Table of Content

31 December 2015, Volume 32 Issue 06

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Establishment of in situ Perfusion Method for Isolation and Culture of Mice Liver Tumor Cells

2015, 32(06):  1-7. 

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Abstract: To estabolish an effective primary culture method for mouse liver tumor cells and provide an effective experimental method for the researches on mechanismes of hepatic tumorigenesis． 9 month-old H-ras12V transgenic liver cancer mouse model was used． Liver tumor cells was isolated based on the improved traditional liver perfusion method，briefly，cut off the blocking arteries and tissues on the surface of the liver tumor to dredge the perfusion liquid for achieving the effective digestion of tumor tissues． The cell viability of per-tumor tissues and tumor tissues were counted，purified and cultured． The morphological characteristics of cells were observed every day until 14 days． Compared to the traditional liver perfusion method，the improved method elvated the viability of cells from 10% to 40%． The primary culture results showed that there was no significant difference between cells isolated from peri-tumor tissues and tumor tissues on the  morphological  characteristics and apoptosis ratio with elongated culture time． The improved traditional liver perfusion method is an effective method to isolate the hepatic tumor cells and significantly improved the viability and quality of the cells which is enough for subsequent experiments．Furthermore，the detailed protocol descripted in this article provides a practicable and feasible operating instruction for related researchers．

Positive Modeling Study on Fertility and Early Embryonic Developmental Toxicity of Cyclophosphamide to SD Rats

2015, 32(06):  8-12. 

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Abstract: Objective In fertility and early embryonic developmental toxicity test for SD rat，different doses of cyclophosphamide were given by subcutaneous injection，the right dose level was chosen to set up positive model，and background data for laboratory was accumulated． Method One hundred and seventy-six SD rats in 7 － 8 weeks were taken，which males and females were in half．Randomly divided into vehicle control group ( Veh) ，low cyclophosphamide dose group ( L: 10 mg·kg － 1 ) ，moderate cyclophosphamide dose group ( M: 20 mg·kg － 1 ) and high cyclophosphamide dose group ( H: 40 mg·kg － 1 ) ． There were 44 rats in each group and males and females were in half． Males were dosed once a week from the 28th day before mating until to be killed． Females were dosed once a week from the 14th day before mating，and were dosed at the 0 day after mating． Totally treated three times for females． Males and unsuccessful mating females were killed after mating，and the other females were killed at the 14th day after mating． Collected the male fertility，female sexual cycle，pregnancy rate，mortality for pregnant rats and stillbirth rate in each group． Result Toxicity on fertility and early embryonic development in SD rats were appeared in all the dose groups of Cyclophosphamide，and the toxicity increased along with increasing dose level． Conclusion Subcutaneous injection to SD rats with different doses of cyclophosphamide，the high dose group had obvious effect to fertility and early embryonic developmental toxicity，so the high dose group was chosen as positive model in this study．

Comparison of Several Separation Media Effect for Detection of Salmonella and Application in Laboratory Animals and Bedding

2015, 32(06):  13-17. 

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Abstract: Objective To improve isolate culture method of Salmonella in laboratory animal，and develop detection method of Salmonella for animal bedding． Method According to variety of domestic and international Salmonella detection methods，10 strains of Salmonella，Escherichia coli and Proteus were cultured by these methods． The best media were selected to detect animal intestines and bedding samples． Result BPW，MKTTn and selective enrichment medium XLT4 were better than other media for isolating Salmonella，its detection limits was 1CFU/g for Salmonella in bedding． In addition，three samples were positive in the 1015 animal samples by this method．Conclusion The improved method of isolation and culture of Salmonella detection was superior to the GB method，and accurate． Therefore，it can be used for detecting salmonella in laboratory animal and bedding．

The Comparative Study on Establishing Blood Lipid Level of Mice Model of Hyperlipidemia with P407 and Fat Emulsions

2015, 32(06):  18-21. 

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Abstract: Objective To compare the blood lipid level of mice with hyperlipidemia induced with P407 and fat emulsions． Method The mice were treated with fat emulsions( IE) ，the others were treated with P407，Finally，the contents of serum total cholesterol ( TC) ，triglyceride ( TG) ，high-density lipoprotein-cholesterol ( HDL-C) ，low-density lipoprotein-cholestrol( LDL-C) ． Result The contents of serum TC、LDL-C were fold-increased，TG was fold-decreasd in mice with fat emulsions，the contents of serum TC、LDL-C、TG were fold-increased，HDL-C was fold-decreasd． Conclusion It is a quite rapid and reliable way to establish hyperlipidemia model with P407．

Study of Tanshinone Ⅱ A Sulfonic Acid Sodium Injection on Rabbit Muscle Stimulation

2015, 32(06):  22-25. 

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Abstract: Observing the rabbits after intramuscular injecting the musculi quadriceps femoris of Tanshinone II A sodium sulfonate ( 60mg) for single dose，whether the local administration muscle tissue appear expansion、swollen、congestion、edema、petechiae、denaturatio、necrosis and other local muscle irritant reactions， synthetic judgment the safety of the test article by intramuscular injection． Use the same amount of isotonic sodium chloride solution as negative control to self-control， set the end group and recovery group to observe the symptom by naked eye and inspect the histopathology． after intramuscular administration the rabbit general symptom observation showed no abnormal findings． Autopsy the observation of naked eye meet the standard，autopsy the observation under microscope showed in the observation time the local muscle stimulation reaction was completely reversible．Tanshinone II A sodium sulfonate injection had no significant stimulation on rabbit muscuil quadriceps femoris，providing a reference for the safety evaluation of drugs．

Study On Modified Transcutaneous Electrical Resistance Test Method

2015, 32(06):  26-28. 

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Abstract: Objective To assess modified in vitro transcutaneous electrical resistantce test method． Method 28 ～30 day-old rats are humanely killed，the dorso-lateral skin of each animal is then removed． Skin discs with a diameter of approximately 20 mm each，are removed． If the resistance value is greater than 20 kΩ，the discs could be used for the test method． Test chemicals ( 150 μL) are applied uniformly to the epidermal surface． Three skin discs are used for each test． Test chemicals are applied for 24 hours at room temperature，and then are removed by washing． The skin impedance is measured as TER． If TER values produced by such chemicals are less than or around 15 kΩ，an assessment of dye penetration should be carried out． The optical density ( OD) of the incubation solution is measured at 565nm． The sulforhodamine B dye content per disc is calculated from the OD values． Result The sensitivity，specificity and accuracy of the modified TER test method are 100%，83% and 88% respectively． The TER test method is considered valid to discriminate corrosive from non-corrosive chemicals．Conclusion The modified TER test method is valid in our Lab．

Pathological Diagnosis and Analysis of Feline Ear Tumors

2015, 32(06):  29-33. 

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Abstract: Objective 8 cases of feline ear tumors were pathologically diagnosed and analyzed to explore the occurrence regularity． Method The feline ear tumors were obtained from China Agricultural University Animal Hospital from 2009 to 2015 by surgical treatment，which were analyzed according to pathological diagnose the occurrence regularity of the location，tissue origin and age． Result The occurrence rate of Squamous cell carcinoma and Ceruminous gland adenoma /adenocarcinoma and mast cell tumor in feline ear were each accounted for 25% and 25% and 37. 5%． Ear tumors of cats occurred in 0 － 4. 9 years old accounted for 13. 5%，5 － 9. 9 years old accounted for 37. 5%，more than 10 years old accounted for 50%． The tumors all occurred in the external ear，but not in middle ear and inner ear． Conclusion Squamous cell carcinoma and Ceruminous gland adenoma /adenocarcinoma and mast cell tumor were the often occurred in the feline ear． There was an upward trend of incidence on feline ear tumors with age increase and most common in older cats． All the tumors were occurred in the external ear．

Cryopreservation of Mice 2-cell Embryos by Vitrification Based on DAP

2015, 32(06):  34-37. 

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Abstract: Objective To explore the frozen effect of mice 2-cells embryo，to establish embryo cryopreservation technique of mice． Method Three strains mice of SPF female in 4 weeks old ( including C57BL /6，ApoE － / － ，NOS3 － / － ) were selected to carry out experiments of superovulation，in vitro fertilization，vitrification，2-cell embryo recovery and in vitro culture technique． Then，observed the experiment effects． Result The average number of superovulation of the three strains including C57BL /6，ApoE － / － ，NOS3 － / － mice was 42. 98，27. 93，23. 11 per mouse，respectively． The mean 2-cell embryo rate of in vitro fertilization was 68. 79%，32. 70%，23. 08%，respectively． The survival rate after freezing-thawing was 56. 92%，54. 84%，70%，respectively． The embryo recovery rate after freezing-thawing was 72. 77%、80. 87%、83. 33%，and the rate of embryos developed to blastocyst in vitro was 72. 37%，60. 78%，25%，respectively． Conclusion Superovulation，in vitro fertilization，embryo cryopreservation，embryo recovery，of 4 week old mice showing a better effect by the same technology method，and C57BL /6 was better than genetically engineered mice． Thus the basic establishment of mouse 2-cell embryos cryopreservation technology．

Establishment and Preliminary Application of ELISA in Detecting Sendai Virus Antibody in Mongolian Gerbil

2015, 32(06):  39-43. 

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Abstract: Objective To develop a ELISA method for determination of Sendai virus( SeV) antibody in Mongolian gerbil． Methods Hatchs chicken embryos，vaccinate the Sendai Virus，prepare the normal chicken embryo allantoic fluid antigen and special SeV antigen，titrate the best working density of enzyme union and the normal or special antigen and carry out the experiments of specificity，sensitivity，accuracy and stability． Result The best working density of normal，special antigen and the enzyme union is 0. 1 μg /mL，2 μg /mL and 1 ∶ 5 000 respectively; The inter-assay coefficient of variation of normal antigen and special antigen is 9. 6% and 8. 4% respectively，the intra-assay average coefficient of variation is 9. 0% and 5. 9% respectively; The detection sensitivity is 1∶ 10 240; There is no cross-reactivity with mouse hepatitis virus( MHV) ，Pneumonia virus of mice ( PVM) ，and Reovirus type 3． The stability test shows the relative deviation is below 25%． Conclusion The ELISA method is good in specificity，sensitivity，duplication，and stability，as a result of which ELISA can be used in detecting the Sendai virus antibody．

Traceability Verification Experiment of Ammonia Concentration Detection

2015, 32(06):  44-46. 

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Abstract: Detection of ammonia concentration is an important indicator of the dynamic detection of laboratory animal facility． The accuracy of detection is related to curving of standard ammonia sample，which is dependent on calibration the concentration of standard sample． So the curve of standard ammonia commercially available was compared with that of ammonia preparation solution using the reagent colorimetric method． The result shows the deviation of curves is － 1. 79%． Detection of ammonia concentration is affected by many factors，to ensure the accuracy and consistency of the detection results，and laboratory verification and calibration should be performed．

The Making of Experimental Animal Specimens and Its Role in the Experimental Zoology Teaching

2015, 32(06):  47-49. 

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Abstract: The paper introduces the method and process of the making of experimental animal specimens，and illustrates the application of the making of experimental animal specimens in experimental zoology teaching．

Promotion and Application of Training and Assessment Management System of Laboratory Animals Professional

2015, 32(06):  50-55. 

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Abstract: The management system of laboratory animal professional training and assessment contains 7 subsystems and was continuous improvement while it was promoted． Among them，the work license examination system of laboratory animal professional was officially promoted to use in Beijing in April 2014． By the end of June 2015，99 examinations were held in Beijing，and 7439 people attended these examinations． The qualified rate was 95. 35%． While Heilongjiang，Inner Mongolia，Tianjin，Liaoning，Shanxi，Sichuan and other provinces and cities have also been prepared to use the system． The laboratory animal professional training contents will be the same level in different provinces and cities after the promotion and application of training and assessment management system of laboratory animals professional in China．

The Pathological Observation and Presentation of the Model of Alveolar Epithelial Cell II in Mouse

2015, 32(06):  56-58. 

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Abstract: Alveolar epithelial cells II ( AT II) is a kind of very important cells of alveolus． They are hard to be observed in microscopic field because of very less number． Here we found an extraordinarily increased number and increased in volume of AT II cells in the alveolus of one kind of gene knockout mice． In this paper，the phenomenon combined with image are introduced，and sums up the physiological function of alveolar type II cells and possible relationship with TSE-1． This type of increase in AT II cells in gene knockout mice can easily be seen in microscopic field; furthermore，it can lay the platform for future experiments about AT II cells and provide us with an animal model and material base for pathophysiological research on lung cells．