection of CFA in rats. The expression of MOR in DRG was detected by immunohistochemistry. Pain threshold before and after i.pl. injection of MOR agonist and antagonist was measured by radiant heat method. Results Rats suffered from an intraplantar injection of CFA developed chronic inflammatory pain,and the painthreshold still reduced on 18 day after CFA injection compared to that in the normal group. Immunohistochemistry staining revealed that compared with the normal group, the expression of MOR in DRG of CFA rats was increased (P<0.01). After the paw dorsal surface injection of MOR agonist, the pain threshold of CFA rats was increased, while that of normal rats exhibited no significant change. After the paw dorsal surface injection of MOR antagonist, the pain threshold of CFA rats was reduced, while that of normal rats had no significant change. Conclusion Under chronic inflammatory pain condition, DRG MOR expressionis enhanced, which participates in the regulation of chronic inflammatory pain, and may contribute to the prevention of further more serious pain.

Abstract:Objective To compare the effects of forming atherosclerosis by conducting ballon injury operation after 1th, 2th and 3th week of Vitamin D₃(VD₃) i.p., exploring the best method for atherosclerosis modeling. Methods 36 male rats were selected for balloon-injured carotid artery. SD rats were divided into 4 groups randomly: control group (n=6), Model group1 (n=10), Model group2(n=10), Model group3 (n=10). Control group were fed up with common diet. Model groups were fed up with high-fat diet and injected 4.0×10⁵IU/kg VD₃ through enterocoelia in the beginning, followed by the balloon-injured left carotid artery operation after 1th, 2th and 3th week respectively and 1.0×10⁵IU/kg VD₃ injection at 0th, 2th week after operation. The rats were killed at 4th week after operation. The serum levels of TG, TC, HDL-C and LDL-C were checked. ELISA was used to detect the content of hsCRP, IL-6 and TNFα. HE staining was used to observe the pathological changes in the thoracic aorta, and the thoracic aorta thickness, plaque area(PA), cross-sectional area of vessel(CVA) and the ratio of PA to CVA(PA/CVA) were analyzed. Results After 4 weeks of operation, levels of TC and LDL-C were significantly increased in Model group2 and 3 compared with that of the control group (P<0.05). Furthermore, contents of hsCRP, IL-6 and TNFα of model groups were also seriously higher than that of the control group (P<0.05), and that of Model group 3 were the highest. Typical AS plagues were observed in different degrees in model groups, and thoracic aorta thickness and PA/CVA were obviously increased than that of control group (P<0.05). Model group 3 turned out masses of lipid foam cells accumulated, and PA, CVA and PA/CVA were significantly increased than that of Model group2 or 3. Conclusion The AS model can be established successfully in rats with ballon injury after 3 weeks of high-fat diet plus VD₃ i.p., which is the ideal method to induced atherosclerosis model.

Abstract:Objective The aim of this study was to explore the effect of testosterone deficiency on serum lipid levels and hepatic lipid accumulation in miniature pigs fed a high-fat diet (HFD). Methods Eighteen sexually mature male Chinese Wuzhishan miniature pigs (6~7 months old) were used in this study. The pigs were divided in three groups (n=6 animals/group) as follows: intact male pigs, castrated male pigs and castrated male pigs with testosterone replacement. They were fed a HFD diet for 12 weeks and body weights were recorded weekly. Serum levels of testosterone, total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and triglycerides (TG) were measured. Hepatic TG and TC levels were also determined, and liver tissues were embedded in paraffin and stained with hematoxylin and eosin (H&E). Results (1) The body weights of pigs in each group were found to be linearly elevated over time. Though castrated pigs gained less weight than did pigs in the other groups, no significant differences were found between them. (2) Castration caused a significant decrease in serum testosterone levels in pigs. This effect was recovered by testosterone treatment. (3) Serum levels of TC, LDL-C and TG were significantly increased in castrated pigs. However, castration had no significant effect on serum HDL-C levels. Testosterone treatment reduced the increased serum lipids in castrated pigs. (4) Hepatic TG and TC contents in castrated pigs were also significantly higher than those in other groups of pigs. Testosterone treatment reduced the increased hepatic lipids in castrated pigs. (5) Compared with other groups of pigs, castrated pigs showed increased steatosis. However, testosterone treatment attenuated hepatic steatosis in castrated pigs. Conclusion Testosterone deficiency caused severe dyslipidemia, and increased hepatic lipid accumulation in miniature pigs fed a high-fat diet.

Abstract:Objective To discuss the effect of in vitro fertilization (IVF) and mouse sperm cryopreservation, to establish a simple and economic frozen system for the genetically engineering mice preservation. Methods Sperm from genetically engineering mice were cryopreserved, IVF was performed using post-thawed sperm, then embryo transfer,to compare the effects of cryopreservation medium、age of male mice and sperm preincubation medium. Results Using CPA as sperm cryopreservation medium, when PM was used thawed-sperm preincubation in IVF, the fertility rates were from 82.49% to 91.43%, when HTF was used thawed-sperm preincubation in IVF, the fertility rates were from14.46% to 27.38%, there was a signification difference between PM and HTF sperm preincubation medium; 10 to 35 weeks male genetically engineering mice sperm were succeed cryopreservation, and positive mice were procreated after 2-cell embryos were transferred; R18S3、CPM and CPA was used to freeze sperm, the fertility rates were 75.85%、88.89% to 94.27%, positive mice were procreated after 2-cell embryos were transferred; 2-cell embryos after IVF were freezed, then thawed and positive mice were procreated after 2-cell embryos were transferred. Conclusion Using CPA as sperm cryopreservation medium, when PM was used thawed-sperm preincubation in IVF, genetically engineering mice sperm were succeed cryopreservation.

Abstract:Objective To screen genotypes of small body in Tibet minipigs by single nucleotide polymorphism analysis (SNP) of growth hormone (GH) gene. Methods The PCR products are sequenced. According to the results, SNP analysis are applied in GH gene 5' fragment of 108 Tibet minipigs. Results Five mutation points were found by comparison. The polymorphism information content of the T45C locus is highest. The results of growth traits of different genotypes showed that the abdomen of 6～8 month old Tibet minipigs of TC genotype at T45C mutation site is small; the weight and height of 3～5 month old Tibet minipigs of AA genotype at G84A mutation site is small; the body length and height of 6～8month old Tibet minipigs of GG genotype at G93A mutation site is small. Conclusion The AA TC, GG genotypes of T45C, G84A, G93A mutation sites in GH gene may be associated with small body. We also found that genetic mutations always occur in the above sites with high heterozygosity and genetic diversity, which can provide rich material for breeding research.

Abstract:Objective To establish a new congenic inbred mouse strain carrying and expressing EGFP and Foxn1^nugene for cancer research including human glioma as well. Methods According to criterion of GB14923-2010, the male Foxn1^nu nude mice backcross the female C57BL/6-Tg (CAG-EGFP) transgenic mice for 10 times, Then identify the phenotype using the methods and equipment as below: fluorescent flashlight and matching glasses; multifunction vivo imager; fluorescence microscopy. Results The congenic inbred mouse strain named Foxn1^nu. B6-CAG-EGFP/SU (Soochow University). All the 14 biochemical loci are homozygous and same with Balb/c mouse in addition to the Pep3 loci ("b" type instead of "a" type). Peripheral blood lymphocyte count shows the lymphocytes occupy 15% of nucleated cells; T lymphocytes occupy 0.3%, meet the requirement of inbred strain of EGFP nude mice. Conclusions Established a new congenic inbred strain -Foxn1^nu.B6-CAG-EGFP/SU which both express EGFP stably, and own immunodeficiency with lack of T lymphocytes. The phenotype "b" of biochemical loci "Pep3" is the unique characteristic that distinguish SU to Foxn1^nu.

Abstract:Objective To establish experimental vascular dementia rat model and evaluate gait behavior. Methods Vascular dementia rat model induced by bilateral carotid artery ligation methods, 50 days for real-time gait behavioral training and testing after surgery. Results Compared with the sham group, Experimental vascular dementia model rats had 19 gait indicators appeared significantly statistical difference, Animal model gait abnormal behavior is mainly reflected in the forelimb step width increased (P < 0.05), each foot walk cycle extension (P < 0.05), Each foot stance time increased (P < 0.05, P < 0.01), and the swing time shortened (P < 0.05), Homologous coupling shortened (P < 0.05), each foot average footprint area and average intensity increased (P < 0.05, P < 0.01). Conclusion Experimental rat model of vascular dementia in real time gait abnormal behavior and seen in patients with clinical symptoms similar, can provide a reference model for the establishment and judgment.

Abstract:Objective To establish multiplex PCR assay for detection of chicken embryo lethal orphan virus(CELO)and egg drop syndrome virus (EDS). Methods According to GenBank gene sequence, two pairs of specific primers designed were amplified CELO long fiber protein and EDS hexon protein gene sequence. The specificity and sensitivity of multiplex PCR were tested. We also use the multiplex PCR to detect exogenous CELO and EDS in influenza virus. Results Two target bands have been successfully amplified and verified by sequencing. The specificity of the method is better, and the sensitivity is 10^-4μg/mL. The results of detecting exogenous CELO and EDS in 12 influenza virus were negative. Conclusion The multiplex PCR assay for detection of CELO and EDS was established successfully, which have good specificity and high sensitivity, and have high value and application prospect for detecting exogenous CELO and EDS in influenza virus.

Abstract:Objective To observe the effectiveness of introducing Diazepam into combined aesthesia of Sumianxin and ketamine hydrochloride. Method A total of 80 rabbits of both genders for operation were randomly divided into A, B and C groups. The A group was injected with Sumianxin intramuscularly (0.3 mL/kg by weight). The B group was injected with Sumianxin and ketamine hydrochloride intramuscularly (0.3 mL/kg by weight). The C group was injected with Diazepam intravenously (1.5 mL/kg by weight) combined with Sumianxin and ketamine hydrochloride injected intramuscularly (0.3 mL/kg by weight). The aesthetic effects, induction time, anesthesia maintaining time, total anaesthetic dose and operation time were observed, recorded and compared. Result The induction time of the C group was significantly shorter than A and B groups (P<0.01). The initial anesthesia maintaining time of the C group was the longest among the three (P<0.01) with least total anaesthetic dose (P<0.01). The operation time of the C group was the least with best aesthetic effects (P<0.01). Conclusion Introducing Diazepam into combined aesthesia of Sumianxin and ketamine hydrochloride can improve the aesthetic effects. Therefore, this is an optional aesthetic method for time-consuming animal operation or sensitive surgical sites of rabbits.

Abstract:Objective Giardia lamblia is an important pathogen of zoonosis giardiasis, it poses a potential threat to the quality of SPF (specific pathogen-free) laboratory animals cannot be ignored. The aim of this study is to establish the method of rapid diagnosis of Giardia lamblia, and analyze the test results of 516 batches form 17 manufactures. Methods Direct microscopy, Giemsa-fast staining and multiplex polymerase chain reaction (multiplex PCR) were applied to detect Giardia lamblia. Results Numerous of Giardia lamblia trophozoites and cysts were detected in SPF laboratory animals by using direct microscopy and Giemsa-fast staining, and multiplex PCR were performed to identify 18S rDNA, β-giardin, TPI and GDH genes of DNA extracted from these trophozoites and cysts identified Giardia lamblia. Direct microscopy, Giemsa-fast staining, and multiplex PCR methods can be used to detect Giardia lamblia. Of the 2562 SPF laboratory animals studied, 22.9% (586/2562) were positive for Giardia lamblia. Conclusions Direct microscopy, Giemsa-fast staining, and multiplex PCR were effective techniques with high sensitivity and specificity for rapid diagnosis of Giardia lambliain. It is not satisfactory that the results of Giardia lamblia examination in 516 batches form 17 manufactures failed to meet the requirements 100%.

Abstract:Objective To improve the fixation method of indwelling needle inserted in the tail vein in rats to keep the inserted needle for a longer indwelling time. Methods One hundred healthy Wistar rats (age 5～6 months, male:female=1:1) were randomly divided into two groups: the experimental group and the control group. Rats in both groups received the same tail vein indwelling needle puncture and cannulation. The control group got the traditional fixation, namely fixed by sticking with 3M transparent dressing paste. The experimental group received in addition to the traditional fixation, a 0.1 mm-thick aluminum tube placed outside the needle fixation site. The indwelling time in the rats were recorded and analyzed. Results The indwelling time was (166.86±9.03) h and (20.24±5.04) h in the experimental and control groups, respectively (t=68.546, P<0.01). Conclusions Our improved method is safe and reliable. It can prolong the indwelling time of the punctured intravenous indwelling needle, and provides a useful rat model in studies on the complications of intravenous indwelling needle kept for a longer time.

Abstract:Breeding and quality control of SPF nude mice involves many links and works. The quality and quantity of animals are directly affected by breeding management and quality control. A standardized breeding system of SPF BALB/c nude mice were gradually established at the center after 2007. This paper introduces specific practices and experience in facilities, stock, breeding management, and quality control of SPF BALB/c nude mice, in order to provide the reference for related breeding and study in SPF BALB/c nude mice.