Table of Content

28 August 2023, Volume 40 Issue 4

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Change of eNOS and ET-1 for Highland Multiple Organ Dysfunction Syndrome Model in Juema pig

FU Xiaoyan, FENG Xiaoming, LI Yuanming, XIAO Pan , WANG Hongyi , LU Lu, ZHANG Mei, NIU Tingxian

2023, 40(4):  1-5.  DOI: 10. 3969 / j. issn. 1006-6179. 2023. 04. 001

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Objective To study the changes of endothelial nitric oxide synthase ( eNOS) and endothelin1 ( ET-1 ) in the development of inflammation in Juema pig model of high altitude multiple organ dysfunction syndrome ( H-MODS ) . Method H-MODS model was successfully established in plateau native Juema pigs induced by endotoxin ( LPS) at three doses of low, medium and high ( groups B, C and D) . Normal saline was used as the control group ( group A) . The expression levels of serum eNOS and ET-1 were detected at different time points. Result The expression levels of eNOS and ET-1 increased sharply after LPS was pumped into Juema pigs in each group, and rose to the highest at 3 h, then decreased rapidly, and basically returned to the normal value at 24 h. There was no significant change in the expression levels at each time point in the control group. The expression of eNOS in group B, C and D was significantly higher than that in group A and group 0 h at 3-12 h ( P<0. 01) ; at 24- 72 h, the expression of eNOS in groups B, C and D basically returned to the normal level, and there was no significant difference compared with group 0 h and group A. The expression of ET-1 in group B, C and D was significantly higher than that in group A and group 0 h at 3-6 h (P<0. 01) ; the expression of ET-1 in group B and C was significantly higher than that in group A and group 0 h at 12 h (P<0. 05) ; the expression of ET-1 in group D was significantly higher than that in group A at 12 h ( P<0. 05) ; the expression of ET-1 in groups B, C and D basically returned to normal at 24-72 hours, and there was no significant difference compared with group 0 h and group A. Conclusion During the formation of plateau mods, the body produces serious inflammatory reaction, and the expression of eNOS and ET-1 increases rapidly, but the expression decreases rapidly under the effect of immune regulation, and its changes reach a balance state after a sharp rise and fall in a short time.

Effects of Heat Stress on Blood Biochemical Indicators in ICR Female Mice

TIAN Yonglu, LI Xiaying, WEI Yusheng, LI Na, CAO Dong, ZHU Desheng

2023, 40(4):  6-9.  DOI: 10. 3969 / j. issn. 1006-6179. 2023. 04. 002

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Objective To explore the effect of heat stress on blood biochemical indicators in mice. Method The control group (25 ℃ ) and heat stress group (42 ℃ ) contained 4 mice, respectively. For each group, we investigated the blood biochemical indicators on the 7 ^th , 14 ^th , 21 ^th and 28 ^th days. The blood biochemical indicators included Na ⁺ , K ⁺ , Cl ^- , TP, ALB, ALT, AST, LDH and ALP, and were tested using Rocky automatic biochemical analyzer. The Na ⁺ , K ⁺ , Cl ^- used the ion-selective electrode method, TP used the biuret method, ALB used the bromocresol green method, and ALT, AST, LDH and ALP used the rate method. Result On the 7 th , 14 ^th , 21 ^th and 28 ^th day of the heat stress group, the serum Na ⁺ concentration was higher than those in the control group, the serum K ⁺ concentration was lower than those in the control group, but there was no significant difference between the two groups. The serum C1 ^- concentration of the heat stress group was significantly higher than the control group only on the 28 ^th day (P<0. 05) . TP level showed no significant difference in each period, while the ALB level of the heat stress group was significantly higher than the control group on the 28 ^th day ( P < 0. 01) . The AST, LDH and ALP concentrations of the heat stress group were significantly higher than the the control group on the 21 ^th day (P<0. 05) . Conclusion Heat stress can increase the concentration of the ALT, AST, LDH and ALP.


Establishment of a Multiplex PCR Assay for Simultaneous Detection Pasteurella Multocida、Staphyloccocus Aureus and Klebsiella Pneumoniae from Rabbit

ZHANG He, TAO Kunsheng, CHEN Jianxing, WANG Haojie, CHEN Hongyan, WANG Yu’ e, XIA Changyou

2023, 40(4):  10-16.  DOI: 10. 3969 / j. issn. 1006-6179. 2023. 04. 003

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Objective This study aimed to establish a multiplex PCR method for detecting three pathogens in rabbits: Pasteurella multocida ( Pm ) , Staphylococcus aureus ( Sa ) , and Klebsiella pneumoniae (Kp) . Method In this study, three pairs of specific primers were designed, targeting the conserved regions of the kmt1 gene in Pm, the nuc gene in Sa, and the kh1 gene in Kp. The optimal annealing temperature ( Tm ) was determined using gradient PCR. The primer concentrations were optimized using the controlled single-variable method. Recombinant plasmid standards ( pMD-Pm, pMDSa, and pMD-Kp) were constructed, and the minimum detection limit was established. The specificity of the multiplex PCR was confirmed using nucleic acid samples of nine pathogens, including rabbit bronchial septicemic Pasteurella and gas-producing clostridia. The repeatability was validated through inter-batch and intra-batch experiments. Clinical samples suspected of infection were tested using the established method and compared with previously reported detection methods to evaluate its clinical application. Result The optimal annealing temperature was determined to be 54. 5 ℃ . The optimal primer concentrations for amplifying kmt1 and nuc genes were 1 μL ( 10 μmol / L) , while for the kh1 gene, it was 0. 5 μL (10 μmol / L) . The lowest detection limits were 20. 6 copies/ μL for pMD-Pm, 18. 2 copies/ μL for pMD-Sa, and 23. 2 copies/ μL for pMD-Kp, indicating high sensitivity. The multiplex PCR method showed specific amplification bands only for Pm, Sa, and Kp, with no amplification bands observed for other pathogens, demonstrating strong specificity. The results of inter-batch and intra-batch tests were consistent, indicating good repeatability. Clinical sample testing revealed individual positivity rates of 62. 92% for Pm, 25. 84% for Sa, and 49. 43% for Kp, which were consistent with the results obtained using previously reported detection methods. Conclusion This study successfully established a multiplex PCR method capable of simultaneously and rapidly detecting Pm, Sa, and Kp in rabbits.

Effect of Daiyu Ointment on Angiogenesis in Rats with Anal Fistula via Nrf2 / HO-1 Signal Pathway

GUO Wei, YAN Lixia, CHANG Shaoqing , LIU Zhenli

2023, 40(4):  17-22.  DOI: 10. 3969 / j. issn. 1006-6179. 2023. 04. 004

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Objective To observe the effect of Daiyu ointment on angiogenesis and Nrf2 / HO-1 signaling pathway in rats with anal fistula. Method 20 SPF-grade SD rats were constructed a post-operative wound model of fistula by comprehensive method, after successful modeling, they were randomly divided into model group and Daiyu paste group, with 10 rats in each group. Rats in the Daiyu paste group applied the wound with drugs, and the model group scrubbed with 0. 9% sodium chloride solution. In addition, 10 rats with only wounds were selected as the control group, and scrubbed with 0. 9% sodium chloride solution, and the rats in each group were treated twice a day, all of which were continuously intervened for 14 days. then the wound healing and local blood flow was observed. Then the rats were sacrificed. ELISA was used to detect the levels of vascular endothelial growth factor ( VEGF) , VEGF receptor (VEGFR) , epidermal growth factor (EGF) , platelet derived growth factor ( PDGF) , interleukin ( IL) - 1β, IL-6, IL-10, tumor necrosis factor α ( TNF-α) in wound tissues of anal fistula; Western blot was used to detect the protein levels of Nrf2 and HO-1 in the wound tissues. Result Compared with the control group, the wound healing rate and blood flow of the model group significantly decreased ( P < 0. 05) . Compared with the model group, the wound healing rate and blood flow of rats in the Daiyu ointment group significantly increased (P<0. 05) ; Compared with the control group, CD31 positive area significantly decreased (P<0. 05) , while significantly increased in Daiyu ointment group compared with model group; The levels of IL-1β, IL-6, IL-8 and TNF-α in granulation tissue were significantly higher in the model group than in the control group ( P < 0. 05) , and the Daiyu cream group was significantly lower than that in the model group ( P< 0. 05) ; wound tissue VEGF, VEGFR, EGF and PDGF in the model group were significantly higher than those in the control group (P<0. 05) , and the Daiyu ointment group was significantly higher than the model group (P<0. 05) ; the levels of Nrf2 and HO-1 protein in the model group were significantly higher than those in the control group significantly higher (P<0. 05) , and the Daiyu ointment group was significantly higher than the model group ( P< 0. 05) . Conclusion Daiyu ointment can promote the healing of wounds and angiogenesis in anal fistula model rats. The mechanism may be related to the activation of Nrf2 / HO-1 pathway. 

Study on the Sensitivity and Specificity of Sniffer Dog on Diagnosis of Human Lung Cancer

GAO Changqing , CHEN Zhiheng , LI Jingjing , ZHOU Xiao

2023, 40(4):  23-27.  DOI: 10. 3969 / j. issn. 1006-6179. 2023. 04. 005

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Objective To evaluate the sensitivity and specificity of sniffer dog on diagnosis of human lung cancer. Method Blood was collected from 25 patients with lung cancer and 127 volunteers in the control group. Serum was used to be identified by sniffer dog under a double-blind setting. Result The sensitivity and specificity of sniffer dog identification of human lung cancer from normal control were 100% ( 95% CI:86. 3% - 100. 0%) , 99. 2% ( 95% CI:95. 7% - 99. 9%) , respectively. The positive predictive value was 96. 2% ( 95% CI:78. 0% - 99. 4%) and the negative predictive value was 100%. The accuracy of sniffer dog on diagnosis of human lung cancer was 99. 3%. Conclusion Accuracy of deteting lung cancer by dog sniffing is over 99%, which can be used as an assistant tool for early screening of lung cancer.

Expression and Immunogenicity Analysis of VP2 Protein of Minute Virus of Mice in Insect Cell-baculovirus Expression System

MA Chang , ZHAO Yingfeng , CHEN Li, LIU Biao, ZHAO Zhigang , YUN Shifeng

2023, 40(4):  28-33.  DOI: 10. 3969 / j. issn. 1006-6179. 2023. 04. 006

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Objective Recombinant MVM VP2 protein was synthesized by baculovirus expression system and the immunogenicity of the recombinant VP2 protein was analyzed. Method The optimized MVM VP2 gene sequence was cloned into the eukaryotic expression vector pFastBac1, then the recombinant vector pFastBac1-MVM VP2 was transformed into DH10Bac susceptible E. coli to form recombinant plasmid. The recombinant plasmid were transfected into insect cells Sf9. Recombinant baculovirus rBacMVM VP2 was harvested after significant cellular lesions were observed. Sf9 cells were inoculated with recombinant virus rBac-MVM VP2 for 48 and 72 h. Immunogenicity of the recombinant protein was tested by IFA and Western blot. Result Recombinant baculovirus rBac-MVM VP2 could successfully express recombinant MVM VP2 protein after infecting Sf9 cells. Western blot and IFA analysis showed that the recombinant protein had good immunogenicity and high expression at 72 h of virus infection. After protein purification, a high purity recombinant protein VP2 was obtained. Conclusion In this study, MVM VP2 protein was successfully expressed by insect cell-baculovirus system and the recombinant VP2 protein has good immunogenicity, which lays a foundation for the study of VP2-related functions and the establishment of clinical diagnosis method.

Breeding and Genotype Identification of PD-1 Humanized Mice

LI Xiaojuan, SUN Yanfeng , XIU Ye , LI Xingjie , LI Ruisheng

2023, 40(4):  34-38.  DOI: 10. 3969 / j. issn. 1006-6179. 2023. 04. 007

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Objective To explore the breeding and genotype identification of PD-1 humanized mice, and provide an animal model for further research on the efficacy evaluation of small molecule inhibitors in vivo. Method Through the construction of PD-1 humanized mice, four F1 generation mice were obtained, which were bred by mating male and female. The number and survival rate of each litter of mice were recorded and observed, and then genomic DNA was extracted from the offspring by tail clipping, the target gene fragment was amplified by PCR, and then the genotype was identified by nucleic acid electrophoresis. The F2 generation homozygous type and homozygous type were used for male and female mating, and wild type and wild type were used for male and female mating. Result The F2 generation mice were identified as wild type, homozygous type and heterozygous type. The genotype of F3 offspring obtained from homozygous and homozygous mating were all homozygous. The genotype of F3 offspring obtained from wild type and wild type mating were all wild type. Conclusion The homozygous genotype of PD-1 humanized mouse was successfully screened, and the population expansion and seed conservation were effectively carried out, providing experimental guarantee for the future application of this mouse model. 

Comparative study on the establishment of hyperlipidemia models in rats induced by different high-fat diets

QI Miao, XU Chenxi, LYU Lingjuan, YANG Zhihong, YANG Runmei

2023, 40(4):  39-43.  DOI: 10. 3969 / j. issn. 1006-6179. 2023. 04. 008

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Objective To discover the hypolipidemic drugs, we established the model of hyperlipidemia in Sprague-Dawley ( SD) rats by feeding the high-fat diets of different recipes. Method Male SD rats were randomly assigned to 6 groups:the control group and 5 model groups. The rats were fed with the control diet and the high-fat diet of five different recipes for 6 weeks respectively. The concentrations of serum lipids were detected at the end of 1, 2, 4 and 6 weeks ’ feeding. The hepatic lipids ’ concentrations were determined at the end of 6 weeks. Result Compared with the control group, after 6 weeks of feeding, the concentrations of serum total cholesterol ( TC) of the rats in model group 2 and 3 significantly increased; the concentrations of serum low-density lipoprotein cholesterol ( LDL-C) of the rats in all model groups significantly increased. And the hepatic lipids ’ concentrations significantly increased in all model groups. Conclusion The model of hypercholesteremia was established by feeding the high-fat diet of recipe 2 and 3 that adding 20% fructose and 10% lard for 6 weeks in rats. this experiment provides a simple, low-cost, and stable animal model for the development of lipid-lowering drugs.

Genetic Analysis of Spontaneous Congenital Cataract Rats

YOU Jinwei, LIANG Lei, ZHANG Xuliang, WU Zhenglin, DONG Min, TIAN Xiaoyun

2023, 40(4):  44-48.  DOI: 10. 3969 / j. issn. 1006-6179. 2023. 04. 009

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Objective To investigate the genetic status of spontaneous congenital cataract rats ( TSC) and evaluate the genetic quality analysis method. Method 11 variables of mandible of TSC rats, BN rats and F344 rats were measured and analyzed by SPSS18. 0 ; choose 6 microsatellite loci commonly. PCR amplification and agarose electrophoresis were used to detect the gene quality. Result There were 9 significant differences in mandibular parameters between TSC rats and BN rats( P < 0. 01) ,5 significant differences between TSC rats and F344 rats ( P < 0. 01 ) . Only 1 band appeared in the amplification products of TSC rats at 6 microsatellite loci of different individuals. 4 loci could distinguish TSC rats from BN rats, 4 loci could distinguish TSC rats from F344 rats, and 1 loci could distinguish three inbred rats. Conclusion The mandibular variables of TSC rats were significantly different from those of BN rats and F344 rats. TSC rats met the requirements of inbreeding lines. The combination of six microsatellite loci can be used to monitor the genetic background of TSC rats and common inbred lines BN and F344. 

Study on Biological Characteristics of Escherichia Coli Isolated from Experimental Rabbits with Diarrhea

DONG Hao, LIU Zhiguo, LI Nan, XING Jin, XU Zhongkan, XING Zhuangzhuang, FENG Yufang, MA Liying

2023, 40(4):  49-54.  DOI: 10. 3969 / j. issn. 1006-6179. 2023. 04. 010

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Objective To reveal the biological characteristics of E. coli isolated from experimental rabbits with diarrhea. Method The genetic lineage, antibiotic sensitivity, disinfectant sensitivity, drug resistance gene and virulence related gene of the pathogenic Escherichia coli strain isolated from SPF rabbits died of diarrhea were studied comprehensively. Result The Escherichia coli strain belonged to B1 type genetic lineage, and carried the virulence gene eaeA, which was the classic virulence genes of enteropathogenic Escherichia coli (EPEC) . In addition, this isolated strain was sensitive to 5 commonly used disinfectants. Besides, it was resistant to 11 antibiotics including penicillin, ampicillin, cephalexin, cephradine, tetracycline, doxycycline, minocycline, erythromycin, midecamycin, vancomycin and cotrimoxazole, and carried 4 drug resistance genes, including aadA1, gyrA, sul1 and tetA. Conclusion The pathogenic Escherichia coli strain isolated from SPF rabbits died of diarrhea is a type B1 genetic pedigree EPEC with multiple drug resistance and it is sensitive to common disinfectants.

Establishment and Evaluation of an Animal Model of Dilated Cardiomyopathy

ZHANG Dong , ZHU Jinyan , ZHANG Min , LI Bin

2023, 40(4):  55-61.  DOI: 10. 3969 / j. issn. 1006-6179. 2023. 04. 011

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Objective To explore the method of establishing and evaluating the animal experimental model of dilated cardiomyopathy (DCM) , and to provide ideas and method for the clinical diagnosis and treatment of DCM. Method Adult beagle dogs were divided into two groups, the experimental group was injected with adriamycin and the control group was injected with 0. 9% sodium chloride solution. Adult beagles were given doxorubicin ( doxorubicin) through the left main coronary artery once a week for 5 weeks after coronary angiography induced by anesthesia. The DCM group ( n = 6) was established, and the sham group ( n = 4) was established. Four weeks after the last injection, the animals in each group were examined by cardiac ultrasound, multichannel physiological recorder and cardiac catheterization technology, hematology and ventricular remodeling evaluation to evaluate the feasibility and effectiveness of DCM modeling. Result Compared with the control group, the heart of the experimental animals in the DCM group was significantly enlarged, and the ventricular contractility was significantly reduced. Compared with the control group, the experimental animals in DCM group showed significant differences in all hematological indexes ( except white blood cell count) . Compared with the control group, and the content of Collagen I / III in DCM group was significantly increased. Conclusion Multiple injection of doxorubicin into the coronary artery is feasible and effective to construct the animal model of DCM. The cycle is short, simple and feasible, and it is more similar to the pathophysiology of human DCM.

Construction of Laboratory Animal Quality Assurance and Service System

ZHANG Jing , LIU Miaomiao, TANG Qianqian , YIN Yutao, CHEN Zhen , XIE Zhongchen, CHANG Zai

2023, 40(4):  62-68.  DOI: 10. 3969 / j. issn. 1006-6179. 2023. 04. 012

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The intersection of multiple disciplines and life sciences has become popular along with the rapid development of modern life science, wherein laboratory animals have been widely used as an important support. The strict quality control of laboratory animals is of vital importance for the repeatability, stability and reliability of animal-based experimental result. In addition, professional technical services will further boost the quality of laboratory animals. After more than ten years of exploration, the Laboratory Animal Center of Tsinghua University has established a relatively complete quality assurance system for laboratory animals and has been continuously improving its professional technical services. Here, Laboratory Animal Center of Tsinghua University aims to provide an operation reference for others by organizing the practical experience over the years, so as to contribute to the development of laboratory animals in China.

Establishment and Application of Fluorescent Quantitative PCR method for Burkholderia Gladioli in Rodent Laboratory Animal

SHENTU Fenqin, LIU Min, WANG Xuemei, WANG Jixing, PENG Gang, WU Shiai, LI Yannan, ZHOU Qian, ZHANG Qiaozhi, HAN Xue

2023, 40(4):  69-72.  DOI: 10. 3969 / j. issn. 1006-6179. 2023. 04. 013

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Objective To establish a fluorescent quantitative PCR ( q-PCR) method for detection of Burkholderia gladioli (B. gladioli) in laboratory mouse. Method the q-PCR method was established with the primers, probe and plasmid of B. gladioli which were synthesized according to the sequence published by Group Standard. And the linearity, sensitivity, repeatability and specificity of the method were verified. 110 samples of mice were tested for B. gladioli by the q-PCR method and bacterial culture method. Result The established q-PCR method for B. gladioli showed good linearity with correlation coefficient R 2 0. 998, and high sensitivity with a detection limit of 1 × 10 2 copies/ 5 μL. The assay could test specifically for B. gladioli with no cross reaction for Staphylococcus aureus, Klebsiella pneumoniae, Klebsiella oxytoca, Pseudomonas aeruginosa and Pasteurella pneumotropica. 110 samples were collected from euthanized mice and tested by bacterial culture and the q-PCR method respectively. The result with the two method were all negative, which were completely consistent. Conclusion The established q-PCR method had good performance and could be used for the detection of B. gladioli in laboratory mouse. 

Safety Analysis of an Electronic Logo Applied to Mice

DU Kai, LU Xinlin, MU Yufeng, LU Cizhang, SHI Xin, WU Deguo

2023, 40(4):  73-75.  DOI: 10. 3969 / j. issn. 1006-6179. 2023. 04. 014

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Objective Skin sensitization test, intradermal reaction test and histopathological examination were carried out for electronic signs to understand their safety. Method The electronic signs was humidified with normal saline, covered with fresh-keeping film, and then covered with gauze on the shaved mouse skin. Record the contact parts after 1, 24, 48 and 72 h. The electronic signs was injected subcutaneously into mice, and the mice were euthanized 24, 48 and 72 h after injection. The erythema and edema of the surrounding tissues were observed, and the tissues were examined pathologically. Result Electronic signs are not irritating to the skin. No abnormal surrounding tissue was found after subcutaneous injection. No abnormality was found in pathological examination. Conclusion No damage to mice was found by the electronic signs.

Current State and Future Perspectives of Biological Models in Pancreatic Cancer

XU Dong , YANG Fei

2023, 40(4):  76-82.  DOI: 10. 3969 / j. issn. 1006-6179. 2023. 04. 015

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Attributable to its highly malignant feature, late diagnosis and early metastasis, pancreatic cancer patients often lost the opportunity of radical surgery due to the local advanced lesion. Although significant progress has been made in the effect of comprehensive treatment, the patients were still with poor overall prognosis and low survival rate. The insidious symptoms of pancreatic cancer limited investigators to acquire early tissue timely for in-depth study, therefore, a suitable biological model should be constructed to investigate the molecular mechanisms underlying pancreatic cancer initiation and progression, and to evaluate new therapies. Spontaneous tumor animal models, transplanted tumor models and pancreatic cancer organoid were the most commonly used preclinical biological models, and we reviewed the current trends in applications of biological models in pancreatic cancer in this paper. 

Research Progress on Effects of Light on Reproductive Performance of Rats and Mice

WU Xianwen , CHEN Ye , XU Guoheng

2023, 40(4):  83-88.  DOI: 10. 3969 / j. issn. 1006-6179. 2023. 04. 016

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Environmental light plays an important role in the sexual maturity and reproductive performance of experimental animals. Different countries and regions have different requirements on the environmental light of laboratory animal rats and mice. This paper reviews the effects of light cycle and wavelength on the reproduction of rats and mice. Other factors related to light, such as light pollution, cage placement and box color, were analyzed and corresponding solutions were put forward. In order to solve the problem of uneven distribution of light source in laboratory animal facilities, the paper proposed the design of wall and ground light belt and the method of moving light source, and discussed the importance of staff training related knowledge.