Laboratory Animal Science

The Expression of NF-κB in Liver of ICＲ Mice at Different Welfare Level

2016, 33(04): 1-5.

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Abstract: Objective To compare the expression of NF-κB in liver of ICＲ mice at different welfare level． Method ICＲ mouse ，6 years old ，welfare were infected by both restraint stress and environmental enrichment ，or only one of them． Body weight gain and food consumption were recorded throughout the study ，IL-2 and COＲT in blood were detected by ELISA ，mＲNA and protein expression of NF-κB p65 in liver were detected after the study finished by real-time quantitative PCＲ and Western blotting．Ｒesult Compared with group C( control) ，the body weight gain of group E( environmental enrichment) was higher ( P ＜ 0. 05) ． The body weight gain of group EＲ ( environmental enrichment and restraint) was decreased ( P ＜ 0. 05) ． COＲT ，mＲNA and protein expression of NF-κB p65 were higher ( P ＜ 0. 05) ． The body weight gain ，FEＲ and IL-2 of group Ｒ( restraint) were decreased ( P ＜ 0. 05) ． COＲT ，mＲNA and protein expression of NF-κB p65 were higher ( P ＜ 0. 05) ． Compared with group EＲ，the body weight gain of group Ｒ ( restraint) were decreased ( P ＜ 0. 05) ． COＲT ，mＲNA and protein expression of NF-κB p65 were higher ( P ＜ 0. 05) ． Conclusion Difference of welfare did exist ，and the difference of expression of NF-κB p65 were similar to it． There were relations between those two ones．

The Effect and Mechanism of Salidroside in Pressure-overload Chronicity Heart Failure Ｒats

2016, 33(04): 6-10.

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Abstract: Objective To explore the effect and mechanism of Salidroside in pressure-overload chronic heart failure rats． Method Totally 30 SD rats were randomly divided into sham operation group，heart failure group and Salidroside group，10 rats in each group． The HF group and SAL group were prepared by abdominal aorta constriction method，and S group only was worn line not constricted． SAL group was given subcutaneous injection from 12 weeks after operation by 60mg / kg·d in 30 days． The S group and HF group received the same dose of normal saline． After 30 days，two lead ECG parameters of limb a was measured and statistically analyzed． After the end of intervention，the rats were anesthetized，and abdominal aortic blood taken from the upper serum after centrifugation． Kit method was used to measure superoxide dismutase ( SOD) ，brain natriuretic peptide ( BNP) ，malondialdehyde ( MDA) ．Ｒesult Twelve weeks after operation，there was a decrease in heart rate，P amplitude and Ｒ amplitude comparing by those of the heart failure group ( P ＜ 0. 05 ) ． ECG showed a significant and ubiquitous J point depression ( P ＜ 0. 05) ，with ST segment notable elevation ( P ＜ 0. 05) ． SOD activity was significantly increased and BNP concentration was significantly decreased in SAL group compared to HF group ( P ＜ 0. 05) ． Conclusion These results show that Salidroside protects pressure-overload rat heart from failure possibly through reducing oxidation．

Isolation and Identification of Two Murine Norovirus Strains

2016, 33(04): 11-17.

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Abstract: Objective The purpose of the study is to investigate the prevalence of murine norovirus ( MNV) in Jiangsu province and analyze their genetic molecular characterization． Method Experimental mice were extracted from three experimental animal centers in Jiangsu area．Ｒectal samples from the experimental mice were tested for partial OＲF2 region ( 547bp，MNV nt 5104-5650) of MNV using ＲT-PCＲ method． We used mouse macrophage cell line ＲAW264. 7 to isolate MNV from the rectal contents of the infected mice． Fragments of VP1genes from MNV isolates were amplified by ＲT-PCＲ and then ligated to pEASY-Blunt Simple CloningVector and cloned to Trans1-T1 chemically competent cells． By kanamycin lysogeny broth plate selection， the positive clones were sequenced and analyzed． Based on the VP1 gene， thephylogenetic analysis tree was created．Ｒesult 4 positive MNV isolates were obtained from 30 mice rectal samples，including ICＲ mice( 2 /8) ，BALB /c mice( 1 /12) and C57BL /6Jmice ( 1 /10) ，infection rate was 13%． On infection，the ＲAW264. 7 cell line inoculated with two ICＲ mice sample showed obvious cytopathic effects ( CPE) after three genetations，as infected cells became rounded and shrunken，with ultimate disintegration of the cell sheet． The ＲAW264. 7 cell line inoculated with BALB /c and C57BL /6J mice samples did not exhibit CPE． Used VP1specific primers，we found the specific bands appeared in the CPE cells sample of two ICＲ mice by ＲT-PCＲ method． The sequencing results showed both of the complete VP1 gene of two MNV strains was 1626bp，which was the same length as that of the reference strain S7-P2 ( GenBank accession number: AB435514) VP1 gene． Phylogenetic analysis showed that the two MNVs of this study were clustered into one group which was mostly related withBJ10-2062 and CＲ4． Conclusion The MNV infection rate was 13. 3% ( 4 /30) in the experimental mice of Jiangsu area． May be MNV transmitted within the breeding facilities． We need to further explore the biological significance of the experimental mice carrying MNV．

Establishment and Identification of a ＲAG2 /IL2ＲG Hybrid Mouse Strain

2016, 33(04): 18-26.

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Abstract: Objective To establish aＲAG2 /IL2ＲG double deficient mice model and the preliminary application．Method ＲAG2 deficient mice were crossed with IL2ＲG deficient mice． The genotype of hybrid mice was determined by PCＲ． The percentages of CD3 + ，CD19 + andCD49b + were detected y FCM in terms of its T，B lymphocyte function and NK cell percentage． The physiological and biochemical parameters of blood and major organs weight were observed．Ｒesult ＲAG2 and IL2ＲG deficient mouse were bred respectively，and ＲAG2 / IL2ＲG hybrid mice model was successfully established． Established stable PCＲ identification methods of ＲAG2 / IL2ＲG double genedeficient mice by PCＲ methods． The ＲAG2 /IL2ＲG hybrid mice lacks T cells，B cells，the percentage of NK cells was very low，and the difference was significant ( P ＜ 0. 05) ． Blood biochemical tests showed that compared with the same week-old C57BL /6， serum biochemical parameters of ＲAG2 /IL2ＲG double deficient mice was not significant changes，the difference was not significant ( P ＜ 0. 05 ) ． In the 18 blood physiological indices of ＲAG2 /IL2ＲG double deficient mice，Ｒed blood cell ( ＲBC) and hemoglobin ( HGB) content is low，and the difference was significant ( P ＜ 0. 05) ，White blood cell ( WBC) ，lymphocytes ( LYMPH) ， lymphocyte ratio ( LYM) ，monocytes，neutrophils count ( NEUT) index were significantly lower，and the difference was significant( P ＜ 0. 01) ． The main organ weight displayed the weights of spleen，thymus and mesenteric lymph node are lower than wild-type C57BL /6 mice，the difference was significant ( P ＜ 0. 05) ． The experiments of humanhepatoma cells ( tissue ) transplants show，ＲAG2 /IL2ＲG double gene-deficient mice tumor transplant success rate and growth rate were significantly higher，compared with the parental single gene-deficient mice and the difference was significant( P ＜ 0. 01) ． Conclusion The mice model of ＲAG2 /IL2ＲG double deficient and a stable system of identification are successfully established． The double deficient mouse provide a good animal model for patient-derived tumor xenograft model．

EAD Saturation Animal Model for Estimating Decompressing Stress of Simulated Complex Submarine Escape Procedure

2016, 33(04): 27-31.

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Abstract: Objective We observed the different responses of animal exposure to simulated submarine escape ( sSE) and direct decompressing after equal air depth ( EAD) saturation to estimate the effects of EAD animal model to decompression load in complex sSE． Method 12 goats were divided into four groups，separately exposed to 150 m sSE after 7m saturation( group 1，EAD = 12． 4 m) ，directly decompressing after 12． 4 m saturation ( group 2) ，150 m sSE after 12m saturation ( group 3，EAD = 17． 1 m) ，and directly decompressing after 17. 1 m saturation( group 4 ) ． The animal behavior，vascular bubble and mortality were observed after exposure． The peripheral blood were collected to blood routine examination，blood coagulation，NO and NOS．Ｒesult there were no significant differences in vascular bubble Spencer classification，blood routine examination，blood coagulation between groups of separate exposure method with same EDA． Large EDA groups had a higher mortality rate ( 50%) ． Group 4 had a higher PT than group 1( P ＜ 0． 01) ，higher APTT and PT than group 2 ( P ＜ 0． 01) ． There was significant difference in NO between different EAD groups ( P ＜ 0． 01) ，but no NOS． Conclusion It was suggested that EAD animal model was available to estimate decompressing stress in complex sSE． Hemorhology and NO may be effective biomarkers in decompressing stress．

The Quality of Chronic Hepatitis B Animal Models Built by Hydrodynamic Transfection

2016, 33(04): 32-36.

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Abstract: Objective The aim of this study was to compare the different features of hepatitis b mice model which were produced by hydrodynamic transfection in different plasmids． Method Inject two plasmids( pcDNA 3. 1 － 1. 3 － C and HBV PAAV － 1. 2 － HBV) to establish two kinds of hepatitis b mice model by hydrodynamic transfection method，and compare the difference and special effects of the models by inspecting the modeling the success rate ，mouse peripheral blood HBsAg and stability，flow detection such as T cells index analysis．Ｒesult The model success rate of pcDNA3. 1 － 1. 3 － HBV -C plasmid was 78. 6%，and after transfection the content of HBsAg could rise to 220 IU/mL in maximum; while the model success rate of Plasmid PAAV － 1. 2 － HBV -A was 56%，and the highest content of HBsAg after transfection was 2300 IU/mL． The immunohistochemical mice liver biopsy was implied that the mice after the transfection of pcDNA 3. 1 － 1. 3 － C plasmid were darker and had a larger area than the mice after the transfection of PAAV － 1. 2 － HBV -A plasmid． Six weeks after transfection plasmid， the peripheral blood of experimental mice was taken to flow cytometry test，and the results showed that comared to normal mice ， the CD4 + and the CD8 + made no difference between the two kinds of mice by the transfection of two plasmids． Conclusion Building hepatitis b mice model by using the method of hydrodynamic transfection could make a significant difference in hepatitis b virus expression level and stability．

The Change of Nerve Growth Factor Genes Expression at Different Time of Kainic Acid-induced Epilepsy in Ｒats

2016, 33(04): 37-40.

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Abstract: Objective To investigate the difference of expression of nerve growth factor( NGF) at different time by rats that induced epilepsy by kainic acid ( KA) ． Method The epilepsy model was used kainic acid intraperitoneal injectioned in rat． The nerve growth factor expression were detected by real-time fluorescent quantitative PCＲ assay with TaqMan probe in different time after intraperitoneal KA．Ｒesult Compared with normal sodium( NS) group， the expression of NGF were lower at 6—12 h ，and were higher at 48 h and 72 h( P ＜ 0. 05) ，but the expression of NGF were risied up at 48—72 h and higher by NS． Conclusion NGF have reparation and protection the hippocampus of rats that induced epilepsy by KA．

Digestion Efficacy of two Types of Trypsin on Sheep Muscles Protein via Comparative-proteomic Analysis

2016, 33(04): 41-47.

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Abstract: Objection To examine the Digestion efficacy of two types of trypsin，one is purchased domestically and the other is from foreign company，on sheep muscles protein via comparative － proteomic analysis． Method Total protein sample of sheep muscles were extracting the by sonication methods． Then，protein samples were digested into peptides by two kinds of trypsin and examined by Liquid chromatography tandem mass spectrometry． Protein sequence databases and bioinformatics tools were used to analyze mass spectrometry data．Ｒesult More than 86% of peptides from sheep muscles digested by with two kinds of trypsin were 6—18 amino acids length． More than 19% proteins or 34% peptides can be identified from sheep muscles digested by trypsin purchased domestically than that by trypsin from foreign company． Conclusion The efficacy of trypsin purchased domestically is much better than trypsin from foreign company． And the price of the domestic is lower than the imports．

The Effect of Survival on Traumatic Hemorrhagic Shock Pigs in Different Temperature of Desert Dry-heat Environment

2016, 33(04): 48-51.

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Abstract: Objective Trauma hemorrhagic shock swines were explored the effect of survival in different temperature of desert dry-heat environment， to provide a theoretical basis for the further research． Method Local male Landrace piglets( 25 － 35kg) were randomly divided into three group: normal temperature group( temperature: 25℃ ± 1℃，humidity 35% ± 5%，n = 10) ; dry-heat groupⅠ( temperature 40. 5℃ ± 0. 5℃，humidity 10% ± 2%，n = 10) ; dry-heat groupⅡ ( temperature 41. 5℃ + 0. 5℃，humidity 10% ± 2%，n = 10) ． They were placed in a cabin ( The Simulated Climate Cabin for Special Environment of Northwest of China，Urumqi of China) ．Parameters of environmental are set according to experimental design，exposuring three hours in their environment，respectively． They were established to model the traumatic hemorrhagic shock，and to observe the changes of vital signs and the time of death．Ｒesult 1． In the normal temperature group，the body temperature was stable at a certain level after traumatic hemorrhagic shock，and the trend was decreased after the stabilization period; the dryheat groupⅠafter the shock rise slowly，and there was no significant difference between the two adjacent time point temperature; dry-heat group II after the shock temperature quickly rose to 42℃，has shown a rapid upward trend until death，and the adjacent time point temperature difference is significant． 2． The average survival time of the three group after shock were ( 567 ± 16. 9) min，( 178 ± 3. 6) min，( 61 ± 2. 8) min，respectively． The survival time of the three groups was significantly different． Conclusion 1． Dry-heat environment traumatic hemorrhagic shock survival time was significantly shorter than room temperature environment，suggesting that dry-heat environment can be accelerated the death of animal in traumatic hemorrhagic shock． 2． The dry-heat group II survival time shorter， suggesting that the environment increases every 1 degrees in this environment，can significantly shorten the traumatic hemorrhagic shock pigs survival time． 3． Temperature rise rate and death rate were positively correlated，the temperature increases quickly，the shorter survival time，suggesting treatment of trauma hemorrhagic shock that effectively control the body temperature may has important clinical significance in dry heat desert environment.

The Technical Study for Establishment，Conservation and Breeding of Genetically Engineered Mice

2016, 33(04): 52-57.

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Abstract: From mice superovulation， embryo collection， zygotes microinjection， embryo transplantation， genotyping，etc．，this text elaborated technical operating essentials for preparing genetically engineered mice． Besides，it also elaborated the technologies about conservation and breeding of genetically engineered mice，including assisted reproductive technology of IVF ( in vitro fertilization) ，frozen and recovery technology of sperm and embryos． It involved the main points of the relevant technical improvement and operation experience． The laboratory successfully built the platform of preparation and conservation of genetically engineered mice and provided a better technical service．

The pathological alteration of rats’brain under acute hypoxia exposure and the expression change of IL-6，TNF-α

2016, 33(04): 58-62.

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Abstract: Objective To discuss under the simulated plateau environment，the rats’brain exposed to the hypobaric hypoxia，the pathological alternation of the rats’brain exposed to the hypobaric hypoxia，and the expression change of IL-6，TNF-a． Method Half male and half female，50 SD rats，and the experiment is divided into five groups． They are atmospheric pressure in the control group，hypobaric hypoxia group，exposure 1 d group，2 d group，3 d group and 7 d group． Test the rats’brain dry to wet weight ratio． The pathological alternation and the content of the TNF-a and IL-6．Ｒesult Compare the normal contrast group． In the contrast， exposure 1 d group，2 d group，3 d group dry to wet weight ratio，the brain tissue of TNF-a and IL-6 go up ( p ＜ 0． 05) ． Exposure 7d group∶ water content of brain tissue and the TNF-a and IL-6 brain tissue both markedly under the exposure 3 d group ( p ＜ 0． 05) ． Pathological analysis showed that normal contrast brain tissue’s cellular forms and structure and inter-cellular space all are normal． Exposure 3 d group brain tissue molecular layer centrum cells，blood vessel choroid plexus，meninx edema，exposure 7 d group brain tissue molecular layer drop compared with 3 d group． Conclusion Under the environment of simulation plateau，SD rats’pathological alternation and dry to wet weight ratio． TNF-a and IL-6 factor content grow significantly at 3d group． But with the extension of exposure time，the injury degree decrease comparatively． This is same with plain people come into plateau， producing hypoxia mechanism． Therefore，research institution chose SD rats brain tissue’s morphological changes and test the content alternation of TNF-a ， IL-6 as the criterion of the hypobaric plateau model whether it will become model or not．

The effect of Taohechengqi Decoction on intestinal flora in the hepatic encephalopathy in rats model

2016, 33(04): 63-66.

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Abstract: Objective To study the influences of Taohe chengqi decoction on intestinal flora in the rats model with hepatic encephalopathy ( HE ) induced by TAA． Method In this experiment，40 SD rats were injected intraperitoneally with TAA and was gavaged with the traditional Chinese medicine decoction，then the effects of Taohe chengqidecoction were observed on neurology test，blood ammonia and intestinal flora．Ｒesult Every dose of Taohe chengqi decoction significantly decreased the staging of HE and blood ammonia． Compared with the model group， the bacterial count of the jejunum and ileum of the traditional Chinese medicine showed a decreasing trend ( P ＜ 0. 05) ，and Taohe chengqi decoction has a callback effect on the bacterial of the jejunum and ileum． At the same time， there still has a callback effect on Bacillus bifidus and Escherichia coli in the colon in each dose group of traditional Chinese medicine， then Both B /E value increased． Conclusion The decoction has a positive effect on the intestinal flora of rats with hepatic encephalopathy， which can increase the colonization resistance of gastrointestinal tract．

Experimental Study on the Influences of Lipid Peroxidation and Antioxidant Capacity of Organism on Traumatic

2016, 33(04): 67-69.

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Abstract: Objective To study the influences of lipid peroxidation and antioxidant capacity of organism for traumatic． Method Choose 11 new zealand rabbits on ordinary level were collected blood samples，bilateral ear edge venules repeated puncture after collecting，every other day，2—3 times in a row，form animal model of trauma，blood samples were taken after the completion of the animal model，to detection serum malondialdehyde ( MDA) ，superoxide dismutase( SOD) ，hydrogen peroxide enzyme( CAT) ，glutathione oxidation reductase( GSHPx) ，total antioxidant capacity ( T-AOC) of the two groups before and after the build animal model，and be statistical analysis．Ｒesult MDA: A group ( 3. 048 ± 1. 259) ，B group( 3. 251 ± 1. 695) ，Group A less than in group B; T-SOD: A group ( 384. 314 ± 50. 122) ，B group ( 348. 942 ± 45. 852) ，A group higher than group B; CAT: A group ( 6. 121 ± 4. 097) ，B group ( 4. 563 ± 2. 223) ，A group higher than group B; GSH-Px: A group ( 377. 808 ± 72. 726) ，B group ( 349. 942 ± 54. 021) ，A group higher than group B; T-AOC: A group ( 5. 537 ± 2. 128) ，B group ( 4. 633 ± 2. 074) ，A group higher than group B; there were no statistically significant difference between the groups( P ＞ 0. 05) ． Conclusion There was certain influence to the lipid peroxidation and antioxidant capacity of organism for trauma ，and there was no significant influence．

Construction and Practice of Training Base for Laboratory Animal Practitioners in Fujian Province

2016, 33(04): 70-73.

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Abstract: The launch of the animal experiment professionals’training is the starting point and foothold to improve the“regularization of laboratory animals”and the“standardization of animal experiments”． Aiming to ( ameliorate) the shortcoming of the bad conditions and limited coverage in previous trainings，this project of“the training base for experiment animal professionals in Fujian province”has been set up and reported basing on the successful experience from other provinces and referring to the ideal of driving license examinations and professional title appraisals． The training and testing system of the“working certificate”has been built． Different teaching materials and outlines have been compiled for different trainees． The training courses for experiment animal professionals have been organized four times and 1021 trainees were awarded“working certificates”． This project investigated and analyzed the experience and deficiencies in the construction and practice of training base and also laid the foundation for the future trainings in Fujian．

Study on Sterilization Effect of High-energy Xenon Light Transfer Window

2016, 33(04): 74-77.

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Abstract: Objective To study the disinfection effect of high-energy xenon light transfer window in the air and object surface bacteria． Method The bacterial suspensions were prepared from the standard strains of Staphylococcus aureus，Bacillus subtilis and Escherichia coli and to simulate the surface bacteria on coated blood plate． The air drop bacteria were cultured on common nutrient agar plate． The experimental group was irradiated separately by xenon light transmission windows and ultraviolet irradiation in 3 min，and then was placed in a constant temperature of 37 ℃ with control group for incubating for 24—48 h，then calculated the killing rate．Ｒesult The average killing rate reached to 93. 2% after irradiation by the xenon light on the bacteria suspension coating liquid blood plate; the average killing rate reached to 99. 1% in air bacteria after irradiation by the xenon light irradiation in 3min． The killing rate of two kinds of plate after 3 min ultraviolet irradiation was less than 50%． Conclusion Xenon light transfer window could efficiently and quickly kill the bacteria on the object surface and in the air，average killing rate could reach to 95%，xenon light transmission window would be the replacement of the routine transfer window．

The Application of Patient-derived Tumorxeno Graft Mouse Models Inprecision Medicine Inoncology

2016, 33(04): 78-83.

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Abstract: Patient-derived tumor xenograft ( PDX) mouse models have been used for nearly half a century，and have been one of the important tools in the area of oncology study． In contrast to the traditional tumor-bearing mouse models，PDX mouse models were demonstrated to keep the characteristics of genome，transcriptome，proteome and aberrant signal pathways of real-world patient tumors to the largest extent in vivo，rendering them ideal Avatar substitutes of tumor patients to be extensively used for personal drug testing．Ｒecently，with the development of the concept of tumor precision medicine，the requirement and utility of PDX mouse models are gradually increased，especially for screening and evaluating the effective anti-tumor drugs on the basis of PDX mouse models．Furthermore，it is on the way from bench to bed in the clinic to direct the personal therapy for tumor patients according to the drug screening results from PDX mouse models． Herein，this paper will summarize the critical factors in successful generation，the clinical applications and potential limitations of PDX mouse models．

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