Table of Content

25 April 2017, Volume 37 Issue 2

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Therapeutic Effects and Mechanisms of Quercetin on Non-alcoholic Steatohepatitis in Rats

LI Jian-bo, YU Chen-huan, WANG Zhi-yuan, FAN Jie, YING Hua-zhong

2017, 37(2):  83-88.  DOI: 10.3969/j.issn.1674-5817.2017.02.001

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Objective To investigate the therapeutic effects of quercetin on non-alcoholic steatohepatitis (NASH) in rats, and the effects of quercetin on expressions of nuclear factor-κB (NF-κB). Methods The rats were fed with a high-fat diet for 6 weeks to induce NASH. The NASH model rats were given by gavage with quercetin 40-160 mg/kg for 4 weeks. The levels of serum total cholesterol (TC), triglycerides (TG), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured by the automatic blood chemical analyzer. The protein expressions of NF-κB in the hepatic tissues were detected by western blotting while tumor necrosis factor-α(TNF-α), interleukin-6 (IL-6) and interleukin-1β(IL-1β) mRNA by RT-PCR. Results Compared with control groups, the serum levels of LDL-C, TC and AST were decreased significantly in the quercetin-treated groups. Oral administration of quercetin improved hepatic steatosis and inflammatory cell infiltration effectively in NASH rats, reduced the mRNA expressions of TNF-α, IL-6 and IL-1β, and down-regulated the protein expression of NF-κB, with a dose-dependent trend which can be observed. Conclusion Quercetin had significant therapeutic benefits to prevent NASH through inhibiting hepatic lipid accumulation, that may be works through regulating NF-κB pathway.



Construction of Afp-cre-lacZ Transgenic Mouse Model

GU Xiao-wen, SUN Rei-lin, FEI Jian

2017, 37(2):  89-93.  DOI: 10.3969/j.issn.1674-5817.2017.02.002

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Objective To study the function of alpha-fetoprotein (Afp) positive cells during tissues growth and repair or in tumorigenesis, Afp-cre-lacZ linage tracing mouse model was established. Methods Afp-CreERT mice were established by microinjection and identified by real time PCR. Afp-CreERT mice were crossed with Rosa26-lacZ mice. Cre/lacZ both positive mice were kept as Afp-cre-lacZ mice. Results Fifty-six transgenic mice were born after microinjection. Four of them genotyped Cre positive were kept as founder. Each founder was established a strain. Cre expression of each strain was identified via real time PCR. Thus Afp-CreERT transgenic mouse was established. The Cre/lacZ both positive offspring of Afp-CreERT mice were crossed with Rosa26-lacZ mice were kept as Afp-cre-lacZ mice. Identified by real time PCR, X-gal staining and immunofluorescence, Afp-cre-lacZ mice were observed that they could be used in lineage tracing and further studies. Conclusions The Afp-cre-lacZ mice established may be used as a tool for lineage tracing studies.



Microscopical Observation on Pigment Cells in Fins and Scales of Zebrafish

LIN Jin-xing, FENG Li-ping, HU Jian-hua, GAO Cheng

2017, 37(2):  94-101.  DOI: 10.3969/j.issn.1674-5817.2017.02.003

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Objective To observe the composition, distribution and morphological characteristics of pigment cells in the fins and scales of zebrafish. Method The microstructure of pigment cells in the fins and scales of AB strain of zebrafish was observed by optical microscope. Results All fins in zebrafish distributed melanophores and xanthophores, without iridophores. According to the distribution of the pigment cells, the zebrafish fins were divided into two types: with or without zebra stripes. There were melanophores, xanthophores and iridophores existed in scales. The melanophores were mainly divided into two types: type I was larger, obviously dendritic and light color; type II was smaller, nodular, dark color. Xanthophores were the smallest, yellow or orange yellow. Iridophores were a long rod like or fusiform, with smallest number. Conclusion There were three types of pigment cells in the surface of zebrafish including melanophores, xanthophores and iridophores. The composition and distribution of pigment cells in the fins and scales were different. The types and morphological characteristics of pigment cells in the scales were more abundant than that in fins.



Impact of 4-Phenylimidazole Combined with Aluminum Hydroxide on Humoral Immune Response Induced by Hepatitis A Virus Attenuated Live Vaccine in Mice

MA Jing, WANG Hai-xuan, LI Si-jin, HE Hui, HU Ning-zhu, HU Yun-zhang

2017, 37(2):  102-107.  DOI: 10.3969/j.issn.1674-5817.2017.02.004

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Objective To investigate the impact of 4-phenylimidazole(4-PI) combining with aluminum hydroxide as an immunological adjuvant on humoral immune response in mice immunized with live hepatitis A virus (HAV) attenuated live vaccine (HepA-1). Methods Seven experiment groups are set up, with 7 ICR mice per group randomly. Use 1×phosphate buffer saline as negative control group, aluminum adjuvant group (HepA-1 200 μL+Al(OH)₃ 24 μL), antigen group (HepA-1), M4 (HepA-1+4-PI 1 mg) as control, in which mice were injected subcutaneously with 300 μL, immunizing one time. The mice of three groups (M1, M2, M3) were immunized with live hepatitis A vaccine HepA-1 mixed with aluminum hydroxide,4-PI at concentrations of 500 μg, 1 mg, 1.5 mg respectively. The anti-HAV specific IgG antibody levels were tested by indirect ELISA method in the serum of mice after the first 4th, 8th, 12th,16th week of injection. The health condition of mice were observed and record during the whole study. Results HAV-specific IgG levels of all mice were detected at four setting time point except negative control group and reached the maximum at 12th week. The IgG titers of group M2 mice were the highest at all detected time point and showed significant difference with those of antigen group (t=4.449, 3.633, 2.565, 6.809; P<0.05), group M4 (t=6.256,4.796, 4.153, 4.113; P<0.05) and aluminum group at 4th week (t=2.877, P<0.05). The IgGs induced by group M4 was comparable to the antigen group in each test phase. The optimal dosage of 4-PI was 1 mg for each mouse. The physiological indexes in all the groups of mice was normal during the course of experiment. Conclusions The humoral immune responses induced by HepA-l in mice was heightened by 4-PI combined with aluminum hydroxide,which had potential for developing a novel and compound HepA-1 adjuvant.



Effects of Indoleamine-2,3-dioxygenase Inhibitor Combined with Aluminum Adjuvant on Humoral Immune Response Induced by Hepatitis A Virus Attenuated Live Vaccine in Mice

HE Hui, LI Yan-han, LI Jian-fang, MA Jing, HU Yun-zhang, HU Ning-zhu

2017, 37(2):  108-112.  DOI: 10.3969/j.issn.1674-5817.2017.02.005

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Objective To investigate the compound effect of indoleamine-2,3-dioxygenase (IDO)inhibitor INCB024360 analogue combined with aluminum adjuvant on humoral immune response induced by hepatitis A virus (HAV) attenuated live vaccine(HepA-l) in mice. Methods The ICR mice were injected respectively with HepA-l (18EU) and Al(OH)₃ (300 μg) and three different doses (150 μg, 100 μg, 50 μg) of INCB024360 analogue as experimental group, 200 μL for each and one immunization. At the same time, set up the blank group, pure vaccine group, aluminum adjuvant control group and single INCB024360 analogue (100 μg) group. The sera from tail vein blood of mice were collected at the end of 4, 8, 12 and 16 weeks after immunization and the specific IgG antibody against HAV were detected by indirect ELISA method. Results In addition to the blank group, mice in various group generated anti-HAV IgG at 4, 8, 12, 16 weeks after the immunization. The antibody levels increased as time went on and reached peak at 12 weeks, then decreased gradually. The antibody level of compound adjuvant 2 group is the highest. At 8, 12 weeks, the antibody level was significantly higher than pure vaccine group, aluminum adjuvant control group and single INCB024360 analogue group (P<0.05). At 4, 8, 12, 16 weeks, the difference between the single adjuvant group and the simple vaccine group was not statistically significant. Conclusions The compound adjuvant of IDO inhibitor INCB024360 analogue combined with aluminum adjuvant can obviously enhance the HepA-l induced humoral immune response in mice, and the immunity enhancement effect is better than aluminum adjuvant control group. But the single INCB024360 analogue does not have significant adjuvant effect.



The Immunity Effect Evluation on Tree Shrew by Hepatitis A Virus Attenuated Live Vaccine, Hepatitis B Vaccine Independently or Combined Adjuvant Heparan Sulfate

WANG Dong-bao, HU Yun-zhang, HU Ning-zhu, HAN Yuan-yuan, KUANG De-xuan, LI Yan-han

2017, 37(2):  113-117.  DOI: 10.3969/j.issn.1674-5817.2017.02.006

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Objective To investigate the humoral immune response of tree shrew after the immunization of Hepatitis A attenuated live vaccine (HepA-l)、Hepatitis B vaccine (Hbv) independently and combined with adjuvant heparan sulfate (HS). Methods The tree shrews were randomly divided into 10 groups by the type of vaccine, adjuvant and schedules. The specific IgG levels in sera of tree shrew were determined by ELISA at 4, 8, 12, 16 and 20 weeks after the last immunization respectively. Results Except the blank control group, the anti - hepatitis A virus (HAV) and anti - hepatitis B virus (HBV) antibody was detected in all the experimental group at 4 weeks after last immunization, the level of immune antibody were gradually increased with time and reached the peak at the 12 weeks, then decreased gradually. At the same time, the humoral immunity effect of HS adjuvant group were better than that of vaccine group. There is a significant difference on immunity among tree shrews with different antigens and adjuvant by different immunization schedules. Conclusion The immune effect of vaccine immunogenicity and immune enhancing effect on tree shrew confirms the feasibility of the tree shrew as an animal model.

Analysis on Cyclooxygenase 2 Expression in Different Tissues from Spontaneous Diabetic Mongolian Gerbils

WANG Fei-fei, GONG Jing-jing, HUO Xue-yun, LU-jing, GUO-Meng, LIU-Xin, LI Chang-long, DU Xiao-yan, CHEN Zhen-wen, LV Jian-yi

2017, 37(2):  118-122.  DOI: 10.3969/j.issn.1674-5817.2017.02.007

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Objective To analyze Cyclooxygenase 2(COX-2) expression level in different tissues of diabetic gerbils and to explore the relationship of diabetes occurrence and development in Mongolian gerbils. Methods Different tissues including skeletal muscle, liver and kidney were collected from 6 diabetic gerbils and 6 control animals respectively. COX-2 mRNA and protein expression level in different tissues were separately detected by Real-time PCR and Western blotting. Results The Real-time PCR showed that compared with control animal, the mRNA expression of COX-2 in skeletal muscle had no obvious difference whereas it exhibited uptrend in liver and kidney. As the results of Western blotting, the difference of COX-2 in skeletal muscle was almost undetectable in diabetes group. The relative expression level in diabetic group exhibited uptrend compared with control in liver and kidney. And there was statistical significance in the kidney. Conclusions COX-2 was high expressed in liver and more remarkably in kidney of diabetic gerbils. It may illustrate that the effect of COX-2 mainly occurred in liver and kidney of diabetic gerbils.



Study on Infectivity of EV71 in Kidney Cells of Tree Shrew

WANG Wen-guang, KUANG De-xuan, YIN Bo-wen, LU Cai-xia, HAN Yuan-yuan, LI Na, TONG Pin-fen, SUN Xiao-mei, CAI Lu-kui, DAI Jie-jie

2017, 37(2):  123-129.  DOI: 10.3969/j.issn.1674-5817.2017.02.008

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Objective To investigate enterovirus 71 (EV71) infection characters on the tree shrew kidney cells. Methods The primary kidney cells from tree shrew were isolated by tissue culture. The cell was passaged and morphology was observed regularly. The indirect immunofluorescence identification was performed by using keratin 18. Tree shrew kidney cells were infected with EV71, cell lesions were observed by inverted microscope, viral load was detected by real-time PCR. Vero cells were used as control. Results Tree shrew primary kidney cells can be rapidly prepared by tissue culture and can be passaged easily. The kidney cells are mainly spindle-shaped and show paving - stones structure after adherent. They were identified as kidney epithelial cell by immunofluorescence assay. Tree shrew kidney cells can be infect with EV71 and showed significant cytopathic effect (CPE) such as rounding, falling off and lysis. The viral protein was observed by immunofluorescence. Viral load of was up to 10⁵ copies/mL. The CPE and proliferation trends were similar to that of Vero cell. Conclusion EV71 can infect tree shrew kidney cells under in vitro condition, and the cell assay can provide a platform for drug screening and infection mechanism research of EV71.



Primary Isolation, Culture, Purification and Identification of Corneal Epithelial Cells in Tree Shrew

MIAO Yu-run, SONG Qing-kai, KUANG De-xuan, CHEN Ling-xia, YIN Bo-wen, LI Xiao-fei, DAI Jie-jie

2017, 37(2):  130-135.  DOI: 10.3969/j.issn.1674-5817.2017.02.009

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Objectives To establish a stable technique for primary isolation, culture and purification and identification of tree shrew (Tupaia belangeri) corneal epithelial cells, and to provide a new experimental material for reserch of human ophthalmic corneal diseases. Methods The primary corneal epithelial cells from tree shrew were obtained by improved double digests method, the cells were purifed by method of cornea peeled off with transforming growth factor(TGF)-β inhibitors, achieve the purpose of identification by the method of immunofluorescence with keratin 3/12 antibody. Results The primary corneal epithelial cells from tree shrew with high activity and purity were obtained by double digests method. The corneal epithelial cells were purified food by TGF-β inhibitors and gradient digestion, and the purified cells was maintained a good epithelial cells shape in the subculture. The result of tree shrew corneal epithelial cells by immunofluorescence staining with keratin 3/12 monoclonal antibodies were shown positive. Conclusion An efficiency, simple and economic method was estabilshed for in vitro tree shrew corneal epithelial cells. The primary corneal epithelial cells from tree shrew with high activity and purity were obtained by double digests method, the subcultured cells was maintained a good epithelial cells shape. It will supply a new materials for eye diseases research .



Inhibition of Paclitaxel-Poly Lactic Acid Phosphate(PLCE) Microspheres on Growth of Ovarian Cancer Transplanted in Nude Mice

RAO Zi-liang, WANG Yi, WANG Nuo, XIONG Ren-qing, ZHANG MING-hao, LOU Cai-xia, KUANG Shao-song, TANG Xiao-jiang

2017, 37(2):  136-139.  DOI: 10.3969/j.issn.1674-5817.2017.02.010

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Objective To observe the antitumor effect of paclitaxel- novel copolymer of poly lactic acid phosphate (PLCE) microspheres on proliferation of ovarian cancer. Methods The human overiam cancer (HO8910) were implanted into the ovarian capsule of 68 nude mice. Three weeks later, 42 tumor bearing nude mice were randomly divided into 7 groups as follow, blank control group, negative control group, paclitarel injection group, paclitaxel-PLCE microspheres 1 high and low dose group, and paclitaxel-PLCE microspheres 2 high and low dose group, with 6 nude mice in each group. The drug was administered 1 time a day, and the growth of the tumor were continuously observed for one month. Results The tumor inhibition rate of paclitarel injection was 57.0%, and tumor inhibition rate of paclitaxel-PLCE microspheres 2 high and low dose group were 77.1% and 58.7% respectively. Conclusion Paclitaxel-PLCE microspheres have a strong inhibitory effect on HO8910 ovarian cancer transplanted in nude mice.



Effects of X-ray Irradiation with Different Dose Rates on Peripheral Blood Cell and Immune Organs in Mouse

YU Chun-miao, WANG He, ZHAO Li-song, MENG Dan, GUO Xu, YU Dong-hua

2017, 37(2):  140-143.  DOI: 10.3969/j.issn.1674-5817.2017.02.011

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Objective To explore the effects of different irradiation dose rate of X-ray on mouse peripheral blood cells and immune organ in mouse. Methods Totally 120 mice were randomly divided into 3 groups: the normal control group, low-dose rate irradiated group and high-dose rate irradiated group. The blood samples were taken respectively from mouse of each group at 24 h, 48 h, 96 h, 192 h after irradiation for detecting red blood cells (RBC), white blood cells (WBC), platelets (PLT) and hemoglobins (HGB), and the thymus glands and spleens were taken for calculating organ indexs and determining the content of superoxide dismutase (SOD) and malondialdehyde (MDA). Result The content of RBC, WBC, PLT and HGB in mice were decreased after the different radiation dosages of X-ray, but no significant difference was observed between the high-dose group and the low-dose group. At the same sampling time, a significant difference was observed on the thymus glands index and spleens index of the high-dose and low-dose mice compared with those of the normal control mice. There was a significant difference in the spleens index of the high-dose group and the low-dose group. The thymus glands index had a significant difference between the high-dose group and the low-dose group at the 24 h after irradiation. The contents of MDA were significantly increased, and the contents of SOD were significantly decreased in thymus glands and spleens of the high- and low-dose mice compared with the normal control mice. There was a significant difference in the content of MDA of thymus glands between the high- and low-dose mouse at the sampling time of 48 h and 96 h respectively, while the SOD was observed at the sampling time of 24 h, 48 h and 96 h respectively. There was a significant difference in the content of MDA of spleens between the high- and low-dose mice at sampling time of 24 h and 48 h respectively, while the SOD was observed at the sampling time of 24 h, 48 h and 192 h respectively. Conclusion No significant difference was observed on the effect of high- and low-dose irradiation on the peripheral blood cell in mice. The high-dose irradiation had more influence than low-dose irradiation on the spleen index, while the thymus glands index was not affected by irradiation. The high-dose irradiation had more influence than the low-dose irradiation on the SOD and MDA of the thymus glands and spleens in mice.

Application of Biochemical Markers on Analysis of Population Genetic Structure in KM mice

WANG Hong, WEI Jie, LI Xiao-bo, GONG Wei, YUE Bing-fei

2017, 37(2):  144-149.  DOI: 10.3969/j.issn.1674-5817.2017.02.012

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Objective To analyze the change of genetic structure of KM mice colonies between 1990 and 2010. Methods KM mice colonies (18 mice in 1990 and 50 mice in 2010) were monitored by biochemical markers under instruction of national standard GB/T 14927.1-2008. POPGENE 1.32 software was used to process the data. Results In 1990, average heterozygosity is 0.262 5, polymorphism information content is 0.206 5. In 2010, average heterozygosity is 0.161 5, polymorphism information content is 0.130 7. By compared with the two colonies, population differentiation coefficient is 0.122 4 and the genetic distance is 0.095 1. Conclusion After 20 years outdred, the KM colony has medium change of population differentiation , but still be considered as same strain from the point of population genetics.

Dynamic Assessment on Microenvironment of a New Type Exhaust Ventilation Closed-System Cage

WANG Gui-ping, XUE Zhi-mou, ZHOU Zheng-yu, WU Qiang, HUA Chen, WANG Jing

2017, 37(2):  150-154.  DOI: 10.3969/j.issn.1674-5817.2017.02.013

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Objective To dynamically assess the micro-environmental changes of the new type exhaust ventilated closed-system cage (EVC)for mice. Methods Based on the measurement of ventilation velocity and volume in EVC cages with mice, the air exchange rate were calculated. The parameters of noise, temperature, differential air pressure, relative humidity, micro-environmental ammonia concentration and macro-environmental ammonia concentration were detected continuously for a 7-day-cycle with wooden shaving bedding and for a 14-day-cycle with corncob bedding. Results The noise in two type EVC were 54.2±3.4 db and 55.6±4.1 db. The differential pressure between EVC and room were -23.1±9.2 Pa and -27.5±11.6 Pa respectively. Since the first day of caging change-cycle, the micro-environmental temperature, humidity and ammonia concentrations has raised gradually in cages. On the sixth day, ammonia concentration in two type of cages reached 1.28±0.08 mg/m³ and 1.33±0.15 mg/m³ respectively with wood shaving beddings, the temperature were 23.5±0.27 ℃ and 22.8±0.15 ℃ respectively, the humidity were 67.8%±2.2% and 70.2%±1.1% respectively. On the twelfth day, ammonia concentration in cages reached 1.95±0.46 mg/m³ and 2.17±0.33 mg/m3 with corncob beddings, the temperature were 23.8±0.4 ℃ and 24.1±0.2 ℃, and the humidity were 70.3%±1.8% and 69.6%±1.8% respectively. Conclusions Based on the conditions of current experiment, during 6-day and 12-day caging change-cycle respectively, the micro-environmental parameters, such as: noise, differential air pressure, temperature, humidity and ammonia concentrations met the GB14925-2010 requirements. EVC system with corncob bedding are highly recommended to maintain a stable micro-environment, that could be used in animal research and animal production.



Comparison on Periodic Acid Schiff Staining Effect on Mice Tissues with Three Different Fixatives

XU Yao-yao, LI Si-qi

2017, 37(2):  155-159.  DOI: 10.3969/j.issn.1674-5817.2017.02.014

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Objective Through comparing the slides qualities and periodic acid Schiff (PAS) staining effects with three different fixatives and fixed time on the normal liver, kidney, small intestine from mice, so as to select the optimum fixed methods. Methods The liver, kidney and small intestine tissue from adult male C57BL/6 mice were harvested, then fixed in three fixatives with different times respectively, the 4% paraformaldehyde fluid with 24 h, 48 h, 72 h, the Carnoy’s fluid with 4 h, 8 h, 12 h, and the Bouin’s fluid with 12 h, 24 h, 48 h. After fixed sufficiently, produced paraffin section and then stained routinely with PAS, to observe the staining effect under microscope. Result Different PAS staining results existed among three fixatives with different fixed time on three kind of tissues. The best staining result was observed in Carnoy’s fluid fixed for 12 h, the 4% paraformaldehyde fixed 48 h can basically met the criteria for observation. the staining result was relatively poor fixed in Bouin’s fluid. Conclusion PAS reaction was selective for fixative and fixed time. Carnoy’s fluid is an ideal fixative, but based on the fixed effect, repeatability, safety, and cost, 4% paraformaldehyde fluid can replace Carnoy’s liquid and widely used in PAS reaction.

Establishment of Rodent Model of Thyroid Diseases

TA La, JIN Shan

2017, 37(2):  160-165.  DOI: 10.3969/j.issn.1674-5817.2017.02.015

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This paper summarized the methods for establishing the rodent model of hyperthyroidism, hypothyroidism, autoimmune thyroiditis and thyroid carcinoma, and proposed a method for establishing the rat model of thyroid-stimulating hormone (TSH) suppression therapy for post-operation differentiated thyroid carcinoma.

Research Progress on Implementation of Laboratory Animal Welfare

YE Dong-yang, SUN Jing, LI Ri-fei, ZUO Ru-Nan, LI Yin-qian

2017, 37(2):  166-170.  DOI: 10.3969/j.issn.1674-5817.2017.02.016

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Animal experiments are indispensable means for biomedical research. Laboratory animals play an important role in the development of science. With the social development, people pay more attention on animal welfare. For the purpose of improving and implementing animal welfare, this article discusses the necessity of the implementation of the laboratory animal welfare, explicit its historical development, compares the current situation in our country with abroad, and discusses the influential factors of the laboratory animal welfare and its improvement measures, the main purpose is to let laboratory animal scientists put more effect on animal welfare, while ensure animal welfare through guide people to implement animal welfare.