Table of Content

31 May 2010, Volume 30 Issue 5

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Expression of N Gene of Mouse Hepatitis Virus in Recombinant Baculovirus

ZHOU Jie1，SONG Yu2，JIANG Hao3，HU Jian-hua1，GAO Cheng1

2010, 30(5):  317-317. 

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Objective To express the MIIV nucleocapsid (N) gene with Bac-to-Bac baculovirus expression system. Method According to N gene sequence of MHV A59 strain in Genbank, a pair of specific primers was designed and synthesized. The ORF of N gene was amplified by RT-PCR, and then inserted into the transfer vehicle pFastbacHTa to obtain the recombinant transfer vectors pFastBHa-N,which was used to transform into E. coli DHlOBac, producing specific transposition with Bacmid to yield the recombinant shuttle vehicles, Bacmid-N. Then the Bacmid-N was further used to transfect Sf9 insect cells by lipofectin to generate recombinant baculovirus expressing N recombination protein. Results Recombinant baculovirus, rBN，was obtained. IFA and Western blotting detection confirnl ed that the gene had been expressed in sf9 cells, and the expression product could specially react to the sera of MHV. Conclusion The MHV N protein is expressed successfully by Bac-to-Bac baeulovirus expression system, which may lay foundation for development of diagnostic technique.

Screening and Functional Analysis of Metastasis-associated Genes of Human Lung Cancer

LIU Lei，WANG Xiao-min，JIA De-shui，LI Jing，YAN Ming-xia，Kong Han-wei，YAO Ming

2010, 30(5):  318-321. 

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Objective To screen metastasis-associated genes by using highly metastatic animal and cell line for the purpose of the biomarker exploration of human lung cancer metastasis. Methods RNA from high metastatic SPC-A-1 sci cells and the low metastatic SPC-A-1 cells were isolated and purified，respectively. Differential profil-ing of gene expression between the two cells were screened by cDNA microarray. Western analyzed the expression of some differentally expressed genes. Results Total of 7649 genes, including 3891 genes up-regulated and 3758 genes down-regulated, were found to differentially express between the high metastatic and low metastatic cells by cDNA microarray. Those genes involved in adhesion, apoptosis, transcription, signal transduction, cell cycle and metabolism. After Western blot analysis, CD44 and CDC42EP3 in the SPC-A-1 sci expression was significantly higher than that of SPC-A-1, while the opposite result in FN1. This is consistent with the results of gene chips.Conclusions More than 7000 differential expressed genes might be associated with metastasis of human lung cancer. This study provided a promising reference for mechanism exploration of lung cancer metastasis and new metastatic related genes discovery.

Generation of TAFA2 Gene Knockout Mouse Model

SUN Lian-hua1，CHEN Yan1，MA Sun-kai2，CHI Jun2，KUANG Ying2，GU Ming-min1，WANG Zhu-gang3

2010, 30(5):  322-328. 

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Objective To establish TAFA2 gene knockout mouse model and to create the condition for further in vivo study of its biological function and its role of development and growth in central nervous system. Methods The mouse genomic DNA sequence of TAFA2 gene was verified through bioinformatic analysis. According to the TAFA2 genomic DNA sequence, the strategy of gene targeting and constructing of knockout vector (pBR322-MK-MCS-TAFA2) were established. The knockout region spans from exon 4 to 5 of TAFA2 gene in mouse genome. The gene knockout vector pBR322-MK-MCS-TAFA2 was constructed and confirmed by restriction enzyme digestion and sequencing.Electroporation of ES cells with pBR322-MK-MCS-TAFA2 and screening of both G418 and Ganciclovoir resistant clones were performed according to common protocol. The homologous recombined ES cell clones were identified by PCR. Results After transplantation of homologous recombined ES cells into blastocysts through microinjection and mating of chimeras with C57BL/6J mice, 41 offspring with Aguoti fur in color were acquired, 14 of them (34.1%) show heterozygous genotype for TAFA2. Some heterozygote mice were intercrossed to generate mutant homozygotes. Finally, the cohort of mutant homozygotes for TAFA2 were established. Any abnormal change was not found by preliminary observa-tion of phenotype for heterozygous and homozygous mice. Conclusions The TAFA2 gene knockout mice have been established. The embryonic lethality in homologous mutant mice was not occurred.

Myocardial Injury in Rabbit Congestive Heart Failure Models and the Relationship Thereof to Nitric Oxide

WANG Xiao1，WANG Ning1，LIN Chuan-quan1，XU Qing-wen2，FU Yun1，LIU Jin-yuan3

2010, 30(5):  329-324. 

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Objective Cardio-cerebrovascular disease is a high incident disease in the world，the research of which depends on the establishment of animal models.The purpose of this study was to observe the myocardial changes with time in rabbit congestive heart failure models（CHF），as well as its relationship to Nitric Oxide（NO）。Method Twenty-four rabbits were randomly assigned into three groups with eight rabbits in each one：normal group，model 24h group and model 7d group The model of CHF with volume overload was set up via catheterization，and then the rabbits were executed after 24h and 7d respectively，whose myocardial tissue was used for light microscopic examination.Serum NO was measured using NO assay kit，and myocardial apoptosis was detected by flow cytometry.Caspase-3 expression was determined by Western Blot analysis.Results（1）The structures of myocardial cells in normal group were intact，and there were no defects.The myocardial injury was found in the group of CHF model after 24 h and 7d，while the injury in the 7d group was more extensive and serious.（2）The contents of serum NO increased significantly in model 24h group （3.33±1.30 μmol/L）compared with the normal group（2.09±0.22 μmol/L）（P＜0.05），and decreased a little in model 7d group（2.81±0.77 μmol/L）。（3） The apoptosis rate were（3.72±1.92%），（7.62±1.70%）and（10.73±2.26%）respectively，which is going up as time goes on.（4）The results of Western Blot analysis showed that there was almost no expression of the active form of Caspase-3 in the normal group.The level of Caspase-3 expression was significant in the 24h group and 7d group，while the latter decreased somewhat.Conclusion As time went on，the apoptosis rate increased and myocardial injury aggravated，and the main function of NO is anti-apoptotic in the model。

Primary Observation on Hyperglycemic Models in Mongolian Gerbils Induced by Different Dosage of Streptozotocin

LIU Feng-hua1，GUO Hong-gang2，LOU Qi2，DAI Fang-wei2，LU Ling-qun2，SA Xiao-ying2，ZHOU Guang-xing1

2010, 30(5):  335-339. 

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Objective To explore the proper dosage of streptozotocin (STZ) in the hyperglycemic models in the Mongolian gerbils induced by STZ. Methods The forty Mongolian gerbils were divided randomly into normal control group: A group intraperitoneally (i.p.) injected with acid citrate buffer solution and hyperglycemic groups: B、C、D、E、F group i.p. injected with STZ at the dose of 150、200、250、400、600 mg/kg body weight, respectively. The levels of blood glucose, the body weights and 24 h urine volume in two months were tested accordingly. Result Twenty-four hours after STZ injection, blood glucose concentration of the gerbils in C and D group are more than 16.65 mmol/L and shortly decreased at no less than 16.65 mmol/L since 72 hours after STZ injection, in the following 4 days，blood glucose rose up to 25 mmol/L，the nonfasting blood glucose level became relatively stable since 7 days after STZ injection in Mongolian gerbils, which were kept under observation for 2 months. These hypeiglycemic Mongolian gerbils in C group and D group which had higher achievement ratio, lower mortality and lower rate of spontaneous recovery had distict diabetic symptoms (Polyuria, polydipsia, hyperphagia and weight loss) with hyperglycemia during the experiment period. Seven days aller STZ injection, the achievement ratio of gerbils in B group was 62.5%(5/8) and the maintenance rate of hyperglycemia during the experiment period was 25%(2/8). The mortality of gerbils in E and F group were 83.30%(5/6) and 100% since 7 days after STZ injection. Conclusion The stable hyperglycemic models in the Mongolian gerbils can be induced by large dose of streptozotocin once?the standard of hyperglycemic model in the Mongolian gerbils is that either the fasting blood glucose or nonfasting blood glucose is above 16.65 mmol/L, the proper dosage of streptozotocin (STZ) may be between 200 mg/kg and 250 mg/kg.

Analysis on Animal Model of Human Viral Pulmonary Injury Induced by Highly Pathogenic PRRS Virus

LI Ji-da，XU Shou-zhen，WANG Jian-lin，YIN Yang-bo

2010, 30(5):  340-343. 

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Objective Histopathologic changes of lung from the pigs infected with highly pathogenic PRRSV was observed to explore the feasibility of pulmonary injury animal model induced by PRRSV.Methods Ten pigs of experimental group were infected with highly pathogenic NVDC-JXA1 PRRSV strain, and the lung tissues were collected for histopathologic observation. The pulmonary injury induced by highly pathogenic PRRSV and SARS was compared. Results Pigs of experimental group were all died after artificial infection of highly pathogenic PRRSV, and the typical changes of interstitial pneumonia can be found in lung tissue. There was a high similarity of pulmonary injuries induced by highly pathogenic PRRSV and SARS after comparison. Conclusion It was feasible that highly pathogenic PRRSV could be used as a model of viral pulmonary injury, which had a definite significance to study the pathogenesis of SARRS.

Research on Urinary Calculus Models on Rats Induced by Melamine

YANG Hua1，LIU Fang1，QIN Bo-yin1，Zhou Wen-jiang2

2010, 30(5):  344-348. 

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Objective In order to establish the animal model for research on the damage of melamine to urinary system, the effects of melamine at different dosages to rats were investigated .Method Sixty days old SPF SD rats were divided into 4 groups，one group was set as a blank control and fed with 10 g/L solution of methyl cellulose every day, the other 3 groups were treated with melamine at a level of 0.25, 0.5, 1.0 g/kg，respectively. All groups were administered continuously for 6 weeks. After 6 weeks， the kidney, ureter and bladder of anatomized rats from all groups were observed with both naked eye and stereo-microscope to determine their shape changes, and then the organ coefficients were calculated.Meanwhile, blood biochemical parameters of rats were measured and their kidneys were scanned by small live animals Micro-CT to evaluate the morphological changes. Results After 6 weeks’treatment of melamine, the average vesical and renal calculus formation rate was 66.6% (20/30) and 56.6% (17/30)，respectively. The kidneys from each experimental group were all bigger than that form control group and with distinguished greyish yellow color. There were yellow, cord-shaped sediments which integrate into flakes or granules in the kidneys, and arc-shaped yellow halo was formed between the cortex and medulla. Renal mass, organ indices and CREA, BUN, phosphorus and magnesium content in serum of rats from each treatment group were also significantly higher than that from control group (P＜0.01). Conclusion Part of the SD rats were induced with different degrees of urinary stones after 6 weeks’treatment with melamine at the level of 0.5～1.0 g/kg. Based on these data，the animal model for research on the damage of melamine to the urinary system was established.

Comparative Analysis on Renal Injury Relevant Indicators From Two Type 2 Diabetic Nephropathy Rats Models

WEN Hang-yuan1，CHEN Hong-yan2，YANG Xin-bo2，HUANG Zheng-ming3

2010, 30(5):  349-353. 

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Objective To compare two methods to establish type 2 diabetic nephropathy rats models and the renal injury indices. Methods Type two diabetic nephropathy rat model was established with three-unite-method (STZ，fat-diet, unilateral nephrectomy) and two-unite-method(STZ, fat-diet). Fast-ing blood glucose, insulin sensitivity index，NAG，AGEs，SCr，BUN，mAlb, kidney index were measured respectively and renal pathological changes were observed. Result ① Compared with normal control group, rats in three-unite-method group and two-unite-method group present hyperglycemia，insulin resistant, some characteristics of diabetic nephropathy and renal damnification was observed.② Compared with the fasting blood glucose，insulin sensitivity index，renal function of three-unite-method group，those in two-unite-method group were unconspicuously different. Conclusion Both three-unite-method and two-unite-method were the suitable methods to establish type 2 diabetic nephropathy rats model.

Monitoring on Cardiovascular and Physiology Characteristics of Beagle Dog by Telemetry

LI Na，WANG Wen-yu，XI Jian-fei，XU Hao，DING Shuang-shuang，ZHANG Zhou

2010, 30(5):  354-358. 

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Objective To monitor the heart rate、temperature、hemodynamics and electrocardio-gram of beagle dog by telemetry. Methods After two weeks of imbedding the sensor in abdomen muscle, the heart rate、temperature、hemodynamics and electrocardiogram were monitored continuously for 24 hours by Dataquest A.R.T. 2.3, and repeated on next week. Results In all of the monitor process, every index maintain the normal level, but the heart rate rise to the highest about 95.58±15.89 beat per minute at 10 ： 00 am, with the systolic pressure、mean arterial blood pressure and diastolic pressure were 152.37± 10.67 mmHg、109.37±5.90 mmHg and 87.73±5.01 mmHg respectively. No differences were found between the two detection(P＞0.05). Conclusions The cardiovascular cardiovascular and physiology Characteristics of Beagle dog could been monitored more exactly by telemetry, and the beagle dog was very excited on diet.

Effect of Long-term Transportation Stress on Autonomic Nervous Control of Heart in Two Strains of Wuzhishan Miniature Pigs

YANG Yu-wei，CHEN Min-li，PAN Yong-ming，CHEN Liang，HE Huan，WANG De-jun

2010, 30(5):  359-364. 

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Objective To observe the efiects of long-term transportation stress on autonomic nervous control of heart in two strains of Wuzhishan miniature pigs. Methods Electrocardiogram (ECG) and autonomic activities were recorded after long-lemi transportation stress for 21 h and recovered, irom conscious and unrestrained miniature pig by using a non-invasive telemetry system and the autonomic nervous ftinction was investigated by power spectral analysis of heart rate variability (ITRV). Results (I) It demonstrates an obvious circadian rhythm in Wuzhishan miniature pig, and all of results suggest that the parasympathetic nervous activity is predominant during dark phase, and sympathetic nervous activity is predominant during light phase in miniature pigs. The RR interval (RRI), the root-mean-square of successive differences (RMSSD)，short term variability (STV), very low frequency (VLF), low frequency (LF) and LF/HF in Black strain were significant lower than those of White strain (P＜0.05，P＜0.01), while heart rate (HR), long term variability (LTV), total power (TP) and high frequency (HF) were significant higher than those of white strain(P＜0.05, P＜0.01); (2) Compared with control group,RRI, SDNN, RMSSD, STV, LTV，TP，VLF, LF and LF/HF in wuzhishan miniature pig were significant decreased alter long-term transportation stress (P＜0.05，P＜0.01). while HR and HF were increased (P＜0.05，P＜0.01).The Black strain of RRI，RMSSD，STV, VLF, LF and LF/HF in the long-temi trans-portation stress group were significant lower than these of White strain (P＜0.05, P＜0.01), while HR,LTV，TP and HF were significant higher than these of White strain (P＜0.05, P＜0.01); (3) It has signifi-cant correlations between activity, heart rate and time and frequency domain indices (P＜0.05, P＜0.01).Conclusion It has significant difference on autonomic nervous control of heart between two strains of wuzhishan miniature swine induced by long-term transportation stress, and Black strain maybe easy to damage the balance. Moreover, long-term transportation stress can induce the sympathetic nervous activity decreasing，it maybe a major reason to cause of changing the biological function of miniature pig induced by long-term transportation stress.

Effect of 1,25(OH)2D3 on Myocardium in NOD Mice

ZHOU Zheng-yu，ZHOU Hui-ying，LI Xin-yin，WANG Yu-bing，XUE Zhi-mou

2010, 30(5):  365-367. 

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Objective To study the heart protective effect of 1,25(OH)₂D₃ on diabetic NOD mice Methods A total of 19 NOD mice were enrolled in the study, all of them were treated to be the model of diabetes by injecting CTX, and then randomly divided into 1.25(OH)₂D₃ group and untreated group.Other 4 BALB/c mice were set as normal controls. Myocardial morphology was observed in all three groups after 4 weeks. Results The Electronic microscopic findings showed that after the treatment of four weeks with 1.25(OH)₂D₃, the heart pathological changes were markedly reduced as compared with those diabetic mice untreated by 1.25(OH)₂D₃. Conclusion Treatment with 1.25(OH)₂D₃ can reduce the injury of the heart and prevent the pathological progress of structure of heart in NOD mice.

Comparative Study on Two Color Systems in Antibody Detection by ELISA

WANG Ji，WEI Li，YUE Bing-fei，HE Zheng-ming

2010, 30(5):  368-371. 

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Objective In order to reduce the harm of OPD(O-phenylenediamine) to the operators and pollution to environment, TMB (tetramethylbenzidine) was used as a color reagent to replace OPD in ELISA. Methods The operation was carried out on the basis of GB/T 14926.50-2001, to compare stability, specificity, sensitivity and repeatability of two color systems in detection of mouse hepatitis virus (MHV) antibody by ELISA. Result The TMB is more stable tiian OPD, the relative deviation (15.3%) is smaller than OPD(17.7%); the special antigen P/N value of TMB is higher than OPD and the difference is significant (P＜0.05); The working concentration of Enzyme of TMB is 0.25 times that of OPD, and the detection sensitivity is twice that of OPD; When using TMB, The inter-assay and the intra-assay average coefficient of variation of normal antigen and special antigen is 3.9%513.0% and 4.7%, 11.3% while the OPD is 2.7%, 11.2% and 4.5%,9.5%. Conclusion TMB is better than OPD in stability，reproduction, sensitivity and specificitys. TMB as a color reagent may replace OPD in detection of mouse hepatitis virus (MHV) antibody by ELISA.

Research on Pharmacodynamic Action of Guo Heng Extract

SHEN Long-hai1，AN Yong-tong1，MENG Qing-tao2，CAO Yu2，YAN Yong-gao1

2010, 30(5):  372-376. 

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Objective To observe the function of anti-inflammation, abirritation, activating blood circulation and alleviating soft tissue injuries of “Guo heng extract”. Methods The ear swelling of mouse induced by xylene, foot edema caused by carrageenan, the pain reaction of mouse by chemical stimulation, traumatic skin ccchymosis of rat and acute soft tissue injury model were adopted to investigate the treatment effects of the drug. Result The three dosage groups displayed significantly anti-inflam-mation and the analgesia function to ear swelling and foot edema of mouse, and writhing response mouse stimulated by acetic acid. The middle and high dosage groups could promote blood circulation and mitigate soft tissue injuries. Conclusion “Guo heng extract” had obvious anti-inflammation and the analgesia function. It also had the inhibitory action to skin ecchymosis and soft tissue injuries, and showed some dose-effect relationship.

Adequate Veterinary Care in Laboratory Animals

ZHOU Yan，WANG Jian-fei

2010, 30(5):  393-393. 

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With the development of laboratory animal science in biomedical research, more and more people pay attention to the care and use of laboratory animals, especially animal welfare. There are several components in an effective animal care and use program，among which, adequate veterinary care is considered internationally to be one of the most essential components. Many countries and non-governmental organizations have promulgated guidelines for adequate veterinary medical care for laboratory animals. The objective of this article is to introduce western notions of veterinary care to Chinese audiences，to identify the gaps between China and other countries on this aspect and to recommend the possible resolutions.