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Table of Content

    25 August 2020, Volume 40 Issue 4
    Practice and Enlightenment of Management Mechanism of Laboratory Animals under Different Systems at Home and Abroad
    LIU Xiaoyu, LU Xuancheng, CHEN Hongyan, LU Shengming, LI Genping, HE Zhengming
    2020, 40(4):  263.  DOI: 10.3969/j.issn.1674-5817.2020.04.001
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    Based on the analysis of the development and formation of the management mechanism of laboratory animals under different systems at home and abroad, the advantages of the management mechanism implemented in China were explained, the existing shortcomings were found out, and the countermeasures and suggestions were put forward to improve and strengthen the management system and mechanism of laboratory animals in China.
    Small Ubiquitin-like Modifier Specific Protease 3 Regulates Autophagy in Mouse Alveolar TypeⅡ Epithelial Cells
    ZHU Huiqin, ZOU Yanqiong, YANG Jie, YI Jing, YANG Jie
    2020, 40(4):  270.  DOI: 10.3969/j.issn.1674-5817.2020.04.002
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     Objective     To explore in vivo the role and molecular mechanism of small ubiquitin-like modifier (SUMO) specific protease 3 (SENP3) in regulating autophagy in mouse lung. Methods   Immunofluorescence was performed to detect SENP3 localization in lung tissues. SENP3+/-       and SENP3+/+ mice were starved to induce autophagy. Immunoblotting was used to evaluate the overall autophagy level. Electron microscopy and immunofluorescence were used to detect autophagy, and to identify which type(s) of cells undergoing autophagy in mouse lung. Co-immunoprecipitation was used to detect the SUMOylation modification of autophagy-related molecule coiled-coil myosin-like Bcl-2-interacting protein 1 (BECN1). Results     SENP3 was highly expressed in alveolar typeⅡ epithelial cells in lung tissues. After starvation, the mouse alveolar type Ⅱ epithelial cells underwent autophagy, and SENP3+/- mice was more pronounced than SENP3+/+ mice. SUMO2/3 modification of BECN1 in lung tissue samples was removed by SENP3. Conclusion     SENP3 plays a role on regulating the degree of autophagy of mouse alveolar typeⅡ epithelial cells during starvation in fine-tuning manner.
    Single Nucleotide Polymorphism and Alternative Splicing Analysis between Fresh and Vitrified-thawed Crossbred Blastocysts of Yak via RNA-Seq
    ZHENG Jie, ZHANG Guozhong, HU Yanmei, WU Xueyi, YANG Zongfu, ZI Xiangdong
    2020, 40(4):  279.  DOI: 10.3969/j.issn.1674-5817.2020.04.003
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       Objective    To compare and analyze single nucleotide polymorphism (SNP) and alternative splicing (AS) between the fresh and vitrified-thawed crossbred blastocysts of the yak. Methods    The crossbred blastocysts were derived from yak oocytes in vitro fertilized with Jersey cattle spermatozoa. Total RNA was extracted from these fresh blastocysts (FRB) and vitrified-thawed blastocysts (VTB), and then amplified and sequenced via the Smart-Seq2 method to construct RNA libraries. SNP and AS were analyzed by SAMtools-0.1.19 and ASprofile software, respectively. Results    A total of 51 099 116 and 54 192 358 clean reads were obtained from the FRB and VTB, respectively. The SNP numbers of FRB and VTB were 116 681 and 224 750, the number of C/T type was slightly higher than that of A/G type for the FRB transition types of SNP (Ts). However, the number of A/G type was slightly higher than that of C/T type for the VTB Ts, and the number of A/T type was the least for all transversion types of SNP (Tv). The value of Ts/Tv in FRB and VTB were 2.57 and 2.45, respectively. The AS events in FRB and VTB were 49 388 and 65 241, respectively. It mainly had five patterns of AS, of which the two largest proportions were transcription start site and transcription terminal site. Conclusion     The results of comparison and analysis of SNP and AS between the fresh and vitrified-thawed crossbred blastocysts of the yak provide valid data and theoretical basis for the following related gene function analysis and mining.
    Establishment and Application of TaqMan Real-time PCR Assay for Detection of VP2 Gene of Aleutian Mink Disease Virus
    YAN Wenzhuo, LU Taofeng, ZHOU Jie, ZHAO Lili, CHEN Hongyan
    2020, 40(4):  289.  DOI: 10.3969/j.issn.1674-5817.2020.04.004
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     Objective    To establish a convenient and sensitive fluorescent quantitative PCR method for the detection of Aleutian mink disease virus (AMDV), aim to implement the rapid quantitative detection of AMDV in clinical samples. Methods     According to the conserved region of VP2 gene of AMDV, a pair of specific primers and a probe were designed. The reaction condition was optimized, and the TaqMan real-time fluorescent quantitative PCR detection method was established. The standard curve was mapped, and the specificity, sensitivity and stability of the method were analyzed. Results    The correlation coefficient (r2) of the standard curve was more than 0.994, and it presented a linear relationship in the concentration of the template DNA ranging from 102 copies/µL to 108 copies/µL. The assay was highly specific for amplification from AMDV, but no amplification from CPV, MEV, CDV and CAV. The sensitivity of AMDV detection was 102 copies/µL, and the intra-group and inter-group reproducibility tests showed that the variation coefficient of the method was less than 3%. The result of 417 clinical tissue samples tested by this method showed that the positive rate of AMDV was 81.5% in some areas of China. Conclusion     A reproducible, specific and sensitive AMDV real-time PCR detection method is established, which can be used for clinical detection and epidemiological investigation of AMDV. 
    Effect of Ubiquitin C-terminal Hydrolase 1 Gene Deletion on Reproductive System Development in Female Mice
    WU Bi, LIANG Shanshan, RUAN Jingling, HUANG Xin, TENG Xiaoming, HONG Ling
    2020, 40(4):  296.  DOI: 10.3969/j.issn.1674-5817.2020.04.005
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     Objective    To study the effect of ubiquitin C-terminal hydrolase-L1 (UCH-L1) gene deletion on the reproductive system of female mice. Methods     Vaginal smears, immunohistochemistry, Western blotting and RNA interference methods were used to analyze the effects of UCH-L1 gene deletion on the ovarian function and follicle development in mice. Results    The adult female mice with UCH-L1 deletion had lower body weight, dyskinesia and thinner uterus. No UCH-L1 protein expression was detected in the ovaries and uteruses of UCH-L1-deleted homozygous mice, and the ovary volume was significantly smaller than that of heterozygous mice. There were some primitive or closed follicles in the ovarian cortex, and there was no mature follicle and corpus luteum. The key reproductive receptors including estrogen receptor (ER), follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR) and progesterone receptor (PR) were expressed in the ovary of UCH-L1-deleted homozygous and heterozygous mice. The expression level of ER in the ovary of UCH-L1-deleted homozygous mice was significantly lower than that of heterozygous mice. By RNA interference, knocking down the expression of UCH-L1 in germinal vesicle (GV) oocytes could significantly reduce the maturation rate of oocytes. 
    Construction of Inhibin Gene Knockout Mice and Preliminary Analysis of the Phenotype
    HONG Shenghui, ZHANG Xuliang, WANG Qianqian, LIU Ping, LIU Diwen
    2020, 40(4):  306.  DOI: 10.3969/j.issn.1674-5817.2020.04.006
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    Objective    To construct an inhibin gene knockout mouse model by using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing technology, and to preliminarily study the phenotypes. Methods     Single guide RNA (sgRNA) plasmids against the exon 1 of inhibin α-subunit were designed and constructed. The sgRNA and Cas9 mRNA were transcribed by T7 RNA polymerase in vitro, then mixed and microinjected into the fertilized eggs of C57BL/6J mice. PCR and gene sequencing were used to detect the mutations of inhibin α-subunit in newborn mice. Male and female mice of F2 generation with inhibin gene knockout were selected, and their testis and ovaries were taken to observe and analyze by HE staining after paraffin sections. Results    Twelve F0 generation mice with inhibin gene knockout by CRISPR/Cas9 were obtained. The No.8 male mouse was selected to mate with female C57BL/6J mice, then F1 generation mice were achieved. F1 generation mice were mated with each other to produced F2 generation, and the ratio of genotypes conforms to Mendel’s law in F2 generation. The testis and ovary of F2 generation homozygous mice were cancerous and infertile. Conclusion    The inhibin gene knockout mouse model was successfully constructed, and the cancerous phenotype of homozygous mice shows that inhibin gene plays an important role in the mouse reproductive system.
    Establishment of Salt-sensitive Hypertension Model in C57BL/6J Mice
    YAO Ding, ZHOU Jing, YAN Guofeng, WANG Huiyang, WANG Yadi, MA Zhengwen
    2020, 40(4):  314.  DOI: 10.3969/j.issn.1674-5817.2020.04.007
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    Objective     To establish a mouse model of salt-sensitive hypertension, and to verify the effectiveness of the model using by nifedipine, an antihypertensive drug. Methods    C57BL/6J mice were divided into the normal control group, high-salt diet group and high-salt diet with nifedipine group. The blood pressure change of mice was measured by non-invasive blood pressure system (CODA system). The biochemical indexes of liver function, renal function, blood lipid, blood sugar and ions were measured by the blood biochemical analyzer, and the histological changes of the liver, kidney and carotid artery were observed after hematoxylin-eosin staining. Results    Compared with the normal mice, the systolic and diastolic blood pressure in the high-salt diet group increased significantly (both P<0.01), and those in the high-salt diet with nifedipine group decreased significantly (both P<0.01), and there were statistically significant differences. The liver function indexes (aspartate transaminase, alkaline phosphatase, albumin, total serum protein, and direct bilirubin), blood lipid index (cholesterol), renal function index (uric acid) and ionic levels (Ca, Mg) in high-salt diet group were significantly different from the control group, and those in the high-salt diet with nifedipine group were gradually recovered after nifedipine intervention. The pathological histology showed that the liver and kidney of the high-salt diet group had different degrees of injury. Conclusion    High-salt diet can successfully establish a mice model of salt-sensitive hypertension, and nifedipine can effectively lower the blood pressure of the model mice.
    Therapeutic Effect of Anti-complement 5a Receptor Antibody Combined with Allicin on Induced Inflammatory Bowel Disease In Rats 
    ZHOU Yani, LIU Dan
    2020, 40(4):  328.  DOI: 10.3969/j.issn.1674-5817.2020.04.009
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    Objective    To explore the therapeutic effect of  anti-complement 5a receptor (anti-C5aR) antibody combined with allicin on inflammatory bowel disease (IBD) in rats. Methods     Eighty IBD male rats induced by 2,4,6-trinitrobenzenesulfonic acid sol (TNBS) were randomly divided into 4 groups: model control group, anti-C5aR antibody treatment group, allicin treatment group and  anti-C5aR antibody combined with allicin treatment group, another 20 normal male rats were used as the normal control group. The changes in body weight and survival rate, serum interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) levels, colonic tissue gross observation and pathological score were measured before and 14 d after drug intragastric administration. Results   The body weight and  survival rate of the rats treated with anti-C5aR antibody combined with allicin were higher than those of other treatment groups, and the expression of inflammatory cytokines IL-6 and TNF-α in the serum of the rats treated with the combination of anti-C5aR antibody and allicin showed a significant regression trend with the time. The histopathological score of the colon also suggested that anti-C5aR antibody combined with allicin had the best effect (P<0.05) in the treatment of IBD. Conclusion     Anti-C5aR antibody combined with allicin is an effective treatment for TNBS-induced colitis, and its efficacy is better than anti-C5aR antibody or allicin alone.
    Inhibting Effects of Cytomegalovirus on Partial Immune Index of Mice Implanted with Methacrylate
    JIANG Rong, ZHANG Qimei
    2020, 40(4):  332.  DOI: 10.3969/j.issn.1674-5817.2020.04.008
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     Objective    To study the effect of cytomegalovirus (CMV) on the immune response after polymethyl methacrylate (PMMA) implantation. Methods    A mouse model of CMV infection was constructed by intraperitoneal injection. After 7 days of inoculation, PMMA was implanted into the subcutaneous of back in mice. The proportion of monocytes / macrophages, CD4+T cells, CD8+T cells and regulatory T cells (Treg cells) in peripheral blood was detected by flow cytometry, and the concentrations of tumor necrosis factor-alpha (TNF-α) and interleukin -1β (IL-1β) were detected by enzyme-linked immunosorbent assay (ELISA). Results    Compared with the implantation control group, the proportion of M1 mononuclear / macrophages in peripheral blood decreased by 73.18% (t=5.896, P=0.004), the proportion of M2 monocytes / macrophages increased by 2.18 times (t=7.971, P=0.001), and the CD4+/CD8+T cell ratio decreased by 42.91% (t=6.468, P=0.003), while the Treg cell proportion increased by 2.49 times (t=4.495, P=0.011). Compared with the implanted control group, the concentrations of TNF-α and IL-1β in peripheral blood of implanted CMV group were reduced by 71.65% and 57.95% (t=7.236, P=0.019; t=7.543, P=0.002), respectively. Conclusion    The CMV can significantly inhibit the immune response after PPMA implantation, which provides theory evidence for the treatment of CMV-infected patients after oral biomaterials implantation.
    Distribution of Adenovirus Type 5 in C57BL / 6J Mice Infected by Different Routes
    CHEN Weisheng, GAO Yaqi, LIANG Ning, CHEN Haiyu, CHEN Xingzhi, ZHAN Chunlie, ZHANG Xiuyan
    2020, 40(4):  334.  DOI: 10.3969/j.issn.1674-5817.2020.04.010
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    Objective    To detect the distribution of adenovirus type 5 (Ad5) in C57BL / 6J infected by four different routes. Methods    One hundred and twelve C57BL / 6J mice were divided into 4 groups, each mouse was given 100 µL of Ad5 solution (1 × 109 PFU / mL) by intranasal drip, intragastric administration, tail vein injection and intraperitoneal injection.  On the 1 st, 2 nd, 3 rd, 4 th, 5 th, 6 th and 7 th day after infection, two males and two females in each group were sacrificed for the real-time PCR quantitative detection of Ad5 content in lymph nodes, testis, epididymis, uterus, ovary, spleen, kidney, liver, lung, heart, brain, blood and bone marrow. Results    Ad5 was detected in lung and spinal cord tissue in intranasal drip group, the content of Ad5 in lung decreased mostly 4 days after infection; Ad5 was not detected in all tissues in intragastric administration group; Ad5 was detected in liver and spleen in tail vein injection group, and the decline rate was slow; Ad5 was detected in spinal cord, spleen, epididymis and testis in intraperitoneal injection group, the content of Ad5 in the spinal cord was about 10 times higher than that in the intranasal drip group, the content of Ad5 in spleen was similar to that in tail vein injection group, and the content of Ad5 in epididymis and testis was only 1% ~ 10% of that in other positive tissues. Conclusion    The lung infection test of Ad5 can be carried out by intranasal drip once every 3 days, the liver infection test by tail vein injection, the spleen infection test by tail vein injection or intraperitoneal injection, and the spinal cord infection test by intraperitoneal injection.
    Practice and Exploration on Laboratory Animal Welfare and Ethical Reviewing and Monitoring
    LU Shuangshuang, SHI Xiaomeng, LIU Xiaoyu, SUN Deming, LI Xiaoyan, LU Xuancheng
    2020, 40(4):  339.  DOI: 10.3969/j.issn.1674-5817.2020.04.011
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     Many policies, regulations and standards related to laboratory animals have been issued in China with more than 30 years of development. The understanding of the laboratory animal welfare has been deepened in researchers. The operation and management of the Welfare and Ethics Committee have become more and more standardized. The welfare of laboratory animals can be guaranteed with the high quality management of the Welfare and Ethics Committee. Based on the work experiences, searching the literature and peer exchange, the authors described the common problems found in the oversight and ethical review process, and proposed solutions for improving the experimental animal welfare.
    Research Progress in Depression-like Rat Model Induced by Chronic Unpredictable Mild Stress
    ZHAO Qian, WANG Anna, GAO Xuesong, LI Li, ZHAO Jingjie
    2020, 40(4):  344.  DOI: 10.3969/j.issn.1674-5817.2020.04.012
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    Depression is a kind of mental illness with typical symptoms of depression, slow thinking, and reduced speech and action, which can cause suicide behavior in serious cases. At present, chronic unpredictable mild stress (CUMS) has been widely used in depression research, and its physiological and pathological changes are similar to human depression. This paper reviewed the modeling methods, model evaluation and pathological changes of CUMS, and thought about the above aspects.
    A Review of Modified Right Heart Catheterization for Measuring Pulmonary Artery Pressure in Rats
    DONG Zhengwei, FAN Guanwei
    2020, 40(4):  354.  DOI: 10.3969/j.issn.1674-5817.2020.04.013
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    As the golden standard for measuring pulmonary artery pressure, the right heart catheter method needs to undergo three important physiological bends to reach the pulmonary artery. In order to measure pulmonary artery pressure rapidly and effectively in rats, it is necessary to improve the material, tip angle and corresponding operation of the right cardiac catheter. In this paper, the modified right heart catheterization method to measure pulmonary artery pressure in rats is summarized in order to provide references and ideas for the researchers in this field.