Table of Content

29 June 2015, Volume 32 Issue 03

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Establishment of Cell Line Expressing Chicken B19 BNK Protein Stably and Production of BNK19 － Specific Monoclonal Antibody

2015, 32(03):  1-5. 

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Abstract: Objective To product BNK19 － specific monoclonal antibody using the B19 haplotype chicken BNK protein expressed by a stable cell line． Method B-NK19 gene was amplified from B19 haplotype chicken and then cloned into the pIＲES vector to construct recombinant plasmid of pIＲES-BNK19． The recombinant plasmid was transfected into CHO cell and identified by ＲT-PCＲ and indirect immunofluorescent assay ( IFA) ． BALB /c mice were immunized using the BNK protein expressed by the recombinant E． coli，and the monoclonal antibody was selected from the CHO-BNK19 cell line． Ｒesult The CHO-BNK19 cell line expressing BNK protein stably was identified and two specific strains of monoclonal antibody were obtained using IFA． Conclusion The study is expected to be useful for researching chicken BNK protein and NK cells．

Analysis of Microsatellite Markers in MHC I Region of HBK-SPF Duck

2015, 32(03):  6-9. 

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Abstract: Avian major histocompatibility complex ( MHC) gene family is a cluster of closely linked and highly polymorphic gene groups that are closely associated with immune response or sensibility． Duck MHC I region has a length of 36. 8 kb，including TAP1，TAP2 and five MHC I gene copies ( UAA ～ UEA) ． Two strains，B and Q，of the HBK-SPF ( specific pathogen free ) duck have been bred to the seventh generation in Harbin Veterinary Ｒesearch Institute of Chinese Academy of Agricultural Sciences． Four microsatellite loci were screened in the MHC I region and polymorphism was detected through the single-strand conformation polymorphism，the polymerase chain reaction ( PCＲ) and the directly sequencing． The A locus was found to be polymorphic and characterized by the repeat motif of ( GT) n ． In both the HBK-B and the HBK-Q strains，31 and 140 individuals of the sixth and the seventh generations respectively were detected by the PCＲ and the directly sequencing，and four homozygous and six heterozygous genotypes were found in the upstream 108 bp sequence of the ( GT) n ; There were no PCＲ product at the B locus; PCＲ product of locus C was highly homologous to the corresponding region between the UAA and the UBA genes，and the genotype of locus C could not be identified; locus D was monomorphic，and characterized by the repeat motif of ( ATA) 15 ． This study provides useful data for further research on duck MHC I genome and family establishment．

Effect of Glutamine on Expression of Immune-related Genes in Plague Virus Infected Duck Intestine

2015, 32(03):  10-12. 

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Abstract: Objective To investigate the effect of Glutamine ( Gln) on the intestine physiological activity and the pathway in normal ducks and the models infected with the plague virus． Method 280 specific pathogen free ( SPF) ducklings were randomly divided into eight groups． Gln was administrated at different doses ( 0，0. 5，1，2 g / kg / feed / d) by oral gavage to the control groups and the model groups infected with 0. 2 mL duck plague virus ( DPV)( 0. 2 mL 2000TCID50) ，respectively． The procedures were performed for 6 days． Effects of Gln in control and model ducklings were detected，and expression of the Toll like receptor 4 ( TLＲ4) pathway genes were determined．Ｒesult Expression of TLＲ4 pathway mＲNAs was increased significantly in normal group ( P ＜ 0. 05) ，but reduced significantly in the model groups ( P ＜ 0. 05) ． Conclusion Gln improves immunity in duckling intestine through the TLＲ4 pathway，and resists the immune-mediated injury caused by DPV．

Organic Distribution of Duck Challenge Hepatitis Virus Strain in Ducklings

2015, 32(03):  13-15. 

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Abstract: Objective To investigate the dynamic distribution of duck challenge hepatitis virus strain ( 161 /79 /V) in experimentally infected ducklings using the Taqman real-time fluorescent quantitative PCＲ． Method Ducklings were infected with the 161 / 79 / V strain at 3 － day age，and the virus ＲNAs were extracted from different organs within 24 hours and quantified using the fluorescent quantitative PCＲ method． Ｒesult DHV ＲNAs were detected in heart，liver，brain，lung，pancreas，ileum，cecum，rectum，bursa and spleen 4 hours after inoculation，and the ＲNA copies continuously increased． The highest copy of the DHV ＲNA was detected in liver ( 108. 81 copies /g) 24 h after inoculation，and the ＲNA copies were somewhat low in heart，lung，kidney and spleen ( about 107 copies /g) ．Conclusion Our study indicates that the challenge DHV strain 161 / 79 / V is tissue or organ ecotropic，such as the solid organ，the immune organs and the digestive system of the infected ducklings．

Detection of Telomerase Activity Using DNA-templated Silver Nanoclusters

2015, 32(03):  16-18. 

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Abstract: Objective To establish a method to detect telomerase activity using DNA-templated fluorescent silver nanoclusters． Method In the presence of sodium borohydride and synthesized oligonucleotide，silver ions in the nanoparticles were reduced into silver nanoclusters with excellent fluorescent properties． When DNA sequences containing G bases connected with the DNA-templated silver nanoclusters，the fluorescence will be improved obviously． Telomerase activity was detected using the oligonucleotide 5'-( TTAGGG) n-3' ，which could be extended by telomerase． Ｒesult and Conclusion The method for telomerase activity detection was established using the DNA-templated fluorescent silver nanoclusters．

Influence of Uterine Ｒectal Pouch Endometriosis on Ｒeproductivity of Wistar Ｒats

2015, 32(03):  19-22. 

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Abstract: Objective To establish the uterine rectal pouch endometriosis model using female Wistar rat，and study the histological structure and pregnancy rates in those rats． Method Autologous uterus were transplanted into 40 female Wistar rats to establish the uterine rectal pouch endometriosis model，and 10 rats were treated as the shamoperated control． Male rats were permitted to approach the female models or controls 8 weeks after transplantation，and the farrowing rates and litter sizes were detected． The eutopic and ectopic endometrium were collected 15 weeks after transplantation for histological analysis． Ｒesult Based on the histological examination，development of the ectopic endometrium is different from that of the eutopic endometrium． The cell morphology of the ectopic endometrium changed from early proliferative phase to mid-secretory phase，but that of the eutopic endometrium changed from early proliferative phase to later secretory phase． The average litter size of model group was 6. 02，which was significant lower than the control group ( 8. 5) ( P ＜ 0. 01) or the sham operated group ( 8. 0) ( P ＜0. 01) ． Conclusion Development of the ectopic endometrium is not the same as that of the eutopic one． It makes the rat body environment abnormal，implantation of fertilized eggs difficult，and so reduces the reproductiveity．

Hypoglycemic Effect of Black Garlic in Experimental Diabetic Ｒats

2015, 32(03):  23-26. 

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Abstract: Objective To investigate the effect and mechanism of the “black garlic”( Black garlic) on the body weight and the blood glucose in diabetic rats induced by streptozotocin． Method The diabetic rat model was established by intraperitoneal injection of 50mg / kg streptozotocin． Black garlic was administrated to the rats by oral gavage for 30 days． Body weight and blood glucose level before and after 30 － day oral gavage were determined respectively． Ｒesult Black garlic at the dose of 10g / kg has no effect on body weight and blood glucose level of the control rats． At the high ( 10 g / kg) and the middle ( 5 g / kg) doses，body weight of the black garlic treated group declined，while the blood glucose level increased continuously compared with the untreated group． But there were no significant differences between the two groups． At the low dose ( 2. 5g / kg) ，weight loss of the treated group was relieved and the blood glucose increased slowly compared with those of the untreated group． And the differences were statistical significant． Conclusion This study indicates that proper consumption of black garlic reduces blood glucose level，and delays the development of diabetes． Further studies are expected to explicate the mechanism of the black garlic．

The Effect of Modified Decoction of Ｒehmanniae on Na +-K+-ATP and Ca2 +-ATP Enzyme Activity in Ｒat Models of Alzheimer’s Disease

2015, 32(03):  27-29. 

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Abstract: Objective To investigate the effect of Modified Decoction of Ｒehmanniae on the Na +-K +-ATP and the Ca2 +-ATP enzyme activities in rat models of Alzheimer's disease ( AD) ． Method Ｒat AD model was induced by sterotaxis injection of amyloid － β1 － 40 in bilateral hippocampus． The aging rats were used as the blank control and the models treated with piracetam were used as the positive control． The Na +-K +-ATP and the Ca2 + -ATP enzyme activities in brain were assayed by biochemical techniques． Ｒesult The Modified Decoction of Ｒehmanniae improved the activities of the Na +-K +-ATP esterase and the Ca2 + -ATP esterase． Conclusion The results suggest that the Modified Decoction of Ｒehmanniae improves the activities of the Na +-K +-ATP esterase and the Ca2 + -ATP esterase，and can be used as an effective drug for Alzheimer's disease．

Observation of Inhibitory Effect on Plasma to Melanoma Transplanted from Human in Nude Mice

2015, 32(03):  30-32. 

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Abstract: Objective To investigate the inhibitory effect of plasma( APNP) on malignant melanoma． Method Twelve nude mouse models were established by transplanting the B16 malignant melanoma，and then were randomly divided into two groups，the plasma treatment group and the control group． Animal health was monitored，and the lengths and the widths of tumors were examined regularly． Tumor volumes and inhibition rates were calculated respectively． Ｒesult The inhibition rate was 88% in the treatment group． Conclusion Plasma treatment at a proper dose can inhibit the growth of melanoma effectively．

Establishment of a SEMI-QUANTITATIVE ＲT-PCＲ Method for the Detection of XDH /XO mＲNA Expression in Difference Organ Tissue

2015, 32(03):  33-37. 

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Abstract: Objective To established of a semi-quantitative ＲT-PCＲ method for the detection of xanthine dehydrogenase /oxidase( XDH /XO) mＲNA expression from tissue of the cardiac muscle，kidney，testicle，skin，liver of rhesus monkey． Method Total ＲNA was extracted from the cardiac muscle，kidney，testicle，skin，liver tissue respectively for amplification of XDH /XO gene fragment by two-step ＲT-PCＲ． GAPDH is as a ‘reference’genes to normalize ＲT-qPCＲ data． The specificity of sequence of amplified nucleotide acids were identified by gel electrophoresis and sequencing． The gray scale rate ( IOD ) of electrophoresis strip were obtained from quantity One of GelDocTM XＲ gel image analyzer． Ｒesult A semi-quantitative ＲT-PCＲ method have been established for the detection of XDH /XO mＲNA in different tissues of the rhesus monkey． Quantitative analysis demonstrated highest expression of XDH /XO in liver，and then cardiac muscle，kidney，testicle，skin． Conclusion The semiquantitative ＲT-PCＲ method will be apply to detect XDH /XO mＲNA expression in difference organ tissue．

Analysis of SNPs Associated with Nerve Pattern Ｒelated Genes in Labrador Ｒetrievers

2015, 32(03):  38-43. 

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Abstract: Objective Analysis of 6 SNPs associated to nerve pattern related 4 genes in Labrador Ｒetrievers to provide a early-stage molecular genetic index for nerve pattern diagnostics． Method According to the behavior evaluation standards of China guide dog training center，the dogs belong to typical nerve patterns ( quiet，active， and timid ( weak ) type，4 dogs each) were selected． The vein blood was sampled and the genomic DNA was isolated from blood cells． The PCＲ/sequencing and PCＲ-ＲFLP method were used to analyze the variants of 6 SNPs in nerve pattern related 4 genes: ( COMT ( G482A) ，MOAB ( T199C) ，ABCB1 ( A985T，T3442C，377 － 378 insC) ，GNB1L( 961 － 962 insG) ) ． Furthmore，the association between the SNP variants and nerve patterns was analyzed by Chi-square Test． Ｒesult G/A variants in COMT ( G482A) SNP locus in the proportion of timid dogs was significantly higher than that in the active and quiet dogs ( P = 0. 014; P = 0. 014) ，A/A variants in COMT ( G482A) SNP locus in the proportion of active dogs was significantly higher than that in the quiet and timid dogs ( P = 0. 025; P = 0. 025 ) ; T /C variants in MAOB ( T199C) SNP locus in the proportion of quiet dogs was significantly higher than that in the active and timid dogs ( P = 0. 025; P = 0. 025 ) ; T /A variants in ABCB1 ( A985T) SNP locus in the proportion of active dogs was significantly lower than that in the quiet and timid dogs ( P = 0. 014; P = 0. 014 ) ; T /C variants in ABCB1 ( T3442C) SNP locus in the proportion of timid dogs was significantly lower than that in the quiet and active dogs ( P = 0. 025; P = 0. 025) ; No ABCB1 ( 377 － 378 insC) and GNB1L ( 961 － 962 insG) SNP loci was found． Conclusion G/A and A/A variants in COMT ( G482A) 、T /C variants in MAOB ( T199C) 、T /A variants in ABCB1 ( A985T) and T /C variants in ABCB1 ( T3442C) are significantly associated to nerve patterns and might be used as a molecular genetic index for nerve pattern diagnostics in Labrador Ｒetrievers． However，further large sample analysis is required．

The Determination and Comparison between MＲL / lpr Mice and C57 Mice Blood，Major Organ Coefficient

2015, 32(03):  44-47. 

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Abstract: Objective To determine blood and organ coefficient values between C57 mice and MＲL / lpr transgenic mice at the ages of 4 months，5 months and 6 months old． Method We used MEK － 7222K automatic blood analyzer to analyze C57 mice and MＲL / lpr transgenic mice blood values． Ｒesult On the blood detection，the percentage of neutrophils ( NE%) ， red blood cell count ( ＲBC ) ， hematocrit ( HCT ) ，mean corpuscular hemoglobin ( MCH) ，red blood cells mean corpuscular hemoglobin concentration ( MCHC) values in different months between C57 mice and MＲL / lpr transgenic mice were significantly different ( P ＜ 0. 01) ; among nine items there was no significant difference compared ( P ＞ 0. 05) ． On organ coefficient aspects，C57 mice coefficient heart，kidneys coefficient is significantly higher than the same month-old MＲL / lpr transgenic mice ( P ＜ 0. 01) ; MＲL / lpr transgenic mouse liver coefficient is significantly higher than the same month-old C57 mice ( P ＜ 0. 01) ;there was no significant difference ( P ＞ 0. 05) on other comparable projects． Conclusion Partial blood and organ coefficient data were significantly different in the same months of C57 mice and MＲL / lpr transgenic mice．

Construction of Obesity in Ｒats with Normal Diet Adding Egg Yolk and Peanuts

2015, 32(03):  48-50. 

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Abstract: Objective To establish high fat diet induced obesity in male SD rat model of nutrition． Method Four weeks old male SD rats were randomly divided into experimental group( n = 24) and control group( n = 6) ． The control group was fed normal diet and the experimental group was fed high fat diet which was composed with normal diet ，egg yolk and peanuts． Two weeks later，the obesity resistant rats were excluded from the experimental group．Weight and Lee's index were observed after eight weeks． After twelve hours fasted ，all rats were sacrificed and tested for TC、TG． The hepatic tissue of rats was analyzed histomorphologically． Ｒesult Experimental group weighted 526. 6 ± 46. 4g and the control group weighted 415. 6 ± 33. 7g， there for the weight of experimental group rats were 33. 1% higher than control group rats． Lee's index of the experimental group was 330. 0 ± 8. 2，while the control group was 307. 0 ± 6. 6． The serum total cholesterol of experimental group was 2. 16 ± 0. 17 mmol /L，serum triglyceride was 1. 40 ± 0. 34 mmol /L and control group，s serum total cholesterol was 1. 66 ± 0. 17 mmol /L，serum triglyceride was 0. 82 ± 0. 13 mmol /L． Pathology results showed the experimental group were induced fatty liver．Conclusion This study showed that feeding high fat diet which was composed with normal diet ，egg yolk and peanuts could successfully and stably established obesity model．

The Important Ｒole of“Project Technician Ｒesponsibility System”in Operation and Management of Animal Experiment Facility in Medical Colleges

2015, 32(03):  51-53. 

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Abstract: There are many practical problems in the operation of animal experimental facility in medical college，such as plenty of projects，various types of projects，a large number of researchers and so on，which make management of the facility challenging． In order to improve the efficiency of facility management and solve the problems in the operation and management of facilities，we put forward the management mode of“Project technician responsibility system”． This paper will mainly introduces the important role of“Project technician responsibility system”in the operation and management of animal experiment facility in medical colleges．

Husbandry of Beagle Compliant to the GLP Ｒequirements

2015, 32(03):  54-55. 

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Abstract: To develop a scientific and standardized management protocol for husbandry of Beagle in a GLP facility，the standard operating procedures ( SOPs) were established，and the management of animal care takers and testing persons was improved．

Advances of Differentiation of Bone Mesenchymal Stem Cells into Hepatocyte-like Cells

2015, 32(03):  56-61. 

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Abstract: The best treatment of severe liver disease caused by a variety of factors have been proved to be orthotopic liver transplantation，but the lack of liver donors and a series of problems caused by immune rejection greatly limits the use of the procedure，while in the screening of liver-related drugs ，primary hepatocyte cell is difficult to culture and easy to mutate in the training process． And at the same times ，with the depth of bone marrow mesenchymal stem cell research，more and more evidence indicate that bone marrow mesenchymal stem cells have potential to differentiate into hepatocyte-like cells． Thus，hepatocyte-like cell differentiated from bone marrow mesenchymal stem cells has good prospects in the field of regenerative medicine and drug screening． In this paper ，we summarize the isolation and biological characteristics of mesenchymal stem cells，inducing culture conditions，biological characteristics and clinical prospects of hepatocyte-like cells．

Progress of Studies on the Mental Disorder Animal Model

2015, 32(03):  62-64. 

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Abstract: Development of science and technology is dependent on animal model studies，so reliable animal models for various diseases have been developed around the world． And animal models have made a great contribution to the development of modern medicine． However，there are still many difficulties to reproduce human diseases in animals by artificial induction，and clinical signs of many diseases can’t be monitored in laboratory animals or only observed in the higher mammals． Therefore，animal selection and establishment of animal model are important for a successful experimental design and achieving its aim．