Table of Content

25 June 2019, Volume 39 Issue 3

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Genetically Modified Rabbit Models for Medical Sciences

XUE Ying, FAN Jiang-lin, LIU En-qi

2019, 39(3):  169-177.  DOI: 10.3969/j.issn.1674-5817.2019.03.001

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Genetically modified (GM) rabbits have been proved to be excellent animal models for biomedical research. GM rabbits expressing human genes have been widely used as models for cardiovascular disease, AIDS, and cancer research. So far, GM rabbits were almost generated by pronuclear microinjection, which randomly leads to additive genes integrated in the rabbit genome. Compared to this technology, gene targeting in ES cells is more powerful tool for deepening understanding of gene function. However, the rabbit lack stable ES cell lines. Thus, gene targeting, dependent on ES cells, is not possible used in rabbit. Instead, RNA interference is quickly becoming a valuable experimental tool that allows investigators to knock down the expression of specific genes, and makes it possible to create GM rabbit models. Recently, with the advent of novel genetic technologies, such as Zinc-finger nucleases, transcription-activator like effector nuclease, the RNA-guided CRISPR-Cas endonuclease, there have been a significant breakthroughs in gene targeting of rabbit. Researchers have successfully created some gene knock out rabbits using Zinc-finger nucleases, or transcription-activator like effector nuclease, or the RNA-guided CRISPR-Cas endonuclease. Based on our study in the area of GM rabbits, in this paper, we review the progress of GM technology in rabbits during the past years and emphasize their applications as a model in biomedicine.



Cloning and Analysis of Tree Shrew Mfsd2a Gene and Detection of Its Expression in Different Tissues

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

2019, 39(3):  178-186.  DOI: 10.3969/j.issn.1674-5817.2019.03.002

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Objective To clone and sequence the major facilitator superfamily domain containing 2a(Mfsd2a) gene of tree shrew and analyze its bioinformatics characteristics, and quantitatively detect its expression in different tissues. Methods Total RNA was extracted from the brain and spinal cord of tree shrews, Mfsd2a gene was amplified by RT-PCR. Bioinformatics software was used to analyze its sequence characteristics, system evolution, phylogenetics, structure and physicochemical properties of the encoding proteins. The expression of Mfsd2a gene in different tissues were detected by qPCR using β-actin as an internal reference. Results The full-length of Mfsd2a gene fragment was 1 796 bp, which was highly consistent with the tree shrew Mfsd2a gene sequence published on NCBI. The coding sequence was 1 464 bp, which encoded 488 amino acids. It was also homologous to the MFS transporter superfamily. The tree shrew Mfsd2a protein had a distinct hydrophobic region, but no signal peptide, and its secondary structural elements were consist of alpha helix, beta sheet, random coil and extended strand. Twelve transmembrane structures were found by OCTOPUS analysis. Two N-glycosylation sites were predicted by modified structure. Subcellular localization revealed that it may be mainly distributed in cell membrane and endoplasmic reticulum. qPCR results showed that Mfsd2a were expressed in different tissues of tree shrew, and the it was relatively high in brain and spinal cord. Conclusion The Mfsd2a gene was successfully cloned, and a quantitative detection method for the expression of Mfsd2a was established, which might provide theoretical and technical support for further study of Mfsd2a gene function using tree shrew neurological disease model in the future.



Preliminary Construction and Analysis of Right Ventricular Overload Model in Newborn Rabbits

DING Lei, WANG Shou-bao, JING Hui, LI Yao

2019, 39(3):  187-192.  DOI: 10.3969/j.issn.1674-5817.2019.03.003

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Objective To construct a neonatal rabbit model of right ventricular overload for studying the pathophysiological remodeling of right ventricle in congenital heart disease patients with right ventricular overload. Methods Thirty neonatal rabbits were selected and randomly divided into two groups. According to different surgical procedures, they were divided into surgery of pulmonary artery banding (PAB) group and sham surgery group, each group with 15 rabbits. The surgical methods of the PAB group were as follows: horizontal thoracotomy was performed through dissecting intercostal muscles and splitting the sternum along the intercostal space. Pulmonary artery was banded by a 6-0 silk thread. At 14 days after surgery, the constriction of pulmonary artery was confirmed by echocardiography. The right ventricular systolic and diastolic pressures were measured by cardiac catheterization. The histological changes of the hearts from sham and PAB group were evaluated after Hematoxylin and Eosin staining. Results Right ventricular overload was successfully established by constriction of the pulmonary artery in neonatal rabbits with the survival rate 73.33% at 14 days after surgery. Relative to a sham operation group, the peak pressure gradient across pulmonary artery constriction, right ventricular systolic and diastolic pressures, and free wall thickness were significantly increased in PAB group at 14 days after surgery (P<0.01). Conclusion The right ventricular overload model was successfully established through pulmonary arterybanding in neonatal rabbits, which may be used for studying the pathophysio-logical changes of congenital heart disease with right ventricular overload.



Preliminary Phenotypic Analysis of Myh13 Knockout Mouse

HE Yi-min, GU Ming-min

2019, 39(3):  193-200.  DOI: 10.3969/j.issn.1674-5817.2019.03.004

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Objective A preliminary phenotypic analysis of the Myh13 knockout mouse model was performed in order to investigate the biological function of Myh13. Methods Expression of Myh13 in mice was determined by qRT-PCR and Western blotting. Validation of Myh13 knockout mouse was analyzed by gene sequencing, qRT-PCR and Western blotting. Genotypic ratio in the offsprings from the self-cross of heterozygotes was calculated. Changes in body weight and serum biochemistry parameters were measured. Histopathological features of mouse extraocular muscles were displayed by H&E staining and transmission electron microscopy examination. Results In mice, Myh13 is specifically expressed in extraocular muscle. It was verified that the target gene in Myh13 knockout mouse was successfully deleted at DNA, RNA and protein levels. Genotypic ratio of heterozygotes in the offsprings was consistent with Mendel’s law. No significant differences existed among different genotypes in body weight or serum biochemical analysis. HE staining revealed no remarkable change in homozygous and heterozygous mouse extraocular muscles compared to wild-type mouse extraocular muscles. Electron microscopy of mouse extraocular muscles showed that fat droplets deposited in heterozygotes and homozygotes. Sarcoplasmic reticulum dilation was also identified in homozygotes. Conclusions Knockout of Myh13 can alter the microstructure of extraocular muscles in mice, which may affect the function of extraocular muscles.



Mechanism of Circulating Endothelial MicroRNA Related to Atherosclerosis Induced by Inflammatory Response of Macrophages in Mouse

SUN Li-hua, ZHANG Ying, CAO Gui-qiu, LI Peng, HU Qiang, ZHANG Ya-ling, XING Shi-feng

2019, 39(3):  201-207.  DOI: 10.3969/j.issn.1674-5817.2019.03.005

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Objective To study the mechanism of microRNA related to circulating endothelial particulates (EMPs) on atherosclerosis (AS) induced by inflammatory response of macrophages. Methods There were 206 male ApoE^-/- mice of SPF grade and 50 control groups. The experimental group was randomly divided into three groups: AS model (AS) group, NC-miRNA group and miRNA-19b inhibitor group with 52 in each group, using oil red staining of atherosclerotic lesions in the histology were observed, calculated plaque area ratio, and detect the serum cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) biochemical index expression level of TNF alpha ELISA to detect vascular inflammation factors, IL-1, the change of IL-6 and IL-10, bcl-2, cleaved-PARP and Bax expression were detected by Western blot, mir-19b expression was detected by real-time quantitative PCR, and the apoptosis rate of macrophages was detected by flow cytometry. Results Micrornas-19b inhibitor group can decrease due to the increase in the thickness of the blood vessels of AS, reduce atherosclerotic plaque area percentage, the blood lipid level of TC, TG and LDL-C in the lower, HDL-C level has increased, promoting apoptosis related proteins Bax, cleaved - PARP expression decreased, and resistance to apoptosis related proteins increased the expression of Bcl-2 and proinflammatory factor in vascular IL-1, IL-6 and TNF-〈 level will be significantly lower, anti-inflammatory factor of IL-10 level is higher, At the same time, the apoptosis rate of endovascular macrophages decreased. Conclusion The presence of circulating endothelial microcore-related mirna-19b can reduce the apoptosis of macrophages, and promote the development of peripheral inflammation by up-regulating pro-inflammatory factors, thus promoting the development of AS.



Comparative Analysis of Rabbit Carotid Atherosclerosis Models Established by Collar And Air-drying Method

WANG Cong, CHEN Xiao-xue, YANG Shao-ling, WANG Feng-ling, FAN Lin-yan, HE Qian-qian

2019, 39(3):  208-212.  DOI: 10.3969/j.issn.1674-5817.2019.03.006

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Objective To compare the effect of air-drying and collar method on establishment of carotid atherosclerotic plaque in rabbits. Methods Rabbits were randomly divided into two groups: an air-drying group and a collar group with 10 subjects each. After operations, they were fed with fat-rich diet. The plasma levels of total cholesterol (TC), triglyceride (TG) and low density lipoprotein (LDL) were measured by ELISA in the 0th, 4th, 8th week, and the carotid artery was scanned by ultrasound in the 4th, 8th, 12th week. All rabbits were sacrificed at the end of experiment time. The morphology of the plaques was observed after HE staining. Results With high fat diet, the injured carotid of both groups were thickened and attached by strongly echoed plaques. HE staining showed a large number of foam cells, and all of these phenomenon showed earlier and more serious in the silicone collar group when compared with the air-drying group. Conclusion Carotid artery silicone collar method is safer and more effective than air-drying way in leading to the formation of atheromatous plaque.



Experimental Study on Proliferation, Apoptosis and Migration of Eyelid Fibroblasts in B6-Co Mice

JING Jin, ZHANG Hai-jun, DU Li-li, JI Gui-qing, SHAO Yi-xiang

2019, 39(3):  213-219.  DOI: 10.3969/j.issn.1674-5817.2019.03.007

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Objective To explore the mechanism of eyelid incomplete closure at birth in C57BL/6J-corneal opacity (B6-Co) mice. Methods The eyelid fibroblasts of B6 and B6-Co mice were isolated, cultured and identified. CCK8 method was used to detect the proliferation of eyelid fibroblasts in B6 and B6-Co mice. Apoptosis of eyelid fibroblasts in B6 and B6-Co mice was detected by Annexin V-FITC. Transwell was used to detect the migration ability of eyelid fibroblasts in B6 and B6-Co mice. Results The eyelid fibroblasts of B6 and B6-Co mice were successfully cultured. The proliferation and migration of eyelid fibroblasts from B6-Co mice were significantly lower than those of B6 mice(P<0.01). The apoptosis of eyelid fibroblasts in B6-Co mice was significantly higher than that of B6 mice (P<0.01). Conclusion The proliferative capacity, apoptosis level and migration ability of eyelid fibroblasts in B6-Co mice were significantly different from those of B6 mice.



Development of a Method for Determination of Cytotoxicity Activity of Mouse Splenic Lymphocytes on Human HepG2 Cells

CHEN Li-Ling, ZHONG You-Bao, LIU Xuan, CHEN Lai, YUAN Ke-Wang, HUANG Li-Ting, LI Shan-Shan

2019, 39(3):  220-225.  DOI: 10.3969/j.issn.1674-5817.2019.03.008

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Objective To develop a method for evaluation of the cytotoxicity activity of mice splenic lymphocytes to human HepG2 cells based on flow cytometry(FCM), in addition to ensure the efficiency of evaluation of the cytotoxicity activity of mice splenic lymphocytes to human HepG2 cells in the process of establishing an animal model of human tumor xenotransplantation, which can preserve the biological characteristics of human tumors, meanwhile, has relatively normal immune function. Methods Human HepG2 cells were labeled with CFSE (carboxyfluorescein diacetate, succinimidyl ester) and treated with anhydrous ethanol to mimic the cytotoxic effect of cytotoxic killer cells, then determined by FCM after propidium iodide(PI) staining, based on which the concentrations of CFSE and PI and time for treatment were optimized. The lymphocytes were isolated from spleen of mice as effector cells and human HepG2 cells were target cells. A procedure was developed based on the optimization of conditions including the action time and ratio between effector and target cells. Results The cells were divided into CFSE⁺PI^-, CFSE⁺PI⁺, CFSE^-PI⁺ and CFSE^-PI^- groups by CFSE/PI staining, and the survival and killed cells were well distinguished. The concentration of CFSE was 2.5µmol/mL, the optimal action time between effector and target cells was 12 h, and the effector to target cell ratios of 10∶1 was adopted. Conclusions A method for evaluation of the killing activity of mice splenic lymphocytes to human HepG2 cells based on FCM was successfully developed, which provided a reference for evaluating the killing effect of animal splenic lymphocytes on xenogeneic cells.



An Modified Eight-arm Maze Experiment Method in Behavioral Research

LIU Bo, WANG Yang, LI Zhi-jie

2019, 39(3):  226-230.  DOI: 10.3969/j.issn.1674-5817.2019.03.009

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Objective To identify a simple and effective experimental method for eight-arm maze study. Methods Twenty adult male SD rats were divided into a traditional method group (control group) and a modified electric heating pad method group (test group) (n=10). The bait was only placed at the end of feeding arm in control group. The test group had the same conditions as control group except that the heating pad was placed at the end of the non-predator arm as a punishment tool. Recorded and compared the moving distance, time, speed and the entrance times of rat in the arms via software. Results Compared with the control group, the test group rats shortened the movement distance and explored time significantly, and reduced the errors times into the wrong arm significantly as well. Conclusion Compared with the traditional method, the modified electric heating pad method can evaluate the spatial memory learning ability of rats after 7-days-training more accurately and effectively, which can be used as an effective experimental method.



Detection of Micronucleus Rate of Mouse Bone Marrow Cells Induced by M-phenylenediamine and P-phenylenediamine with Acriding Organe- Flow Cytometry

CHEN Xiu-juan, JIANG Yi, LI-Min, LI-Peining, SUN-Xia, CHEN Zi-ling, Qiu Zhi-feng, HUANG Yu-feng

2019, 39(3):  231-235.  DOI: 10.3969/j.issn.1674-5817.2019.03.010

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Objective To detect the micronucleus rate of m-phenylenediamine and p-phenylenediamine in mouse bone marrow cells using the single-laser flow cytometry, and an automated experimental method for rapid detection of micronucleus rate in the laboratory was established to investigate the mutation detection of flow cytometry in cosmetic raw materials. Methods Mice were treated with m-phenylenediamine and p-phenylenediamine at doses of 25.0mg/kg、12.5mg/kg、6.25mg/kg、3.12 mg/kg respectively. Flow cytometry was used to detect the polychromatic erythrocyte micronucleus in mouse bone marrow compared with the results of traditional microscopy. Results The number of Micronucleated polychromatic erythrocytes in the test group was increased correspondingly, and the micronucleus rate was increased with the concentration, showing a good dose-effect relationship. Flow cytometry showed a good correlation with the micronucleus rate obtained by conventional microscopy (r = 0.956, P<0.01). Conclusions Flow cytometric analysis is objectivity, specificity and high efficiency. It can replace the traditional microscope to detect the incidence of cosmetic raw materials on the micronucleus of mouse bone marrow cells and evaluate its mutagenicity.



Diagnosis and Treatment for Intussusceptions in One Case of Cynomolgus Monkey

LI Liang-shun, PAN Shi-you, ZHI Yuan-fang, BAI shuai-zhou, SUN Jian-hua, GONG Li-kun, REN Jin

2019, 39(3):  236-238.  DOI: 10.3969/j.issn.1674-5817.2019.03.011

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Intussusception occurred in a cynomolgus during a repeat dose toxicity study. Intussusception of cynomolgus was diagnosed by using the methods of clinical observation, hematology and plasma chemistry analysis, palpation and bultrasonic. Reset the intussusceptions by manually in vitro. The actions of supplying energy and antispasmodic drug and dietary restriction were performed for nursing. The animal recovered very well, and the toxicity study proceeded as scheduled. The results indicated that the inchoate intussusception could be correctly diagnosed by using the methods of palpation and bultrasonic; and the inchoate intussusceptions could be effective treated by manually in vitro, and the repeat dose toxicity study wouldn’t be influenced.



Research Progress on Zebrafish Model of Retinoblastoma and rb1 Gene

WANG Li-mei, ZHANG Jian, LIU Zhong-hua, YANG Wei

2019, 39(3):  244-248.  DOI: 10.3969/j.issn.1674-5817.2019.03.013

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Retinoblastoma (RB) is a common primary malignant tumor that occurs in the eyes of infants and young children. A large number of studies have shown that the change of rb1 gene is the main cause of RB occurrence. The zebrafish RB model is a novel animal model for studying RB. In this paper, the latest research results of rb1 gene, as well as the zebrafish RB model produced by xenograft method, gene mutation method, and TALEN gene editing method are reviewed. Furthermore, the RB zebrafish model was compared with the four kinds of reported RB animal models that were raised on mice, rat, rabbit or xenopus.

Review of Spontaneous and Drug-related Islet Fibrosis in Rats

ZHOU Fei, WANG Hao-an

2019, 39(3):  249-252.  DOI: 10.3969/j.issn.1674-5817.2019.03.014

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Islet fibrosis is a common spontaneous disease in rats. The incidence increased with age as well as that of male was higher than female. The pathogenesis and mechanism of this disease have not been conclusive yet. Many drugs could also induce or increase fibrosis in pancreatic islets in rats. Vascular injury in islet was considered to be associated with the occurrence of the disease. This article summarizes the research of spontaneous and drug related pancreatic islet fibrosis in rats, and provides some references for experimental animal research and toxicological pathologists.