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Table of Content

    25 April 2020, Volume 40 Issue 2
    The Effects of Bone Morphogenetic Protein 4 Overexpression on Proliferation and Chondrogenesis of Rabbit Bone Marrow Stem Cells
    ZHANG Fei, DANG Yuan, XUE Laien, WEN Fuli, ZHENG Heping, HU Deqing
    2020, 40(2):  87-94.  DOI: 10.3969/j.issn.1674-5817.2020.02.001
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    Objective To investigate the effects of bone morphogenetic protein 4 (BMP-4) overexpression on the proliferation and chondrogenesis of rabbit bone marrow stem cells (BMSCs) infected by adenovirus. Methods The BMSCs were isolated and cultured by whole bone marrow adherence method, the purity of BMSCs was improved by subculture, the cells were identified by cell morphology and surface molecular marker by flow cytometry, and the optimal multiplicity of infection (MOI) of adenovirus was detected by inverted phase contrast microscopy and flow cytometry. The effects of BMP-4 overexpression on the secretion of type Ⅱ collagen (COLL Ⅱ), sry-related high mobility group-box gene 9 (Sox9) and BMP-4 in chondrocytes were analyzed by enzyme linked immunosorbent assay (ELISA) and Western blotting. The effect of BMP-4 overexpression on the proliferation of BMSCs in vitro was detcted by cell counting kit-8 (CCK-8). Results Primary cells were round, elliptical and spindle-shaped. The second generation cells were typical fibroblast-like long spindle-shaped, vortex-like distribution, with low expression of hematopoietic stem cell surface molecular markers CD34(1.94%) and CD45(2.13%), and high expression of CD44(98.2%), CD29(99.7%) and CD90(98.8%). MOI for 100, with higher infection efficiency and smallest damage to cells, is the most appropriate MOI. Compared with the control group, the overexpression of BMP-4 could significantly promote the proliferation of BMSCs and secrete a large number of COLL Ⅱ、Sox9 and BMP-4 proteins (P<0.05). Conclusion Overexpression of BMP-4 can significantly promote the proliferation and differentiation of BMSCs into chondrocytes in rabbit.
    Establishment of Real-time Fluorescent Quantitative PCR Method for Mouse Hepatitis Virus and Application in Some Rodents
    WANG Ji, WANG Shasha, FU Rui, WANG Shujing, LI Wei, QIN Xiao, HUANG Zongwen, LI Xiaobo, GONG Wei, YUE Bingfei, HE Zhengming
    2020, 40(2):  95-103.  DOI: 10.3969/j.issn.1674-5817.2020.02.002
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    Objective To establish a real-time fluorescent quantitative PCR (Q-PCR) method for detection of mouse hepatitis virus (MHV) in naked mole-rats and other rodent animals. Methods The specific primers and probe were designed and synthesized by selecting conserved regions of MHV E gene to establish the Q-PCR method. Then the linearity, specificity, sensitivity, repeatability and stability of this method was verified. Meanwhile, the established method was used to detect 79 clean grade mice, 63 naked mole-Crats, 35 SPF mice, 10 Mongolian gerbils, 20 golden hamsters and 4 ground squirrels for MHV. Results The real-time fluorescent quantitative PCR method for MHV was successfully established. The linear range of the method was 101 copies/μL to 109copies/μL. The method has no cross reaction with Sendai (SV), reovirus type 3 (Reo3), pneumonia virus of mice (PVM), bovine coronavirus (BCV).The detection sensitivity could reach 10 copies/μL, the results of repeatability and stability showed that the coefficient of variation between experiments was less than 5%.The positive rate of MHV in 79 clean grade mice was 22.78%, and that of 63 naked mole-Crats, 35 SPF mice, 10 Mongolian gerbis, 20 golden hamsters and 4 ground squirrels were all negative. Established method was compared with RT-PCR, the coincidence rate was 97.47%. Conclusion The established Q-PCR method for MHV is good in linearity and specificity, it can be used for the detection MHV in multiple varieties rodents.
    Intraperitoneal Injection in Different Dosages of Cisplatin to Establish a Rat Model of Premature Ovarian Failure Induced by Chemotherapy
    Dong Ruoxi, Zhu Xiaodan, Fan Bozhen, Xu Wenjuan
    2020, 40(2):  104-109.  DOI: 10.3969/j.issn.1674-5817.2020.02.003
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    Objective To investigate a method for establishing a rat model of chemotherapy-induced premature ovarian failure (POF) in rats by intraperitoneal injection with different dosages of cisplatin. Method Fourty-eight unfertiled female Wistar rats were randomly divided into 6 groups, with 8 rats in each group. Different doses of cisplatin was injected intraperitoneally at different time points incluing: ① control group (group A) without treatment, ② cisplatin intraperitoneal small dose injection group 1 (group B) (1.5 mg/kg once daily for 5 days), ③ cisplatin intraperitoneal small dose injection group 2 (group C) (2.0 mg/kg once daily for 5 days), ④ cisplatin intraperitoneal small dose injection group 3 (group D) (2.5 mg/kg once daily for 5 days), ⑤ cisplatin intraperitoneal high dose injection group 1 (group E) (4 mg/kg once in a week, after a week, re-injection, on total for twice) and ⑥ cisplatin intraperitoneal high dose injection group 2 (group F) (4 mg/kg once in a week, after a week, re-injection for 6 mg/kg, on total for twice), to establish a rat model of chemotherapy-induced injury POF. The general condition, estrous cycle, body weight changes, serum anti-Müllerian hormone (AMH) levels, ovarian pathological changes, and number of follicles at various levels were observed. Result Compared with the control group (group A): the estrous cycle of the rats in each group was significantly disordered, the body weight and serum AMH levels were significantly decreased (P<0.05), in which, the group E was decreased most obviously. The number of follicles in the ovary of each group was also significantly decreased (P<0.05), but the number of follicles in the groups E and F was more significantly decreased. Conclusion Rats can be injected intraperitoneally with 4 mg/kg cisplatin once a week, at intervals of one week, for two weeks. This model method of chemotherapy-induced POF is more effective, and the method of modeling is simple.
    Effects of Ozonated Autohemotherapy on Melatonin and Oxidative Stress in Rats with Sleep Deprivation
    SHUI Yunhua, ZHANG Lan, LEI Guanglei, ZHU Yuanjiang, LI Peiyu
    2020, 40(2):  110-115.  DOI: 10.3969/j.issn.1674-5817.2020.02.004
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    Objective To observe the effects of ozone major autohemotherapy on melatonin and oxidative stress in rats with sleep deprivation, and to provide theoretical basis for the treatment of ozone major autohemotherapy. Methods Forty adult male Wistar rats were selected as the study subjects. A rapid eye movement (REM) sleep deprivation model was subsequently developed using the “modified multi-platform sleep deprivation method”. Rats were randomly divided into the control group and the observation group according to the principle of random number table, with 20 rats in each group.The rats in the observation group were given autologous blood transfusion after ozone treatment, and the control rats were given air instead of ozone. Serum melatonin levels were measured at tail vein blood at 0 h, 24 h, 48 h and 72 h after intervention. The venous blood oxidative stress index [superoxide dismutase (SOD), glutathione peroxidase (GSH-px) and malondialdehyde (MDA)] levels were measured at 0 h and 72 h after intervention. Results There was no significant difference in wake (W), slow-wave sleep (SWS), and fast-wave sleep (FWS) time between the two groups of rats at the 0 h intervention (P>0.05). At 72 hours of intervention, W time in the observation group was shorter than that in the control group, and SWS and FWS time were longer than those in the control group. The differences were statistically significant (P<0.05). At 48 h and 72 h after intervention, the serum melatonin levels in the observation group were higher than those in the control group (P<0.05). At 72 h after intervention, the levels of SOD and MDA in the observation group were lower than those in the control group, and the GSH-px level was higher than that in the control group (P<0.05). Conclusion Ozone major autohemotherapy can promote melatonin release in rats with sleep deprivation, decrease serum SOD level and MDA level, and increase serum GSH-px level in rats.
    Protective Effect and Mechanism of Nanoliposomal Quercetin on Focal Cerebral Ischemia-reperfusion Injury in Rats
    KANG Yu, YANG Xiaofang
    2020, 40(2):  116-122.  DOI: 10.3969/j.issn.1674-5817.2020.02.005
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    Objective To study the neuroprotective effects and mechanism of nanoliposomal quercetin (nLQ) on focal cerebral ischemia-reperfusion injury in rats. Methods Eighty male SD rats were randomly divided into sham-operation group, model group, nLQ low-dose group, nLQ middle-dose group and nLQ high-dose group, with 16 rats in each group. The middle cerebral artery occlusion model was established by modified suture method. Fourty-eight hours after cerebral ischemia-reperfusion, the behavioral score was observed by using neurological deficit score method, the cerebral infarction volume was measured by TTC staining test, the water content of brain tissue was measured by wet and dry weight method, the contents of tumor necrosis factor α (TNF-α) and interleukin-10 (IL-10) in serum were determined by ELASA assay, the neuronal apoptosis was detected by flow cytometry, and the expressions of Bcl-2 and Bax in brain tissue were detected by Western blotting. Results Compared with the model group rats, nLQ low, middle and high dose groups could significantly improve the neurological deficit symptoms, reduce the behavioral score, decrease the cerebral infarction volume and alleviate cerebral edema, reduce the contents of TNF-α and IL-10 in serum, reduce the rate of neuronal apoptosis, decrease Bax protein expression and increase Bcl-2 protein expression in brain tissue. The differences were statistically significant (P<0.05 or P<0.01), also with a dose-dependent manner. Conclusion The nLQ has a significant protective effect on the focal cerebral ischemia-reperfusion injury in rats. The nLQ may improve the neurological function, reduce the apoptosis of neuronal cells and decrease the content of inflammatory factors TNF- and IL-10 in blood.
    High-fat Diet Induced Cynomolgus Monkey Model of Non-alcoholic Fatty Liver Disease
    GAO Shiping, LI Feng, ZHA Sifan
    2020, 40(2):  123-127.  DOI: 10.3969/j.issn.1674-5817.2020.02.006
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    Objective To establish non-alcoholic fatty liver disease (NAFLD) animal model induced by high-fat diet, and to analysis the effect of high-fat feeding time for animal NAFLD formation, exploring the correlation between feeding time of high-fat diet and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG), total cholesterol (TC), high-density lipoprotein (HDL), low density lipoprotein (LDL), fatty liver, and fibrosis formation. Methods Before the experiment, basic blood samples were collected from 700 healthy cynomolgus monkeys, then fed with high-fat diet, some of the monkeys were sampled at 1, 2, 3, 4, 5, 6 and 7 years respectively after being fed with high-fat diet and anaesthetized with ketamine hydrochloride, and the blood were collected for detection of ALT, AST, TG, TC, HDL and LDL indicators, and liver tissue biopsy was performed. Results After 2 years of high-fat feed, the serum ALT, AST, TG, TC and LDL levels of cynomolgus monkeys were significantly increased (P<0.01), and HDL level was significantly decreased (P<0.01). The feeding time of high-fat feed was significantly correlated with ALT, AST, TG, TC, liver adipose and fibrosis at the level of 0.01 (bilateral) (r=0.127, 0.121, 0.246, 0.128, 0.306, 0.220), but negatively correlated with HDL (r=-0.298, P<0.05), and significantly correlated with LDL at the level of 0.05 (bilateral) (r=0.081). Histopathology showed that serious fatty degeneration and balloon-like degeneration occurred in the liver over time, fatty liver followed by hepatitis and fibrosis. Conclusion The high-fat diet can significantly increase blood lipid and liver enzyme activity indexes in cynomolgus monkeys, the liver lipid aggregation and inflammatory infiltration are obvious, indicating that high-fat diet can successfully induce NAFLD model and promote the development of NAFLD.
    Analysis of Incidence of Spontaneous Histopathological Lesions in Young SD Rats
    HUANG Jisheng, WU Shuyi, ZHAN Jinhe, NI Qingchun
    2020, 40(2):  128-135.  DOI: 10.3969/j.issn.1674-5817.2020.02.007
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    Objective To study the incidence of spontaneous histopathological lesions of young SPF SD rats at different growth and development stages, to provide information for drug nonclinical safety evaluation especially for pediatric drugs. Methods The samples were collected from SD rats in day 51 (PND51), day 65 (PND65), day 111 (PND111) and day 139 (PND139) after born as control groups in safety evaluation studies in recent 2 years. The pathological sections and histomorphological features of SD rats were observed under an optical microscope, and the incidence of pathological changes was also observed. Result The spontaneous lesions of young SPF SD rats at different times were analyzed and reported. The high incidence lesions occurred mainly in heart, liver, kidney and other organ lesions. The incidence and levels of spontaneous histopathological lesions of liver, heart and kidney were increased and developed with the rats growing. Conclusion The spontaneous lesions and their incidence in young SPF SD rats in drug toxicology research can provide some reference for relevant scientist.
    Pathological Mechanism of Conjunctival Injury in Diabetic Model Rabbits
    GE Jian, ZHU Caihong, WU Yanlin, WU Yongjie
    2020, 40(2):  136-140.  DOI: 10.3969/j.issn.1674-5817.2020.02.008
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    Objective To analyze matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases (TMPs) in the conjunctival tissue cells of diabetic rabbits, and to analyze the relationship between MMPs and TMPs. Methods Twenty Japanese white rabbits were randomly divided into 2 groups, the rabbits in control group were fed with the normal experimental rabbit diet, while the diabetic experiment group was feed with high fat and high sugar diet for 8 weeks, and then injected with streptozourease by auricular vein for establishing diabetic model rabbits. Two weeks after the model was made, two groups of rabbits were anesthetized with general anesthesia, and one piece of 2 mm×3 mm bulbar conjunctiva tissue of each eye was sampled respectively. The right conjunctiva was used for pathological observation, and left eye conjunctiva was used for detecting the MMP-1, MMP-3, MMP-9 and TMP-1, TMP-3. Results The expressions of MMP-1, MMP-3, MMP-9 in the experiment group were high compared with those of the control group (P<0.05), and the expression of TMP-1 in the experimental group was slightly higher compared with that of the control group (P<0.05), but TMP-3 was significantly lower (P>0.05). In the diabetic experimental group rabbits, the conjunctival epithelium was arranged in order, subepithelial tissue was obviously reduced, conjunctival tissue was loose, atrophic fascia tissue was observed, the cells of the lamina propria of conjunctiva were loose, and the inflammatory cells between the cells were increased. Discussion Break down of the balance between MMPs and TMPs expression may produce elastic fibers in bulbar conjunctiva, and pathological changes of hypoconjunctiva tissue, the fascia atrophy changed the microenvironment of diabetic rabbit.
    Observation of Pulse Wave Velocity and Arterial Wall Structure in Spontaneously Hypertensive Rats at Different Ages
    GUO Jing, GU Wenyong, WU Yongjie
    2020, 40(2):  141-143.  DOI: 10.3969/j.issn.1674-5817.2020.02.009
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    Objective To reveal the physiological changes of blood vessels in different growth stages of spontaneous hypertensive rats (SHR) by measuring the pulse wave velocity (PWV) and analyzing the structure of aortic media cross-sectional area (MCSA) in the same period of SHR. Methods Color Doppler ultrasonography and vascular pathological analysis were used to determine PWV and MCSA of SHR at 4, 6, 8, 10 and 12 month of age. Results The PWV of 12-month-old SHR was significantly higher than that of 8-month-old, 6-month-old and 4-month-old SHR respectively(P<0.05). The PWV of 10-month-old SHR was significantly higher than that of 6-month-old and 4-month-old SHR respectively (P<0.05). Meanwhile, the MCSA analysis of SHR showed that the aortic MCSA of SHR increased gradually with age. Conclusion With the increase of age, the arterial compliance of SHR is worse, the vascular elasticity is decreased, and the degree of atherosclerosis is enhanced.
    Analysis of Nutritional Components and Amino Acid Composition of SPF Golden Hamster Milk
    FAN Fangling, XIA Shuang, GAN Lu, XIA Fang
    2020, 40(2):  144-148.  DOI: 10.3969/j.issn.1674-5817.2020.02.010
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    Objective To determine the main nutritional components of SPF golden hamster milk and compare with other rodents milk (mice, rats, guinea pigs), rabbit milk, cow milk and infant formula milk. Methods According to the national standard, the essential nutrients of golden hamster milk were detected by Kjeldahl and ICP-MS. The content of amino acids was detected by an automatic amino acid analyzer. Results The main nutrient contents of 100 g golden hamster milk were as follows: water 86.4 g, protein 6.94 g, fat 2.70 g, lactose 2.21 g, mineral elements calcium 120.00 mg, magnesium 20.10 mg, phosphorus 82.05 mg, zinc 1.470 mg, etc. The total content of hydrolyzed amino acids was 61mg/g, and the content of essential amino acids was 24.4 mg/g, accounting for 0.4 of the total amount of amino acids. Conclusion The milk of golden hamster is characterized by high protein, low fat and low lactose, which provides a scientific basis for the formulation of sterile golden hamster formula milk.
    A Brief Introduction About Key Points of Management in ABSL-2 Laboratory
    YANG Hua, ZHAO Yong, SONG Zhigang, LI Shun, ZHOU Xiaohui
    2020, 40(2):  149-153.  DOI: 10.3969/j.issn.1674-5817.2020.02.011
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    Laboratory animals represent the important tools which were widely used for medical researches. The biosafety of laboratory animals and animal experiments have provided key base of the development of biomedical science and technology for human society. Currently, the animal biosafety level 2 (ABSL-2) laboratory are widely used in the fields of life sciences and medical researches. This review introduces the key points of management of ABSL-2 laboratory, including the structure designing, the administrational approving, the facility running, the management of human resources, the experimental operation, the emergency handling, and how to setup effective biosafety management system to avoid the leakage of bio-hazardous factors, to improve the animal laboratory biosafety level.
    Summary of the Information Management System of Laboratory Animal in Colleges and Universities
    LI Wei, CHEN Xiaojuan, KE Xianfu, WANG Lie
    2020, 40(2):  154-158.  DOI: 10.3969/j.issn.1674-5817.2020.02.012
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    In view of the complex situation of the management of the laboratory animal in colleges and universities, this paper analyzes the key elements of the management, explores the information management system suitable for the experimental animal in colleges and universities, gives full play to the standardization and intelligence of the management, and provides guarantee for the teaching and scientific research in colleges and universities. By introducing the design ideas and functional modules, this paper summarizes the new laboratory animal information management system .
    Research Progress on Animal Models of Osteoarthritis
    XIA Mengxiong, HAN Haihui, LIANG Qianqian, ZHAI Weitao, XU Hao
    2020, 40(2):  159-165.  DOI: 10.3969/j.issn.1674-5817.2020.02.013
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    There are plenty of animal models of osteoarthritis (OA). Researchers all over the world are trying to identify different pathogenic characteristics with different animal models. Focusing on primary OA and posttraumatic OA, this paper utilizes the genetically modified models and the experimental induction models to analyze their advantages and disadvantages, to support the research of the pathogenesis, the change and the process, and the outcomes of OA.
    Comparison of Microbiological and Parasitological Monitoring of Rats and Mice
    TAO Lingyun, ZHOU Jie, HU Jianhua, GAO Cheng
    2020, 40(2):  166-172.  DOI: 10.3969/j.issn.1674-5817.2020.02.014
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    This article introduces common health monitoring recommendations for microbiological and parasitological detection of mice and rats in China and abroad. The content includes the monitoring items, methods and frequency. The authors put forward some suggestions for the solution of problems in infectious agents to monitor of mice and rats in China.