Table of Content

25 December 2021, Volume 41 Issue 6

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Animal Models of Human Diseases

Effects of Diabetes on Colorectal Cancer/Breast Cancer Progression and Intestinal Flora Based on a Diabetes-tumor Mouse Model

WANG Xiaoyu, JIANG Shengyao, LIN Zhibing, CUI Li

2021, 41(6):  469-479.  DOI: 10.12300/j.issn.1674-5817.2021.066

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Objective To investigate the effects of diabetes on tumor progression, and the effects of diabetes combined with colorectal or breast cancer on the intestinal flora by constructing a diabetes-tumor mouse model, and the differences in body mass, tumor volume, survival rate, and initial flora of the mice in different treatment groups were statistically analyzed. Methods Sixty mice were divided into control, diabetes, colorectal cancer, diabetes + colorectal cancer, breast cancer, and diabetes + breast cancer groups. The diabetic mouse model was established by streptozotocin injection, and solid tumor models of colorectal and breast cancer were further established on this basis. Changes in the body mass, tumor volume, and survival rate of the mice in each group were observed and analyzed. The mouse feces were collected, and then the microbial community sequencing data were analyzed using the QIIME2 platform based on the DATA2 and Vesearch methods. Results The body mass (P < 0.05) and survival rate (P < 0.01) of mice with diabetes combined with colorectal or breast cancer were significantly reduced, but there was no significant difference in tumor volume in mice. Compared with the control group or the colorectal cancer group, the species richness of the intestinal flora in mice in the diabetes + colorectal cancer group was significantly altered (P < 0.05); while the diabetes + breast cancer group did not significantly change (P > 0.05). The beta diversity and bacterial species composition of the intestinal flora in mice with diabetes combined with colorectal or breast cancer were also affected. Conclusion Diabetes promotes the progression of colorectal or breast cancer. Diabetes combined with colorectal or breast cancer has a greater impact on the intestinal flora of mice than colorectal or breast cancer alone.



Establishment of a Mouse Abdominal Aortic Aneurysm Model and Histological Characteristics

XIA Congcong, LIU Haole, WEI Panpan, CHENG Daxin, XU Baohui, LIU Enqi, ZHAO Sihai

2021, 41(6):  480-485.  DOI: 10.12300/j.issn.1674-5817.2021.115

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Objective To investigate the method of establishing C57BL/6J mouse abdominal aortic aneurysm (AAA) model by porcine pancreatic elastase (PPE, 1.5 U/mL in PBS) perfusion and evaluating its histological characteristics. Methods Thirty male mice were divided into two groups, the model group (n=20) and solvent control group (n=10). After opening the abdominal cavity in all mice, the infrarenal abdominal aorta to the iliac artery segment was isolated and perfused with PPE solution at a dose of 1.5 U/mL for 5 minutes. After the operation, the abdomen was closed and sutured. The aorta segment of the control group was perfused with PBS. The diameters of the abdominal aorta were measured before and after 14 days of the operation, and perfusion aorta segments were collected 14 days after operation for following histological analysis. The histological characteristics of the aneurysms were analyzed and graded by histological and immunohistochemical methods. Results Compared with the preoperative or PBS solvent control mice, the abdominal aortic diameters were significantly increased in the PPE perfused group on day 14 after operation (0.53±0.03 mm, 0.78±0.06 mm vs 1.20±0.12 mm, respectively). Histological analysis revealed that the PPE-infused AAA segments had elastic fiber breakage, smooth muscle cell depletion, and increased inflammation response, which resembles the typical pathological features of AAA, suggesting the mice model of AAA was successfully induced by PPE infusion. According to the histological characteristics of the AAA lesions, we graded the aneurysms and established a revised practical method for evaluating the aneurysm lesions in the experimental AAA mouse model. Conclusion A methodological system for establishment and histopathological evaluation of PPE-induced AAA in mice has been successfully established.



Effects of Carbon Tetrachloride-induced Acute Liver Injury on Cardiac Pathological Changes in a Rat Model

ZHANG Lili, WAN Xin, JI Jingquan, LI Ting, FAN Yimin, LI Baohong, TIAN Shuaiyan

2021, 41(6):  486-492.  DOI: 10.12300/j.issn.1674-5817.2021.033

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Objective To investigate the effects of carbon tetrachloride (CCl₄) on cardiac function and morphology in rats with acute liver injury. Methods Rat models of acute liver injury were established by intraperitoneal injection with different concentrations of CCl₄, while an equal volume of vegetable oil was injected intraperitoneally as a control group. Electrocardiogram (ECG) was performed at 24 and 48 hours after inducted by CCl₄, and the animals were sacrificed after blood collection from the abdominal aorta. The levels of serum alanine aminotransferase (ALT) and cardiac troponinⅠ(CTNⅠ) were measured by an enzyme-linked immunosorbent assay. Liver and heart tissues were sectioned and stained with hematoxylin-eosin (HE) and the pathological changes were observed by optical microscopy. The levels of malondialdehyde (MDA), superoxide dismutase (SOD), and creatine kinase (CK) in myocardial tissues were measured by visible light spectrophotometer. Results The changes in serum ALT level and liver morphological in the model groups proved that the acute liver injury model induced by CCl₄ was successfully established, and the degree of liver injury was dose-dependent. The ECG of the model groups revealed electrophysiological changes, such as ST-segment elevation, T-wave sharp, and Q-T interval prolongation in different degrees. HE staining showed that obvious pathological changes in the heart tissue, and the degree of inflammatory reaction, edema and necrosis of myocardial cells gradually aggravated with the increase of CCl₄ injection concentration. Serum CTN Ⅰlevels were significantly increased (P<0.01), CK and MDA levels in the myocardial tissue were significantly increased (P<0.05), while SOD levels in the myocardial tissue were significantly decreased (P<0.05), and the changes in various indexes were more significant in the high concentration CCl₄ group and 48 h after induction by CCl₄. Conclusion CCl₄ can induce cardiac function and morphological changes in rats with acute liver injury, and the degree of myocardial injury is dose- and time-dependent.



Application of Laboratory Animal Models in Cancer Precision Medicine Research

SONG Weijie, ZHOU Yan, NIU Ruifang

2021, 41(6):  493-500.  DOI: 10.12300/j.issn.1674-5817.2021-027

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Laboratory animal disease models play an important role in clinical translational research, and have been widely used in life sciences, medical chemistry, and biological health. In addition, animal models are often used as effective tools to study cancer occurrence, development, and metastasis. In recent years, with the increasing demand for precision medicine, the patient-derived tumor xenograft (PDX) model has been widely used as an important model for drug screening and translational research in the development of anti-cancer drugs and the introduction of personalized treatment plans for individuals. With the perspective of the development of experimental animal tumor models and the application of immunodeficient animals, this review summarizes the status of PDX models in precision medicine, defines the characteristics of different types of animal models, and provides prospects for future development trends and applications.

Construction and Preliminary Application of a Rncat Congenital Cataract Mouse Model

ZHANG Yan, YAN Shunsheng

2021, 41(6):  501-504.  DOI: 10.12300/j.issn.1674-5817.2021.015

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Congenital cataract is the main cause of blindness in children. The Center for Disease Control and Prevention of Xinjiang Uygur Autonomous Region successfully constructed mouse models of congenital cataracts. The biological characteristics, behavioral characteristics, gene sequences, and mutant proteins of the model mice were studied, yielding some notable achievements. These novel cataract mouse models may serve as reliable animal models for drug screening and cataract prevention. This paper reviews the research progress on the biological characteristics, behavior, genetics, and mutant proteins of the Rncat congenital cataract mouse model.

Construction Methods and Comparative Research Progress of Premature Ovarian Failure Animal Models

LI Hongxuan, LI Sihui, FENG Jiaxin, TANG Kai, LU Rui, HAN Siyin

2021, 41(6):  505-514.  DOI: 10.12300/j.issn.1674-5817.2021.056

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Currently, the factors associated with premature ovarian failure animal models mainly include iatrogenic, immune, genetic, metabolic, and environmental factors. The animal models constructed based on these factors have their own characteristics. Here the construction methods and comparison of animal models of premature ovarian failure have been reviewed to provide useful modeling information for researchers with different needs.

Research Progress Related to Candidate Treatment Methods and Modeling Factors for Diabetic Animal Models with Skin Injury

LIN Jiang, LUO Fei, LIU Peng, HAN Siyin, CHEN Zhenxing, LIANG Zhongxiu, LAN Taijin

2021, 41(6):  515-520.  DOI: 10.12300/j.issn.1674-5817.2021.025

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Currently, multiple drugs and other therapies are used for diabetic wounds, including Chinese herbs, Western medicine, biological agents, and physical therapy, these agents have been shown to be associated with favorable clinical outcomes, however, their long-term efficacy needs to be further improved. A diabetic wound is the result of an interaction among multiple cells and cytokines, and therefore a single treatment method is often ineffective. On the basis of establishing stable and reliable animal models, the mechanism underlying each treatment method can be clarified through pharmacological research, providing a basis for combined clinical application and improved clinical outcomes. In this paper, the scientific literature related to diabetic animal models with skin injury from the last 24 years was collected, and the factors involved in animal model preparation and treatment data were statistically analyzed to provide references for improving the modeling efficiency and scientifically evaluating the effectiveness of treatment methods.

Technology and Method

Analysis of Seven Performance Evaluation Program Results for Pathogenic Bacteria from Laboratory Animals in 2013—2020

XING Jin, FENG Yufang, WANG Hong, ZHANG Xueqing, GAO Qiang, YUE Bingfei, FU Rui

2021, 41(6):  521-527.  DOI: 10.12300/j.issn.1674-5817.2021.104

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Objective To promote the development of domestic laboratory animal detection proficiency testing activities and testing levels by assessing self testing capabilities. Methods The National Institutes for Food and Drug Control participated in the International Council for Laboratory Animal Science (ICLAS) laboratory animal detection proficiency test (i.e. Performance Evaluation Program for Diagnostic Laboratories, PEP) in 2013—2020. PEP pathogenic bacteria samples were tested by culture, biochemical identification, PCR, and sequencing. Finally, the results were analyzed and summarized. Results There were 40 samples in seven international PEP activities, involving 27 kinds of pathogenic bacteria. The coincidence rates of the seven results were 8/8, 2/2, 5/8, 9/9, 5/5, 6/7, and 1/1, respectively, including 33 pathogen detection items and 7 antibody detection items. Nine pathogens were missing from the national standards: Bordetella hinzii, Serratia marcescens, Corynebacterium mastitis, CAR (Cilia-associated respiratory) bacillus, Actinobacillus muris, Citrobacter freundii, Klebsiella oxytoca, Streptococcus agalactiae, and pathogenic Escherichia coli. Conclusion Through participating in PEP, we can identify the differences between domestic and foreign standards in laboratory animal detection and the testing capabilities that we need to improve, expecting to be in line with international standards as soon as possible.

Population Genetic Quality Analysis of Microsatellite DNA in Wistar Rats Based on T/CALAS 21—2017

WEI Jie, ZUO Qin, WANG Hong, LI Huan, ZHOU Jiaqi, GUANG Jiaona, FAN Tao, LIU Zuomin, FU Rui, YUE Bingfei

2021, 41(6):  528-534.  DOI: 10.12300/j.issn.1674-5817.2021.073

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Objective To analyze the genetic quality of an outbred stock of Wistar rats in different periods by T/CALAS 21—2017 method, and to evaluate the applicability of the association standard. Methods Wistar rats selected from the same outbred stock in 2015 and 2019 were named as group A and group B, respectively. Twenty-five pairs of microsatellite primers were used to get genetic parameters for quality analysis of the outbred stock Wistar rats according to the association standard T/CALAS 21—2017. Polymorphism information content (PIC) was used to analyze the polymorphism of the loci. Results One hundred alleles in group A and 69 alleles in group B were obtained. The average heterozygosity of the two groups was 0.574 and 0.447, while the average PIC was 0.541 and 0.393, respectively. Conclusion The association standard T/CALAS 21—2017 has good applicability in the genetic quality analysis of outbred stock rats, and the genetic diversity of group A is better than that of group B.

Factors Affecting Production Efficiency of Bacterial Artificial Chromosome Transgenic Mice

WANG Qianqian, WANG Wei, LIU Rui, WEI Zhen, LIU Chong

2021, 41(6):  535-542.  DOI: 10.12300/j.issn.1674-5817.2021.032

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Objective By comparing some key factors in generating bacterial artificial chromosome (BAC) transgenic mice, such as the background of strains, the concentration of the BAC DNA used for injection, the size of the BAC DNA, and the storage time of the BAC DNA injection, to optimize the generation of BAC transgenic mice. Methods The same procedure of BAC DNA preparation was used with two mouse strains, i.e. C57BL/6J and FVB/N to produce embryos; four concentrations of DNA for injection (0.75, 1.0, 1.5, and 2.0 ng/µL, respectively) were utilized to microinject into the embryos. The size of BAC DNA fragment and the time from BAC preparation to injection were recorded, and the birth rate and positive rate of transgenic mice were compared. Results The positive rate of transgenic mice with FVB/N background was higher than that of C57BL/6J background (P < 0.05). When different concentrations of BAC DNA were adopted, the birth rates of C57BL/6J and FVB/N mice from high to low were 0.75 ng/µL>1.5 ng/µL>1.0 ng/µL>2.0 ng/µL. When the injection concentration was 1.5 ng/µL, the positive rates transgenic mice in C57BL/6J and FVB/N newborns were the highest, while the positive rate was 0 in C57BL/6J newborns when the injection concentration was 0.75 ng/µL and 2.0 ng/µL. When different concentrations of BAC DNA were adopted, the positive rate of transgenic mice in FVB/N newborns from high to low was 1.5 ng/µL>1.0 ng/µL>2.0 ng/µL>0.75 ng/µL. When the BAC fragment size was 197 kb, the mice birth rate was highest at (22.49±9.41)%, and when the fragment size was 99 kb, the birth rate was lowest at (13.61±15.65)%. When the BAC fragment was 197 kb, the transgenic mice had the highest positive rate of (13.56±12.88)%, extremely significantly higher than that for 114 kb (P < 0.01) and significantly higher than that for 99 kb (P < 0.05). The storage time of BAC injection was not linearly associated with the positive rate of transgenic mice. Conclusion Compared with C57BL/6J, mice with FVB/N background appear more suitable for the generation of BAC transgenic mice. When the concentration of BAC DAN injection is 1.5 ng/µL, the positive rate of transgenic mice is the highest. BAC fragment size is not a constraining factor for positive transgenic mice. The highest efficiency is expected when the BAC DNA is injected within one week after preparation.



Improvement and Effect Evaluation on Method of Tracheal Instillation in Rats

YU Yixiang, CAO Shanshan, FENG Yi, LI Jiarui, WANG Rong

2021, 41(6):  543-546.  DOI: 10.12300/j.issn.1674-5817.2021.031

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Objective To establish a new method for observable, easy and accurate non-invasive intratracheal instillation in rats. Methods Disposable medical syringes and capillaries were used to make the sampler. Optical fiber was used to achieve visualization. Soap water was used to check if the intubation is correct. Sampler was introduced into rat trachea through endotracheal tube. Twenty rats were randomly divided into two groups and instilled with 0.9% saline solution or fine particulate matter (PM_2.5) suspension, respectively. Results The operation were performed successfully for 120 times in the 20 rats. HE staining was observed after 24 h of the last instillation. The pathological morphological changes of the lungs were induced by intratracheal instillation of PM_2.5, which were similar to those of chronic obstructive pulmonary disease (COPD). Conclusion The improved method of non-invasive intratracheal instillation is simple, rapid, and safe, and requires no special equipment, which can be widely used in experiments for respiratory tract administration.



Research and Application Progress in Visualized RPA-LFD Nucleic Acid Detection Technology

YU Lingzhi, TAO Lingyun, WEI Xiaofeng

2021, 41(6):  547-553.  DOI: 10.12300/j.issn.1674-5817.2021.019

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Recombinase polymerase amplification (RPA) is a novel isothermal nucleic acid amplification technology. Compared to PCR, it features ease of use, high efficiency, high sensitivity, high specificity, and does not need specific instruments, which allows it as an alternative to PCR and the most promising tool for rapid molecular diagnosis. RPA combined with lateral flow dipstick (LFD) (RPA-LFD) enables visual detection of amplified products and has promising applications for rapid nucleic acid detection of pathogens on site. The novel technique provides a new method for quality supervision and inspection of laboratory animals. In this paper, we reviewed the principle, research status, and technical difficulties of RPA-LFD, as well as the progress in rapid extraction of nucleic acid on site.



Laboratory Animal Management

Exploration on Informatization of Laboratory Animal Management Process in Colleges and Universities: The Case of Zhejiang University

CHEN Xiaojuan, LI Wei, WANG Lie

2021, 41(6):  554-558.  DOI: 10.12300/j.issn.1674-5817.2021.045

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Laboratory animal husbandry management, biosafety risk assessment, and ethical review of laboratory animals are core tasks of university laboratory animal centers. The Laboratory Animal Center of Zhejiang University reformed the informatization of the laboratory animal management process based on the framework of information management system combined with its own operation characteristics. It has established a full-process informatization system for supervising from project review, project implementation, to project final approval. The system has improved the efficiency of husbandary management, and strengthened the biosafety and welfare ethics of laboratory animals, providing a new idea for the management of laboratory animals in colleges and universities