Table of Content

25 June 2017, Volume 37 Issue 3

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Preliminary Establishment and Research on Form Deprivation Myopia Model in Tree Shrew

YANG Dong-mei, ZHU Qin, LI Na, GUO Li-yun, ZHANG Xiao-fan, ZHANG Jie-ying, HU Min, DAI Jie-jie

2017, 37(3):  171-178.  DOI: 10.3969/j.issn.1674-5817.2017.03.001

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Objective To establish the adolescence and early adulthood form deprivation myopia (FDM) model in tree shrew and to observe the retinal morphology, in order to explore the role of age in the development of myopia and the effection of local retinal mechanism for FDM. Method Thirty tree shrews at age of 4 months and 5 months without congenital myopia and other eye diseases were respectively selected. All tree shrews were randomly divided into: control group, and cover group. In the cover group, right eyes served as the experimental eye, left eye as control eyes. Experimental eye were covered with handmade semi-translucent film. then measure the diopter and ?axial?length of tree shrews after being covered for 3 weeks and 6 weeks. The retinal thickness and the number of cells in each layer of retina were observed by electron microscopy after being covered for 6 weeks. Results The tree shrews were born 4 months and 5 months form deprivation after 3 weeks, hyperopia was alleviated but not statistically significant compared with control eyes, and two groups of tree shrews cover eye diopter and eye axis are different obviously compared with control eyes after 6 weeks. At deprivation period, axial continue to extend and gradually increase to myopia, and they have a good negative linear relationship. Form deprivation can lead to thinning of the retina of the tree shrew, can also lead to number decrease on photoreceptor cell layer, inner nuclear layer, and ganglion cell layer cells. Conclusion Form deprivation can induce myopia formation and retinal morphological change in adolescence and early adulthood tree shrews.



Antagonistic Effect of Selenium on Change of Mitochondrial Membrane Potential of NRK-52E Cells Induced by Sodium Fluoride

CHANG Kai, WANG Yu, PANG Wen-biao, GAO Ji-ping, CHEN Zhao-yang, SONG Guo-hua

2017, 37(3):  179-184.  DOI: 10.3969/j.issn.1674-5817.2017.03.002

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Objective To study effects of fluoride on the change of mitochondrial membrane potential in rat renal tubular epithelial cells (NRK-52E cells). Methods The experiments were divided into 4 groups: the control group, the fluoride group, the selenium group and the selenium intervention group.NRK-52E cells were cultured with fluoride and selenium respectively in various concentration, and fluoride and selenium combination for 48 h. (5 mg/L or 20 mg/L NaF、0.0171 mg/L or 0.0342 mg/L Na₂SeO₃). The mitochondrial membrane potential were detected by flow cytometry. The activity of enzymes and the content of free radicals were examined by relevant reagent kit, such as superoxide dismutase(SOD) reagent kit. Results Compared with the control group, the mitochondrial membrane potential decreased (P<0.05 or P<0.01) and SOD, total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-PX) and catalase (CAT) enzyme activities decreased (P<0.05) with the increase of the concentration of sodium fluoride.The mitochondrial membrane potential of the selenium intervention group was increased (P<0.01) compared with that of the fluoride exposed group.Compared with the fluoride group of 20 mg/L, the relative selenium intervention group SOD, T-AOC, GSH-PX and CAT activities were increased (P<0.01). Compared with the selenium group of 0.0171 mg/L, the relative selenium intervention group the mitochondrial membrane potential decreased (P<0.01) and SOD, T-AOC, GSH-PX and CAT enzyme activities decreased (P<0.05 or P<0.01). 5 mg/L the fluoride group compared with the selenium group, the mitochondrial membrane potential changed significantly (P<0.01). But for SOD, T-AOC, GSH-PX and CAT activities ,there were no significant changed. 20 mg/L the fluoride group compared with the selenium group, the mitochondrial membrane potential changed significantly (P<0.01). SOD, T-AOC, GSH-PX and CAT activities also changed significantly (P<0.01). Conclusion The certain concentration of selenium could induce the decrease of mitochondrial membrane potential in NRK-52E cells induced by fluoride, that may be achieved by restoring the activities of activities.



Effect and Mechanism of Ubiquitin-specific Peptidase 19 on Muscle Atrophy of Chronic Obstructive Pulmonary Disease Induced by Cigarette Smoke Exposure in Rats

LIU Qian, LIU Song, XU Wei-guo, GUAN Si-bin, GUO Xue-jun

2017, 37(3):  185-190.  DOI: 10.3969/j.issn.1674-5817.2017.03.003

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Objective To investigate the effects of ubiquitin-specific peptidase 19 (USP-19) on rats bearing chronic obstructive pulmonary disease (COPD) induced by cigarette smoke (CS) exposure. Methods Rats exposed to chronic CS was chosen for the study. For histological examination, lungs and quadriceps femoris muscle were stained with hematozylin and eosin. Total RNA and protein were extracted for Real-time PCR and Western blot analysis to assess the MHC, USP-19 and MAPKs gene expression. Results Twelve weeks CS exposure produced lung lesions that morphologically resembled human emphysema, leading to the enlargement of alveolar ducts. Skeletal cell numbers per high-power (HP) lens increased after 12 weeks by 40% in comparison with the control group, suggesting muscle wasting. Chronic CS exposure decreased the mRNA level of MHC. MHC protein content in the quadriceps femoris muscle was decreased in the 8- and 12-week groups. CS significantly stimulated phosphorylation of ERK1/2, p38 without altering the total ERK1/2, p38 content. Conclusions Cigarette smoke-induced skeletal muscle atrophy is associated with up-regulation of USP-19, which via MAPKs probably.



Comparison on Hepatocarcinoma Model Induced by Diethylnitrosamine in Apc-mutant Rat and F344 Rat

XIE Bei, ZHAO Lei, SUN Jing, ZHANG Li-juan, KURAMOTO Takashi, WEI Hu-lai

2017, 37(3):  191-197.  DOI: 10.3969/j.issn.1674-5817.2017.03.004

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Objective To establish hepatocarcinoma models induced by diethylnitrosamine (DEN) in F344 rats, KAD (Kyoto Apc Delta) rats and compare the advantages and disadvantages between them. Methods Twenty-five KAD rats were randomly divided into 2 groups. The 5 rats in control group were given the normal water. The 20 rats in experimental group were given the water containing 40 μg/mL DEN for 5 weeks, and the remaining 15 weeks they were given the normal water. Every 4 weeks, part of KAD rats was sacrificed to undergo pathological examination and make pathological sections of liver tissues and tumor tissues. Twenty-five F344 rats were treated the same as KAD rats. Results Hepatocarcinoma could be induced successfully in KAD rats and F344 rats. The same pathological developing process was found in them, which was similar with human. The average of 1.00±0.82 grey-white lesions could be found in 3 KAD rats (3/4) when they were induced in the 16th week, while no lesions could be found in F344 rats (0/4) at that time. Until the end of the experiments (20 weeks), 3.50 ±1.29 cancers and gray-white lesions had been developed in KAD rats, while only 1.25±0.96 could be found in F344 rats. The success rate of hepatocarcinoma induction in KAD rats was better than that in F344 rats significantly (P<0.05). Conclusion Comparing to F344 rats, KAD rats could be a better hepatocarcinoma induction model, with shorter induction time, higher sucess rate and numbers of hepatocarcinoma. KAD rat may be an ideal model for dynamically researching the hepatocarcinoma induction.



Research on Reovirus III (Reo-3) Infection in ICR Mice

LUO Yin-zhu, ZHANG Yu, HE Li-fang, HUANG Bi-hong, WU Rui-ke, Min Fan-gui, PAN Jin-chun, YUAN Wen, WANG Jing, Guo Peng-ju, HUANG Ren

2017, 37(3):  198-203.  DOI: 10.3969/j.issn.1674-5817.2017.03.005

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Objective To establish reovirus type 3 ( Reo-3) infection model of ICR mice and observe clinical syndrome, investigate virus load in target organs and serology dynamic change in infected mice. Method Animal adaption method was used to increase the virus’s virulence. Systemic infection model was established with tail vein and intraperitoneally inoculation to ICR. The clinical sign was observed. The tissues and serums were harvested on 0 d before and 4 d、7 d、18 d、25 d、35 d、72 d、129 d after inoculation. Blood samples of three mice were randomly collected on 47 d、81 d、103 d. qPCR and ELISA were respectively used to detect the virus nucleic acid at target tissue and the antibody level. Results Virus virulence was increased after four times adaption trial. Mice death was observed at d7, d9, d10, d11, d16 post inoculation. The highest load of the virus was found in liver through qPCR, followed by heart, spleen, and lung. The peaks of viral load emerged from 6 to 11 d in all organs, negative results appeared at 129 d in the most organs. The earliest antibody detect time was on 18 d. The titer of antibody hit the peak on 25 d, then maintained a high level till to 129 d. Conclusion virus virulence can be enhanced significantly through mouse in-vivo inoculation, multiple organs inflammation and early death in mice can be identified by the systemic infection way of Reo-3. This experiment provides an approach to establish Reo-3 infected mice animal model and provides good experimental data for further research on Reo-3 infection mechanism .



Development of Detection Method for Sendai Virus by Nucleic Acid Sequencing

ZHANG Huan-huan, YU Chen-huan, DAI Fang-wei, MA Yue, LANG Ju-lia, WANG Yu, YING Hua-zhong

2017, 37(3):  204-208.  DOI: 10.3969/j.issn.1674-5817.2017.03.006

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Objective To develop the method for detection of Sendai virus by nucleic acid sequencing. Methods General primers according to Sendai virus genome sequences were designed and optimized to sequencing primers. Then the best condition of creating library was established. This method was verified by nucleic acid sequencing and analysis. Results A pair of highly specific general primers was designed. The sequences are respectively 5'-GCTGCAAAACGCTGTGGG-3' and 5'-TGGRACYT-CAGAAAGAATRGG-3', and the sequencing primers were established by ligated adaptor sequences to the 5'-end of general primers. The established condition of creating library and library products were obtained by the first round amplification using general primers and the second round using sequencing primers. Sendai virus was effectively distinguish from other organisms by nucleic acid sequencing and analysis, and was precised to TianJin strain. Conclusion A detection method was developed for Sendai virus by nucleic acid sequencing, which may be as the base for microbiological detection in laboratory animals.

A Comparison on Hyperlipidemia Rat Models Induced by High-fat Diet with Different Proportions of Sugar and Oil

QIN Hui-yan, WANG Shao-long, WEN Ping-jing, ZHAO Peng, LI Bin, ZHANG Jie-hong

2017, 37(3):  209-213.  DOI: 10.3969/j.issn.1674-5817.2017.03.007

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Objective To compare hyperlipidemia rat models induced by high-fat diet with different proportions of sugar and oil. Methods Fourty rats were divided into normal control group and three high-fat diet (I, II and III) groups with 10 rats per each. The normal control group was fed with normal diet, and the other three groups were fed with high-fat diet with different proportions of sugar and oil for 6 weeks respectively. The body weight and the total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) in serum were detected weekly. Results No significant difference were observed on body weight among different groups. The TC, LDL-C and TC/HDL-C ratios in all high-fat diet groups were higher than those of normal control group at each time point. The TG in high-fat diet II group was higher than normal control group at each time point, but was only higher at the first 4 weeks in high-fat diet I group, and no significant difference in high-fat diet III group except at second week. Conclusion Both high-fat diet I and II can induce combined hyperlipidemia rat models rapidly and steady, and high-fat diet II is more superior. High-fat diet III may be applied to induce hypercholesterolemia in rat.

The Sub-chronic Toxicity of Sodium Chloride in Rats

LI Guang-xian, LIU Xiang-mei, LIU Dong-hong, LIU Yin, SUN Xia, CHEN Han-jin, GUO Xin-dong, HUANG Yu-feng

2017, 37(3):  214-219.  DOI: 10.3969/j.issn.1674-5817.2017.03.008

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Objective To explore the sub-chronic toxicity of sodium chloride on rat by oral gavage. Methods Eighty SPF SD rats were randomly divided into control, low-dose, middle-dose and high-dose groups with half male and half female. The treatment groups were exposed to sodium chloride (low-dose group with 100 mg/kg, middle-dose with 500 mg/kg and high-dose group with 1 000 mg/kg) and the control group were exposed with pure water by oral gavage for continuous 90 days. Clinical observations, daily diet consumption and body weight of rats in each group were recorded during exposure. After exposure completed, the rats were anesthetized and blood samples were collected from abdominal aorta to test the routine blood indexes, serum biochemistry and plasma electrolytes. The main organs were dissected and weighed to calculate the organ coefficients. Histopathology changes of main organs were observed by microscopy. Results There were no difference in body weight, blood routine index, daily ration and organ coefficients between all groups (P>0.05). Serum biochemistry examination showed that, compared with the control group, the ALT and AST were decreased (P<0.05) while the TBIL and calcium content were increased (P<0.05) in middle-dose and high-dose groups. In the pathological examination of rats, pathological changes of spongy degeneration, congestion, necrosis and infiltration of liver were found in some rats in middle-dose and high-dose groups. And some male rats in middle-dose and high-dose groups also found renal?tubular calcification, thymic hemorrhage and bleeding tendency of lymph nodes. No changes of biological significance were found in the serum biochemistry examination in the control and low-dose group. No pathological lesion can been seen in the control and low-dose group. Conclusion The sub-chronic toxicity of sodium chloride by oral administration can cause blood circulatory disorder and renal deposition of calcium salt in male rat.

Composition and Diversity of Fecal Microflora in SPF Chickens at Different Growth Stages

ZHOU Yan, DIAO Chen-xi, ZHANG Yuan-yuan, YU Hai-bo, LU Tao-feng, ZHAO Li-li, CHEN Hong-yan

2017, 37(3):  231-239.  DOI: 10.3969/j.issn.1674-5817.2017.03.013

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Objective To provide the composition and diversity of intestinal flora at different growth stages of SPF chickens. Methods The feces of SPF chickens at 10 d , 12 weeks , 23 weeks and 52 weeks old were collected. The two hypervariable regions (V3-V4) of the 16S rRNA of the samples were sequenced using the Illumina HiSeq 2500 platform, and the alpha diversity, beta diversity and fecal community characteristics were analyzed. Results The fecal flora sample at four time points had similar high abundance and good uniformity and there was no significant difference in species diversity between the two groups (P>0.05). More than 95% of abundance were found in Firmicutes, Proteobacteria and Bacteroidetes; Lactobacillus, Streptococcus, Bacteroides, and Enterococcus were dominant species, and the abundance of different periods were significantly different (P<0.05). Conclusions The feces flora of SPF chiken at different growth stages are diverse, and the dominant bacteria are mainly beneficial bacteria. The experimental results are expected to provide the basis for assessing the quality, health monitoring and safety evaluation of SPF chickens.



Preparation of Goose Parvovirus VP3 Antiserum and Identification of Propagation in Duck Embryo and Duck Embryo Fibroblasts

NIU Yin-jie, LIU Bai-han, ZHAO Li-li, SUN Chang, CHEN Hong-yan

2017, 37(3):  240-243.  DOI: 10.3969/j.issn.1674-5817.2017.03.014

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Objective To prepare antiserum against VP3 protein and propagate goose parvovirus (GPV) H in duck embryo and duck embryo fibroblasts (DEFs). Methods The prokaryotic expression vector pET30a-VP3 were constructed based on VP3 seqeucne of GPV H. VP3 protein was induced, expressd and purified, then immunized the New Zealand white rabbit. The VP3 antiserum was collected, purified and verified by Western blotting. GPV H fluid was inoculated into shaoxing duck embryo and DEFs, and the propagation of GPV was identified by PCR and indirect immunofuorescence. Result The VP3 antiserum was successfully prepared, and GPV H was well propagated in shaoxing duck embryo and DEFs. Conclusion VP3 antiserum preparation and GPV replication in duck embryo and DEFs may be provided basis for GPV molecular biological characteristic and pathogenic mechanism research.



Differential Expression Analysis of miR-200b-3p and miR-200b-5p in Marek’s Disease Resistant and Susceptible SPF Chickens

WANG Rui-qi, LIAN Chuan-jiang, YI Cheng, HAN Ling-xia, YANG Chun-wen, CHEN Hong-yan

2017, 37(3):  244-248.  DOI: 10.3969/j.issn.1674-5817.2017.03.015

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Objective To validate high throughput sequencing results by fluorescence quantitative PCR technique and analyze the expression of miR-200b-3p and miR-200b-5p in bursa, and explore possible molecular mechanism associated with Marek’s disease (MD) resistance/susceptibility. Methods Through optimization, the ideal amplification conditions and reaction system of real-time fluorescence quantitative PCR were identified. Results miR-200b-3p and miR-200b-5p fluorescence quantitative PCR results and high throughput sequencing results were consistent in down-regulation, but not significant difference. miR-200b-3p and miR-200b-5p two homologous miRNA showed similar expression after infected by Marek’s disease virus (MDV). Conclusion Although the difference is not consistent with the results of significant analysis, miR-200b-3p and miR-200b-5p may be associated with resistance / susceptibility of MD.

Application of PCR Method for Rapid Sex Determination in SPF Jinding Duck

XU Li-jing, SUN Chang, LU Tao-feng, CHEN Hong-yan, ZHAO Li-li

2017, 37(3):  249-251.  DOI: 10.3969/j.issn.1674-5817.2017.03.016

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Objective To determine the sex of SPF Jinding Duck, the universal primer gCHD of avian chromosomal DNA-binding protein 1 (CHD1) gene on avian chromosomes were synthesized. Methods Blood of 85 SPF Jinding Duck were collected and genomic DNA was extracted for PCR amplification. The sex of the birds were determined by analyzing the number of bands after gel electrophoresis. Results The CHD1-Z and CHD1-W were found in the PCR products of agarose gel electrophoresis from females and CHD1-Z was from males. Conclusions The PCR can be used as an effective method for sex identification in birds.



Advances in Research on Relationship between Transporter Associate with Antigen Processing Gene Polymorphism and Diseases

WANG Xing-tong, CHEN Hong-yan, HAN Ling-xia

2017, 37(3):  252-256.  DOI: 10.3969/j.issn.1674-5817.2017.03.017

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Transporter associate with antigen processing (TAP) is a heterodimer which plays a crucial role in antigen presenting process by transporting endogenous antigen peptide from the cytoplasm to the endoplasmic reticulum where can be bound by the major histocompatibility complex (MHC) class I molecules and further be recognized by CD8+T lymphocyte. Polymorphisms of TAP gene and the expression level of TAP protein are associated with susceptibility to certain specific diseases including tumor, autoimmune disease, viral disease and infectious diseases. Here, some similar comparative medicine researches of TAPs in laboratory animals to diseases associations are reviewed.