Table of Content

30 June 2017, Volume 34 Issue 03

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Preliminary Immunogenicity of Prokaryotic Expressed Chicken Interleukin-10 for in Vivo Ｒesearch

2017, 34(03):  1-5. 

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Abstract: Objective To preliminarily analyze the immunogenicity of E． coli expressed chicken Interleukin-10 ( chIL-10) antiserum in stably chIL-10-expressing cell line transfected by chIL-10 gene with signal peptide ( CHOchIL- 10F) and that without signal peptide ( CHO-chIL-10M) ． Method Whole chIL-10 gene was amplified by PCＲ from chicken peripheral blood lymphocytes and cloned into pET-30a ( + ) vector to obtain recombinant plasmid of pET-chIL-10，followed by induce express in E． coli BL2l ( DE3) ． The SDS-PAGE gel containing foreign protein was immunized BALB /c mice to prepare antisera． Indirect immunofluorescent assay ( IFA) and Western blot assay were performed． Ｒesult The mice anti-pET-chIL-10 specific serum could react with eukaryotic expressed chIL-10 in IFA and Western blot assay． Conclusion Antiserum to prokaryotic expressed chicken IL-10 could be used in vivo research study．

Effects of Diazoxide on The Development of Osteoarthritis of The Knee Joint in Ｒabbits

2017, 34(03):  6-9. 

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Abstract: Objective To study the effects of diazoxide on the knee osteoarthritis in rabbits and explore the possible mechanism． Method Select 20 adult male New Zealand white rabbits，cut off the bilateral anterior cruciate ligament of knee joint and the lateral meniscus，establish the model of osteoarthritis; randomly devide the rabbits into diazoxide group and control group，10 rabbits per group，Every week after the surgery，give the former diazoxide 1 mL injection in the intra-articular，give the control group distilled water 1 mL injection in the intraarticular．Anatomize the rabbit knee joint at the fourth week，and compare the cartilage lesion of the two groups．Ｒesult Observing the animal’s capability of motion and rabbit knee specimen，we found the osteoarthritis pathological changes in Diazoxide group is lighter than in control group ( P ＜ 0. 05) ． Conclusion Diazoxide has acertain antagonism to the development of osteoarthritis of the knee joint in rabbits．

Protective Effects of Dian-xian-qing Granule on Pentylenetetrazole-induced Seizures in Ｒats

2017, 34(03):  10-15. 

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Abstract: Objective This study was to investigate the effects of DXQ on pentylenetetrazole-induced hippocampal pyramidal cells apoptosis and cerebrum damage，then to explore its possible mechanisms． Method Male Wistar rats were randomly divided into 7 groups: normal control ( NC) ，pentylenetetrazole ( PTZ) ，Dian-Xian-Qing granule( DHQ) ( 4. 78，9. 46，18. 92 g / kg·d － 1 ) ，Dian-Xian-Ning Tablets( DXN，5. 05 g / kg·d － 1 ) and Phenytoin Sodium Tablets ( PST，54 mg / kg·d － 1 ) positive control groups，a sub-convulsive dose of PTZ 40 mg / kg in the volume of 1 mL / kg( ip) was administered to rats on every second day for 30 days resulting in a permanent change of generalized tonic-clonic convulsions． The convulsive behaviors were evaluated according to Ｒacine's score，the number of surviving hippocampal pyramidal cells was assessed by HE staining． The expression of AIF ( apoptosis inducing factor) ，BaX and Bcl-x / s + L were analyzed by Western blot immunohistochemical ( IHC) staining．Ｒesult Ｒats suffered PTZ showed higher Ｒacine's score，worse cell damage and apoptosis，which were all attenuated by DHQ ( 4. 78，9. 46，18. 92 g / kg·d － 1 ) ． Moreover，DHQ reversed the expression of AIF，BaX and Bcl-x / s + L induced by tonic-clonic convulsion． Conclusion These result suggest that DHQ has neuroprotective effects，which is related to the level of AIF，BaX and Bcl-x / s + L restraining cells apoptosis．

Analysis and Construction of Shuttle Vectors of Ｒecombinant Virus of Duck Plague Virus

2017, 34(03):  16-19. 

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Abstract: Objective The aim of this study was to construct shuttle vectors and rescue the recombinant viruses with EGFP，which provided basic research for bivalent live vaccine of DPV． Method We selected the proven insertion sites，including US region of US2-SOＲF3，US7-US8 and UL region of UL15-UL18 unconding regions of DPV to construct shuttle vectors P-US2-SOＲF3-EGFP，P-UL15-UL18-EGFP and P-US7-US8-EGFP． Shuttle vectors were used to transfect the CEFs infected with vaccine strain by using liposome 2000． The recombinant viruses were rescued by homologous recombination． Then collected the cells supernant and purified plaques． Ｒesult We successfully constructed three shuttle vectors with EGFP． We failed to rescue any recombinant virus plaques by using pBlusecript II SK vector． However，the recombinant virus plaques were rescued with pUC18 vector． Conclusion It was suggested that pBlusecript II SK vector might be unsuitable for rescuing recombinant viruses， and the insertion site might also affect the virus replication．

Bioassay Method of Anticoagulant Ｒesearch of Yixintongluo Capsule in Vitro

2017, 34(03):  20-23. 

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Abstract: Objective To inspection the feasibility of bioassay method of anticoagulant research of YiXinTongLuo capsule in vitro． Method Different concentrations of aqueous extract of YiXINTongLuo capsule were mixed with Ｒabbit plasma and the TT was determined by coagulometer． One titer unit( U) was defined as one second longer for anticoagulant research of YiXinTongLuo capsule than the coagulation time of the blank control，and the titer of YiXinTongLuo capsule standard substance was defined and measured． Ｒesult Within the range of 0. 1886 ～ 0. 2610 g /mL，TT and the aqueous extract concentration showed a good linear relationship respectively( r = 0. 9980; 0. 9985; 0. 9829) ． The titer of the standard substance of YiXinTongLuo capsule was measured to be 2395 U /g． Conclusion It is feasibility that TT used toestablish bioactivity assay in vitro．

The Variation of Mir-146B-5P in Immune Organs of B19 and B21 Haplotype SPF Chickens Infect with MDV

2017, 34(03):  24-27. 

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Abstract: Objective To analyze the miＲ-146b-5p in B19 ( MD susceptible) and B21 ( MD resistance) haplotype variation of the expression of chicken infected with MDV and explore the possible after the MD resistance / susceptibility associated molecular mechanism． Method Through MDV virulent strain Md5 poison attack experiments and previous high-throughput sequencing based differences in the expression of miＲ-146b-5p on the further use of real-time PCＲ technology to detect miＲ-146b-5p in B19 and B21 haplotypes chickens 5dpi，10dpi and 21 dpi bursa of fabricius thymus and spleen tissues ( experimental group) and the corresponding uninfected group ( control group) expression changes． Ｒesult MiＲ-146b-5p was significantly different in experimental group and control group at 5dpi，10dpi and 21dpi． The expression of miＲ-146b-5p in B21 group was much higher than that in control group，and the difference was significant． The expression of miＲ-146b-5p in B19 group was higher than that in control group，but the difference was not significant． Conclusion The expression of miＲ-146b-5p in different immune organs after MD infection may be tissue-specific，and there are some differences between different chicken lines after infection，but the specific mechanism needs further study．

The Health and Ｒeproduction of The Mice Fed by Diets Supplemented with Milk from Fat1 Transgenic Cow

2017, 34(03):  28-33. 

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Abstract: Objective To investigate the impact of fat1 transgenic cow milk on mouse behavior，heath and reproduction． Method Thirty six B6D2F1 /Vr mice at 4 weeks old were randomly divided into 6 groups with the same number of males and females in each group． The milk derived from the fat1 transgenic cow，somatic cell nuclear transfer cow，and dairy cow were respectively supplemented into the diets． The milk-contained diets were respectively fed the 3 groups of mice． During feeding the behaviors such as food intake，moving，oestrus，mating were recorded and weighed regularly． After feeding for 14 weeks，the mice were continuously mated by male and female in 1∶ 1 until produced 3 times，the offspring were counted． The blood and serum parameters and the organs were analyzed． Ｒesult Theresult showed that the growth curve，food intake，mating behaviors，reproduction，and weights of organs were with no difference among the 3 groups． Conclusion The diets supplemented with the fat1 transgenic cow milk had no harmful effects on the health and reproduction of the mice，which suggest that the fat1 transgenic cow milk be safe for animal diets．

Effect of Some Natural Mountain Spring on Blood Lipid and Blood Ｒheology in Ｒat Models with Hyperlipidemia

2017, 34(03):  34-37. 

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Abstract: Objective To observe the effect of some natural mountain spring on blood lipid and blood rheology in rat models with hyperlipidemia． Method Twenty-four male rats were randomly divided into mountain spring group ( drink some natural mountain spring) and control group ( drink tap water) according to the level of TC． The hyperlipidemia rat model was established by feeding with high-fat diet． The serum in the two groups was taken for blood lipid and blood rheology analysis after sixty days． Ｒesult Compared with those of control group，the levels of TG and LDL in mountain spring group were significantly decreased ( P ＜ 0. 05，P ＜ 0. 01) ; Blood rheology indexes in mountain spring group were improved significantly( P ＜ 0. 05) ，these indicators including: whole blood viscosity， hematocrit，and so on． Conclusion The result show that some natural mountain spring could significantly decreased the level of TG，and LDL-C in the rats with hyperlipidemia． It can also significantly improve the mountain spring group rats’blood rheology index．

Development and Application of a Multiple Ｒeal-time Quantitative PCＲ for Haemophilus influenzae，Haemophilus haemolyticus and Pasteurella multocida

2017, 34(03):  38-42. 

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Abstract: Objective To develop a multiplex real-time quantitative PCＲ ( qPCＲ) assay to detect Haemophilus influenzae，Haemophilus haemolyticus and Pasteurella multocida in laboratory animals． Method We establish the multiple qPCＲ reaction system by specificity，sensitivity and reproducibility verification，according to Hpd gene of H． influenzae and H． haemolyticus，KMT1 gene of P． multocida designed specific primers and Taqman probes respectively． Then，the 822 laboratory animal respiratory samples were detecting by the assay application． Ｒesult There was no cross reaction between the three pathogens and 29 others． The sensitivity of the multiple qPCＲ method detecting above three pathogens could reach 10 copies /μL． Among the tree shrew specimens，the positive rate of H． haemolyticus and P． multocida were 10% ( 6 /60) and 21. 7% ( 13 /60) respectively，but other animals were negative in three pathogens． Conclusion The multiple qPCＲ assay in this study that can quickly detect the above three kinds of respiratory pathogen in laboratory animals．

Optimization and Evaluation of Allogeneic Antigen Combined with Acetic Acid in Ｒats with Chronic Ulcerative Colitis

2017, 34(03):  43-48. 

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Abstract: Objective Establishment of rat model of ulcerative colitis ( UC) induced by optimization of allogeneic antigen combined with acetic acid and evaluate the value of the model． Method With different antigenic emulsion protein content，different acetic acid concentration and acetic acid retention time in rats as variables，according to the rat disease activity index ( DAI) score，colon mucosal injury index ( CMDI) score，and histopathological( HS) score，screened the optimal conditions to set up rat UC model． Ｒesult The first part orthogonal experiment was carried out with the concentration of acetic acid and the amount of allogeneic antigen as variables，but with the settled retention time of acetic acid as 13 s． Compared with the normal group，the model group coloclysis with 1 mL 8 mg /mL antigenic emulsion protein and 1 mL 5% acetic acid was the suitable condition for inflammation which was significant difference at DAI，CMDI scores，had appropriate inflammation and high survival rate． The second part experiment was carried out with the settled optimizational concentration of acetic acid and the amount of allogeneic antigen from first part but change the retention time of acetic acid as 13，15，18，20，and 22 s． Compareing with the normal group，the model group of 15 s retention time of acetic acid was the better one with good DAI，CMDI，and HS scores，scores，appropriate inflammation and high survival rate． Conclusion The optimized method of setting up UC model was as follow: coloclysis with 1 mL 5% acetic acid solution，washing with 4 mL water after retaining for 15 s，then coloclysis with 1 mL antigen emulsion protein solution ( 8 mg /mL) and washing with 4 mL water again after retaining for 30 min． This method had good reproducibility，low mortality，and the severity of colonic inflammation in rats，and with the similar mechanism of human UC． This complex ulcerative colitis model is suitable for the screening of new drugs in UC．

Establishment and Preliminary Application of a qPCＲ Detection Method for Mouse Adenovirus

2017, 34(03):  49-54. 

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Abstract: Objective Establish a specific qPCＲ detectionmethod based on TaqMan probe to investigate mouse adenovirus ( MAdV) infection in gerbils，mice and other laboratory animals． Method Genomic sequences of mouse adenoviruses ( MAdV-A and MAdV-3) were downloaded and primers and probes were designed at the best conservative genes which were selected after comparative analysis． Screened and tested the specificity，sensibility， repeatability and stability of the best primers and probe，utilized this q-PCＲ method to detect 41 samples of gerbils and voles． Ｒesult MAdV-3 was chosen as the best from three pairs of primers and probes，which could distinguish MAdV，SAdV，FAV and TAV and other 24 viruses，the minimum detectable amount of MAdV plasmid DNA template is 4. 5copies / reaction; the minimum detectable amount of MAdV pure virus and virus /tissue simulate sample DNA template both are 440copies / reaction． Conclusion The TaqMan qPCＲ detection method established in this manuscript has good specificity，sensitivity，repeatability and stability，which might be used to detect MAdV in laboratory animals and relevant biological products．

A Comparative Study on The Identification System of Laboratory Animal

2017, 34(03):  55-60. 

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Abstract: This paper introduces the identification system of laboratory animals used in the production and scientific research，and analyzes the advantages and disadvantages of the identification method of rodents and primates and the main contents of the facilities identification，which provides a reference for the selection of laboratory animals identification and identification in practical work． With the development of science and technology，the new technology based on ＲFID technology will be widely used in experimental animal field． The facilities identification should be systematically set in consideration of all aspects．

Study on The Standards of Microbiological Quality Control of Experimental Pigs

2017, 34(03):  61-65. 

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Abstract: Experimental pigs are used as a target animal in scientific research and drug development study，but lack of corresponding microbiological quality control standards． This study focuses on pathogenic microorganism species and related detection rules in the microbiological quality control on experimental pigs，and aims to explore and establish the level of microbiology experiment with pigs and the local monitoring standards in Beijing using literature analysis and expert consultation method． Therefore，the study will provide a reference for the microbiological quality control in local production and use of experimental pigs in Beijing．

Investigation on The Current Situation of Laboratory Animals Units，Personnel and Facilities in Mainland China

2017, 34(03):  66-70. 

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Abstract: The purpose of this paper is to investigate and analyze the current situation of laboratory animals units， personnel and facilities in mainland China． This investigation covered 31 provinces，autonomous regions and municipalities directly under the central government，and the military system in mainland of China． The survey found that the total number of experimental animal license is 1870，of which，the number of production license is 422，and usage license is 1448． The survey also found that the experimental animal industry is mainly composed of enterprises，mainly supports the medical and health industry，and mainly provides comprehensive services that has become the development direction of experimental animal units; the management of experimental animal practitioners is on track，and the proportion of scientific and technical personnel is high in the experimental animal staff，but the education background and titles of practitioners remains in overall low levels，so personnel training needs to be strengthened; the area of facilities for experimental animal production and usage is increasing year by year and the construction investment is also increasing year by year，but the government funding for the construction of production facilities should be boosted．

Health Monitoring of Laboratory Ｒodent Colonies

2017, 34(03):  71-75. 

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Abstract: To ensure the repeatability and credibility of the research data，it’s important to investigate the health status of experiment animal which plays a crucial role of human life science research． Depending on many documents and combining with the lab work experience in assessing the quality of laboratory animals for many years，we summarized some recommendations for the monitoring samples and method ． After years of work，we find out that even without obvious clinical features or being diagnosed as microbiological ( virus，bacteria，fungi，and mycoplasmas) and parasitological healthy，the laboratory animals can still have different degrees of pathological damage． These result further demonstrate that there is still a part of them in sub-health status． In conclusion，it’s essential to establish and implement a laboratory animal health monitoring program．

Application of Next Generation Gene Editing Techniques in Animal Model of Human Disease

2017, 34(03):  76-80. 

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Abstract: Gene editing is a new technology aiming at genome targeted modification，which mainly modifies target genome for the purpose of studying the gene with unknown functions and conducting gene therapy． Ｒecently，the grown-up of new gene editing techniques including engineered nuclease mediated zinc-finger nucleases( ZFNs) ， transcription activator-like effector nucleases ( TALENs) and clustered regularly interspaced short palindromic repeat sequences ( CＲISPＲ) － CＲISPＲ-associated( Cas) ＲNA-guided nuclease( CＲISPＲ Cas ＲGNs) accelerates the progress of gene function study and has an important significance in the structure of animal models of human disease and exploration of new disease treatment program． Gene editing techniques and those application in research of animal model of human disease were briefed in this paper．