Table of Content

25 April 2019, Volume 39 Issue 2

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Endoscopy Guided Tracheal Injecting Bleomycin Model of Pulmonary Fibrosis in Mice

YU Hua-jun, WU Shang, HUANG Hui, LIN Bi-yun, WU Jun, OU Hua-jun, ZHANG Hai-tao

2019, 39(2):  94-98.  DOI: 10.3969/j.issn.1674-5817.2019.02.005

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Objective To establish intratracheal intubation under direct vision of multifunctional endoscopic, and adopted to establish the model of bleomycin induced pulmonary fibrosis in C57BL/6J mice. Methods Establish method and steps of endotracheal intubation under direct vision of multifunctional endoscopic with five C57BL/6J mice. Ten mice were randomly divided into two groups (5 in each). Control group were given 50 µL of saline and experimental group were given 50 µL of bleomycin (3.5 mg/kg) to observe the death rates of mice. At the 28th day, the mice were sacrificed by cervical delocation. By compared the vitality of superoxide dismutase (SOD), glutathione peroxidase(GSH-PX), the content of malondialdehyde (MDA) and hydroxyproline (HYP) in the two groups of mouse lung tissue. Lung tissue fibrosis degree were observed by HE and Masson stain. Results All C57BL/6J mice were successfully intubated by using multifunctional endoscopy under direct view and no mice died after intubation. The 5 mice given India ink had black materials evenly distributed from trachea bottom until the lungs. Compared with those of control group, HYP and MDA contents in mouse lung tissue experimental group elevated(P<0.01), while SOD and GSH-PX level in mice lung tissue decreased (P<0.01). The lung tissue was obtained and stained by HE and Masson. By comparison with control group, collagen deposition in experimental group mouse lung tissue is serious. The pathological changes were in accordance with pulmonary fibrosis. Conclusion The method of endotracheal intubation under direct vision of multifunctional endoscopic is safe, effective and convenient. This method was successful to establish pulmonary fibrosis in mice model with high efficiency induced by bleomycin, and worthy of popularization and application.



Establishment and Comparison of Rat Models of Insulin Resistance Combined with Hyperuricemia

LIU Meng-yuan, FENG Xue-xuan, LI Li-si, ZHANG Mei-yi

2019, 39(2):  99-104.  DOI: 10.3969/j.issn.1674-5817.2019.02.006

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Objective To prepare successful, stable and optimized insulin resistance combined with hyperuricemia (IR-HUA) models, the preparation methods of IR-HUA model, HUA model and IR model in rats were compared. Method Totally 40 male SD rats were randomly divided into the normal control group (A), the HUA model group (B), the IR-HUA model group (C), and the IR model group (D). The group A and group B were fed with the ordinary feed and drinking water, the group C and group D were fed with the high fat feed and high sugar diet and drinking 10% fructose water. During 8 weeks for modeling, at sixth weeks, rats in group B and group C were fed with yeast extract (16 g/kg/per day) 21d, At the end of sixth, eighth week, the observation index were detected. Result At the eighth weekend of the experiment, compared with group A, the concentrations of serum uric acid (SUA) and urine uric acid (UUA), total uric acid excretion and the fraction excretion of uric acid (FEUA) in group B increased; The levels of fasting insulin (FINS) and HOMA-IR index, and urination volume in group C decreased; the concentration of UUA and FEUA in group C increased; the levels of Lee 's index, FINS, urination volume,and total uric acid excretion in group D declined, the concentrations of fasting plasma glucose(FPG), UUA, serum creatinine (SCr) and uric creatinine (UCr) in group D increased. Conclusion HUA model could be prepared in SD rats by fed with yeast extract 16 g/kg/per day for 21d. The IR model in SD rats induced by high fat and high sugar diet for 8 weeks was not ideal, it could be due to the shorter molding time. The rat model showed no obvious characteristics of IR. The preparation method of IR-HUA model needs to be further improved. In addition, high-fat and high-sugar diet can reduce the volume of urine and the excretion of uric acid in rats, and reduce the secretion of insulin in rats, leading to the increase of FPG.



Isolation, Identification and Subculture of Pulmonary Fibroblasts in Tree Shrew

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

2019, 39(2):  105-110.  DOI: 10.3969/j.issn.1674-5817.2019.02.007

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Objective To establish a reliable method for isolation, identification and subculture of pulmonary fibroblasts in tree shrew. Methods Pulmonary fibroblasts of neonatal tree shrew were isolated and cultured by tissue adherence culture and trypsin digestion respectively. The fibroblasts was observed by inverted microscope and identified by immunofluorescence with Vimentin. Then the pulmonary fibroblasts was subcultured, and cryopreservation and recovery experiments were carried out. The cell growth curve was drawn by MTS method, and human embryonic lung diploid fibroblasts (KMB-17) were used as control. Results Tree shrew pulmonary fibroblasts could be obtained by both tissue adherence culture and trypsin digestion. The cells were mainly spindle-shaped, and vimentin immunofluorescence assay were generally positive, which were consistent with KMB-17. Tree shrew pulmonary fibroblasts can be subcultured 4-5 times continuously. After cryopreservation and recovery, the cells can still remain viable and enter the logarithmic growth phase about 3 days later with a cell density of 5×10⁴ cells/mL. Conclusion The pulmonary fibroblasts in tree shrew can be effectively obtained by tissue adherence and trypsin digestion, which will be very helpful for the study of lung-related diseases in vitro.



Mouse Genetic Quality Monitoring Method Establishment Based on Next-generation Sequencing through Multiplex PCR

QIAN Qiang, XU Yuan, WANG Ya-heng, ZHOU Yu-xun, XIAO Jun-hua, HAN Ling, BAO Shi-ming, LI Kai

2019, 39(2):  111-117.  DOI: 10.3969/j.issn.1674-5817.2019.02.008

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Objective To establish a multiplex PCR targeting next-generation sequencing for mouse genetic quality monitoring. Methods Firstly, 112 single nucleotide polymorphisms (SNPs) on 20 chromosomes were screened from common inbred mice. Then, multiplex PCR amplification was performed on the fragments near the SNPs. After the library was constructed, high-throughput sequencing on Illumina platform was performed. The data was analyzed in a bioinformatics pipeline to obtain SNP information. Results The sequencing results showed that the uniform depth of amplicon was obtained, and the success calling rate of each site was over 90%. Secondly, under high specificity and high-depth sequencing conditions, the allele ratio of SNP sites approached 1 or zero, which is consistent with homozygous conditions. After 4 batches of mouse samples (98 in total) were analyzed, the proportions of SNP sites successfully identified were 99.82%, 92.00%, 99.10% and 90.35%, respectively. All mouse individuals were found to be homozygous and were successfully identified as the target strain. Compared differences between each pair strains, the maximum number of difference was 73, the minimum number was 3, and the average number was 53. The median number of difference was 60, showing our approach has a higher resolution for common inbred mouse strains. Conclusion The SNP typing scheme of the multiple PCR protocol is an accurate, rapid and efficient genotyping program for genetic quality testing and strains identification.



Effects of Sugar Concentration Control on the Longevity and the Mid-gut Stem Cells of female Drosophila Melanogaster

TANG Run-dong, SONG Si-yuan, WU Wei

2019, 39(2):  118-123.  DOI: 10.3969/j.issn.1674-5817.2019.02.009

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Objective To assess the effects of sugar concentration control on the longevity and the mid-gut stem cells of female Drosophila Melanogaster and then to find a optimum eating habits for women. Methods Detect the variance of body weight, survival rate, longevity and mid-gut stem cell’s form with gradient sugar concentration treatment (the sugar concentration in normal medium as 100%). Results With the increase of the age, all the fly groups’ body weight raised slowly first and then rapidly after 21 days. Body weight in the high sugar concentration treatment group increased faster than that of low sugar concentration treatment group. With the experiments of gradient sugar concentration treatment after high sugar concentration treatment, the optimum sugar concentration is 50%. With the extended of the treat time, the survival rate increased rapidly after 14 days. All the groups’ survival rates were increasing with the gradient sugar concentration treatment after high sugar concentration treatment group. Drosphila in the 50% sugar concentration treatment group had the highest survival rate. With the sugar concentration control, all the fly groups’ average longevity raised and the optimum sugar concentration was 50%. With the extended of the treat time, all the groups’ mid-gut stem cell had large morphological changes. They had be rescued by the sugar concentration control and the optimum sugar concentration was 50%. Conclusion The sugar was a important nutrient for D.melanogaster. It had large effects on D. melanogaster’s body weight, longevity and mid-gut stem cell. The body weight decreased and the longevity increased with the diet control. And they could rescue the damage on mid-gut stem cell.



Phototoxic Effects of Ultraviolet Irradiation with Different Time Course on Skin of SD Rats

SUN Xia, LIU Xiang-mei, PANG Zeng-xiong, LIU Dong-hong, XU Ying-yu, JIANG Yi, LI Min, QIU Zhi-feng, HUANG Yu-feng

2019, 39(2):  124-130.  DOI: 10.3969/j.issn.1674-5817.2019.02.010

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Objective To compare the skin phototoxicity damage of SD rats induced by ultraviolet radiation at different radiation time. Methods The skin phototoxicity damage rat models were established by ultraviolet radiation of UVA (4.5mJ·cm^-2·s^-1)+UVB(0.036 mJ·cm^-2·s^-1). The models were irradiated twice a week for 4 weeks, and the cumulative radiation time of model 1, model 2 and model 3 were 342 min, 440 min and 520 min, respectively. At the end of the experiment, the skins were histopathologically observed, as well as the expression of melanoma specific marker (HMB45), Vimentin and endothelial cell marker (CD34) in skins were examined by immunohistochemistry. Results Compared to normal control, there showed partial hyperkeratosis of epidermis, uneven thickness of epidermis, deepening of wrinkle, enlargement of hair follicle, and hyperplasia of sebaceous gland in model 1, model 2 and model 3. In addition, with the increase of radiation time, the epidermal lesions were more serious, the skin thickness and sebaceous gland cross-sectional area were larger, and the skin injury scores were higher. Compared with normal control, the expressions of Vimentin of skin flbroblast and CD34 of Dermal capillaries increased tremendously in model 1, model 2 and model 3, and the increasement were more obviously in model 2 and model 3. At the same time, the expressions of HMB45 of skin increased distinctly in model 2 and model 3. Conclusion With the extension of ultraviolet radiation time, the degree of skin damage is gradually increased. At the same time, this study may provide an appropriate animal model for evaluating the efficacy of sunscreen.



Effects of X-ray Irradiation with Different Dosages on Immune Organs in Mouse

YU Chun-miao, FU Jia-qi, ZHAO Li-song, GUO Li-dong, GUO Xu, YU Dong-Hua

2019, 39(2):  131-135.  DOI: 10.3969/j.issn.1674-5817.2019.02.011

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Objective To explore the effects of different irradiation dosages of X-rays on mouse immune organs. Methods Totally 192 mouse were randomly divided into 6 groups: the normal control group and single irradiation 2.0 Gy group, 3.0 Gy group, 4.0 Gy group, 5.0 Gy group and 6.0 Gy group. The samples were taken respectively from mouse of each group at 24 h, 48 h, 96 h, 192 h after irradiation,and the thymus glands and spleens were weighed and taken for calculating organ index and determining the content of superoxide dismutase (SOD) and malondialdehyde (MDA). Results At the same sampling time, the thymus glands and spleen index and the content of SOD in each dose group were significantly less than control group, The content of MDA was significantly higher than control group. The trends of change in thymus glands and spleen index of the mouse in each group increased and decreased first at the same irradiation dosages and different sampling time. The content of MDA in thymus glands increased with the prolongation of sampling time, and the content of MDA in spleen was a trend of reducing the rise first. The content of SOD in thymus increased first and then decreased with the prolongation of sampling time. The content of SOD in the spleen of irradiated 2.0 Gy, 3.0Gy and 4.0 Gy mouse increased with the sampling time. And in the irradiation of 5.0 Gy, 6.0 Gy group, the content of SOD in the spleen of mouse increased first and then decreased with the prolongation of sampling time. Conclusion The effect of X-rays on immune organs in mouse was positively correlated with the dose of irradiation during the observed dose and measurement time. Different tissue weight recovery times are different, and low-dose irradiation begins to recover time. Higher dose irradiation begins to recover earlier.

Effect of Vitamin D₃ on Kidney of Rat at Toxic Dose

SHI Xiang-hua, YANG Wen-bin, XUE Run-miao, ZHAO Jing-yu, XU yan, ZHANG Cai-feng, CHAI Qiu-yan

2019, 39(2):  136-139.  DOI: 10.3969/j.issn.1674-5817.2019.02.012

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Objective To observe the effects of vitamin D₃ (VitD₃) at toxic dose on the blood and renal biochemical indicators of rats. Methods The specific pathogen free SD rats were randomly divided into five groups: control group, low dose VitD₃ group (1 875 IU), medium dose VitD₃ group (3 750 IU ), high dose VitD₃ group (7 500 IU), and super high-doseVitD₃ group (15 000 IU). They were intragastric administration once a day for 3 weeks.The relevant indicators were detected after treatment finished. Results Serum blood urea nitrogen (BUN) and Creatinine (Cre) decreased significantly with the dose decreasing, and the content of serum Ca²⁺ increased significantly with the dose increasing. The content of superoxide dismutase (SOD), and serum malondialdehyde (MDA) decreased with the dose increasing. In renal tissue, the content of Ca²⁺ gradually increased with the dose increasing, and there was no significant difference in MDA at the dose of 1 875 IU and 7 500 IU. Conclusions At the dose of 1 875 IU to 15 000 IU, VitD₃ has significant effects on the renal biochemical indicators such as BUN, Cre and serum Ca²⁺, indicate that the dose range 1 875 IU to 15 000 IU can induce renal toxicity in experimental rats.

SNP Genotypes of Olfactory Receptor Genes Associated with Sniffing Ability in Working-dogs Based on Sequenom MassARRAY SNP System

GAO Yi-long, HE Xing-liang, QIANG Jing-ning, SONG Zhen-hua, WEN Hai, TAO Xiao-ran, SUN Yong, QIN Hai-bin, BAO Xi-jun, MENG Ge, ZHU Qian

2019, 39(2):  140-145.  DOI: 10.3969/j.issn.1674-5817.2019.02.013

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Objective To find out the relationship between single nucleotide polymorphism (SNP) genotypes of canine olfactory receptor (OR) genes and odor detection performance in working-dogs. Method The multiplex Sequenom MassARRAY SNP system was adopted to detect the several SNP sites of OR genes and to analyze the correlation between different SNP genotypes and olfactory ability of working-dogs. Results The representative 68 SNP sites from canine OR genome were selected. The detection rate of this system in 3 breeds of Spinger Spaniel, Labrador Retriever and German Shepherd dog were all above 90%. The results showed that 23 SNP sites were found to be significantly correlated with the sniffing ability of working dogs (P< 0.05). But after the Bonferroni correction and FDR_BH correction, only 4 SNP sites including COR52AC1_436, rs850523383, CfOR0149_364 and CfOR0055_265 were found to be significantly correlated with sniffing ability (P< 0.05, BONF) and extremely significant (P< 0.01, FDR_BH). Conclusion Four major SNP sites of OR gene were screened, which significantly affected the sniffing ability of working dogs. A molecular genetic detection method was preliminary established to identify working-dogs potential sniffing ability, which was appropriate for Spinger Spaniel, Labrador Retriever and German Shepherd dog.

Sperm Cryopreservation and Recovery of C57BL/6J Background Genetically Modified Mice

NIU Bo-wen, CHEN Li-xiang, ZHU Meng-min, PENG Xiu-hua, QIN Bo-yin, LI Feng

2019, 39(2):  146-150.  DOI: 10.3969/j.issn.1674-5817.2019.02.014

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Objective To establish a high-efficiency sperm cryopreservation and in vitro fertilization (IVF) method on C57BL/6J background gene-modified mice. Methods This study compared the effects of different methods on sperm cryoprotectant, sperm concentration, treatment after thawing and IVF medium on 2-cell cleavage rate, and then used technologies developed to cryopreserve and recover sperm of knockout mouse lines on inbred C57BL/6J backgrounds. Results Sperm were frozen with R18S3, which containing 18% gossylose and 3% skimmed milk powder, then thawed for IVF in HTF medium and their fertility (two-cell cleavage) was 8.4%. Sperm were frozen with M-R18S3, a cryoprotective solution that containing 477 µmol/L monothioglycerol (MTG) in R18S3 and then IVF in HTF medium, their fertility (two-cell cleavage) was 20.6%. Sperm cryopreserved in M-R18S3, IVF in HTF with 1mmol/L reduced glutathione (GSH), have better IVF rate than when cryopreserved in R18S3 alone (43.5% vs 25%). High concentration sperm was cryopreserved in M-R18S3, then thawed for IVF in HTF medium with 1 mmol/L GSH and their fertility (two-cell cleavage) was higher than low concentration sperm (64.2% vs 43.5%). Sperm from 3 knockout and 1 transgenic mouse lines on C57BL/6J backgrounds cryopreserved using this techniques were all thawed successfully to obtain IVF fertility (two-cell cleavage) between 34% and 90%, and after embryo transplantation to get genotypically confirmed offspring. Conclusion High concentration sperm was frozen in M-R18S3, screened in IVF drop after thawing, and then IVF in HTF with 1 mmol/LGSH, can be used as an effective method for sperm preservation and recovery of C57BL/6J background genome-modified mice.

Advances on in Vitro Culture of Female Reproductive and Development Toxicity Based on Animal Welfare

LU Qin, ZHOU Yue-hua, TANG Hai-fei, WANG Zhi-wen, ZHANG Ting

2019, 39(2):  155-162.  DOI: 10.3969/j.issn.1674-5817.2019.02.016

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The aim of reproductive and developmental toxicity study is to identify the effects of exogenous substances on reproductive function and development of mammals by animal experiments, predicting the potential adverse effects on reproductive cells, pregnancy, pregnancy, delivery, lactation and other parental reproductive functions, as well as on the development of offspring embryos/fetuses and postnatal development. The methods of studying the reproductive and developmental toxicity of whole animal in females have been widely used in the evaluation of reproductive and developmental toxicity of drugs, food, chemicals and cosmetics. However, its main problems are insensitivity, long cycle, and large sample size. Especially, as the reproductive function of in vivo experiments is mainly based on parameters related to reproductive and developmental consequences, it is difficult to reveal the toxic action site and mechanism. Alternative method of female reproductive and developmental toxicology refers to any method or procedure which can replace animal experiments whenever possible, reduce the required number of animals or optimize and reduce animal suffering in animal experimental program, especially in preliminary evaluation of exogenous toxic substances as a powerful complement for animal experiments. In this paper, the technique and research progress of in vitro culture of female reproductive developmental toxicity were summarized in this paper, the advances in vitro culture of female reproductive toxicity were summarized from cells and tissue culture in female reproductive system, embryonic cells and tissue culture, breast cell culture so as to provide references for the study of in vitro culture of reproductive and developmental toxicity in females.

Research Progress of Rabbit VX2 Tumor Model

MI Jin-xia, FANG Zhao-qin

2019, 39(2):  163-168.  DOI: 10.3969/j.issn.1674-5817.2019.02.017

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The rabbit VX2 tumor model is commonly used as a xenograft tumor model in large animals, which is also widely used in surgical operation, imageology and interventional therapy because of its body type and easy replication. The methods of establishing rabbit VX2 tumor model include injection of cell suspension and inoculation of tumor tissue block. The tumor models can be established differently depending on the site of the transplant. Researchers can select suitable tumor models for their own purposes. With the popularity of imaging equipment and the application of the emerging local treatment methods, more and more studies have been conducted using the rabbit VX2 tumor model. This paper reviewed the source of VX2 cells, biological characteristics, methods of tumor models and research applications.