Table of Content

25 February 2019, Volume 39 Issue 1

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Advantages and Challenges of Tree Shrews in Biomedical Research

JIA Jie, DAI Jie-jie

2019, 39(1):  3-8.  DOI: 10.3969/j.issn.1674-5817.2019.01.002

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Animal model is an important method to study the mechanism of human disease, to screen drug, and to evaluate the safety and efficacy of drugs. With the development of molecular biology technology and the study of molecular genetic mechanism of human diseases, it is difficult to study the complex symptoms of human diseases in biomedical research using rodents as a result of the differences in genetic background, physiological and metabolic characteristics between rodent and human, the limitation of rodent in life science research is increasingly prominent. Primates play an irreplaceable role in biomedical research due to its evolution and biological characteristics. However, the limitation of genetic manipulation, long breeding cycle and high cost restrict the application of primates in biomedical research. The evolution of tree shrews are more closer to the primate than rodents. Tree shrews has been increasingly applied in the field of biomedical research since its characteristics of easy breeding, fast reproduction, the low cost and the key factors of neurological dysfunction and infectious diseases are conservative compared with primates. Based on the characteristics of physiological metabolic process and anatomical structure of tree shrews, this paper analyzes the advantages and problems of tree shrew in biomedical research.

Behavior Detection Method of MPTP Induced Tree Shrew Parkinson’s Disease Models

ZHANG Zhi-cheng, SONG Qing-kai, LI Xiao-hui, LI Na, YUAN Yuan, HUANG Xin, WANG Xuan, LI Ming-xue, DAI Jie-jie

2019, 39(1):  9-14.  DOI: 10.3969/j.issn.1674-5817.2019.01.003

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Objective To establish the acute Parkinson’s disease(PD) model of tree shrew, the Catwalk and force plate actimeter (FPA) detection system was used as a method to evaluate PD behavior. Methods Totally 14 adult conventional grade tree shrew(Tupaia belangeri), weighing (147.6±17.07) g, were randomly divided into model group (n=10) and control group (n=4). The PD model of tree shrews was established by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) drugs, and control group was injected with equal volume of saline. Results The acute PD model of tree shrews was successfully prepared. Compared with control group, the model group spatial utilization index of tree shrews decreased and the bout of low mobility (BLM) index increased significantly (P all<0.001) after 1 day dosing, indicate that the spatial utilization ability of the model group was impaired and the activity degree was decreased; The maximum contact mean intensity of the right front paws(RF), right hind paws (RH), left front paws (LF). the mean intensity of RF, RH, LF and force average of limbs are decline (P all<0.05).That indicate the increased muscle stiffness of tree shrews, which can be used as a quantitative indicator of PD gait analysis; Tremor indices 1 (0~15 Hz) was significantly enhanced (P<0.01), which could better simulate the clinical manifestations of PD patients with rest tremor. Conclusion MPTP induced tree shrews acute PD model can better simulate the early PD clinical manifestations. Catwalk and FPA analysis systems provide systematic, Objective. and reliable PD behavioral data.



Isolation and Identification of Spinal Astrocytes from Tree Shrews

WANG Xuan, WANG Wen-guang, LI Na, YUAN Yuan, ZHANG Zhi-cheng, SUN Xiao-mei

2019, 39(1):  15-20.  DOI: 10.3969/j.issn.1674-5817.2019.01.004

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Objective To establish an effective method of isolation, identification and primary culture method for astrocytes derived from the spinal cord of tree shrews in vitro, providing experimental materials for in vitro studies of spinal astrocytes. Methods Neonatal tree shrews were euthanized to collect spinal cord tissue, the spinal cord membrane and blood vessels were removed under a dissecting microscope to reduce the effects of fibroblasts, vascular endothelial cells and red blood cells. Then, the spinal cord tissues were digested with trypsin and DNase I, and cultured. According to the difference in the adherence time of astrocytes, fibroblasts and microglia, the fibroblasts were removed by differential adherence speed. After the cells were confluent, the non-staple gel was removed by shaking with a constant temperature shaker. Neurons were removed by three consecutive purifications. The cultured cells were identified by immunofluorescence staining of glial fibrillary acidic protein (GFAP). Results The astrocytes were isolated by differential adherent culture after trypsin and DNaseI digestion. The isolated cells had typical protoplasmic and fibrous characteristics, the protoplasmic type was polygonal, the protrusions were few and strong, the fibrous protrusions are abundant and spread outward. The cellular immunofluorescence results showed that GFAP expression is positive, and the purity could reach 95%. Conclusion The isolation and purification of spinal cord astrocytes from tree shrews are successfully established.



The Construction of Ring Finger Protein 126 Gene Knockout Mouse by Using CRISPR/Cas9 Technique

GAO Meng-qiao, AI Dong-xu, LI Yu, SUN Fei, WANG Jin, FAN Jun-wen, YUAN Zheng, LIU Yuan, SUN Zhao-zeng

2019, 39(1):  21-25.  DOI: 10.3969/j.issn.1674-5817.2019.01.005

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Objective To establish ring finger protein 126 (RNF126) gene knockout mouse model by CRISPR/Cas9 technique and predict the biological function of RNF126 gene. Methods The knockout primers were designed for exon 4 of Rnf126 gene of C57BL/6 mice to construct sgRNA recombinant expression vector. The mixture of sgRNA and Cas9 mRNA , obtained through transcription in vitro, was injected into the fertilized eggs of C57BL/6 mice by microinjection and the fertilized eggs were transferred into surrogate mice. The DNA of offsprings were screened by PCR identification. Results SgRNA for Rnf126 gene and Cas9 mRNA were successfully obtained. A total of 82 fertilized and microinjected zygotes were successfully transferred into 3 surrogate mice. 11 offsprings were obtained in F₀ generation. One of the positive mouse with 62 bases missing as the founder was selected for propagation and the mutation was also detected in F₁ and F₂ generations. Conclusion Rnf126 gene knockout C57BL/6 mouse model was successfully derived, it may be used as a tool for studying the biological function of RNF126 gene.



The Silence Effect of siRNA Sequences Targeting on Vegf/Vegfr Genes in Rat Endothelial Cells

CHEN Lin-zhong-ri, YIN Dong-lai, ZHAO Ze-ting, WEI Qian-han, CHEN Xin, ZHANG Yu-mei

2019, 39(1):  26-33.  DOI: 10.3969/j.issn.1674-5817.2019.01.006

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Objective The effective siRNA sequences targeting on Vegf/Vegfr were screened for interference the expression of Vegf and Vegfr genes. Methods In this study, three siRNA sequences each gene were designed based on the gene of rat Vegf, Vegfr1 (Flt1) and Vegfr2 (Flk1). The effects of gene silence of these siRNA nucleotides on its target gene were evaluated by Western Blot and RT-PCR after transfection in rat endothelial cells. Result VEGF can be silenced by two siRNA sequences target on Vegf gene, FLT1 and FLK1 can be effectively depressed by one sequence target on Flt1 and Flk1 genes respectively. Conclusion The effective sequences were screened and could be used in angiogenesis research of VEGF/VEGFR signaling.



Establishment of Loop-mediated Isothermal Amplification for Detection of Feline Panleukopenia Virus

MA Qin, YAN Wen-zhuo, ZHOU Jie, GAO Cheng, LIU Tie-long, ZHAO Li-li, CHEN Hong-yan, LU Tao-feng

2019, 39(1):  34-38.  DOI: 10.3969/j.issn.1674-5817.2019.01.007

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Objective To establish a rapid, convenient detection method of feline panleukopenia virus (FPV) by loop-mediated isothermal amplification (LAMP). Methods Two pairs of primers were designed according to the conserved sequences of FPV in Genbank. The efficient LAMP amplification conditions were determined by optimizing temperature and time. Results The results showed a highly efficient amplification for FPV nucleic acid which performed at 65 ℃for 60 min. It also showed a high sensibility with a detection limit of 5.01×102 copies/µL, 10 times higher than the conventional PCR detection in the sensitivity. There was no cross reaction with feline herpesvirus type 1(FHV-1), canine parvovirus(CPV) and vanine adenovirus (CAV). Conclusion The established LAMP method is good in duplication, stability, specificity, and sensitivity. This method can be used for the fast detection of FPV nucleic acid in clinical samples from cats.



Establishment of Two Kinds of Lung Cancer Models in Mouse and Evaluation by Micro PET-CT

SHEN Yan, WANG Yun, ZHANG Ru, RUAN Zheng, MAO Jian-hua

2019, 39(1):  39-45.  DOI: 10.3969/j.issn.1674-5817.2019.01.008

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Objective To compare the difference of two kinds of lung cancer models in mouse, which were established by injecting with the human non-small cell lung cancer (NSCLC) cells through tail vein or orthotopically inoculating in the lung using a microsyringe through the intercostals space. Methods The NCI-H1975 cells of NSCLC were injected into the NOD-SCID mouse via tail vein or intercostals space. After 15 days, mice were scanned by micro PET-CT to monitor the tumor growth and metastasis. Then mice were sacrificed and their lungs were dissected, fixed with phosphate-buffered neutral formalin and prepared for standard histological examination. Results Mice that were injected with the NCI-H1975 cells through tail vein for 15 days did not form tumor nodules on the lung. However, the mice that were orthotopically inoculated in the lung through intercostals space significantly had tumor nodules in their lungs. Conclusion A NOD-SCID mouse lung tumor model can be successfully established by transplanting NCI-H1975 cells into the lung through intercostals space. This model is simple, repeatable, and efficient.



Effect Evaluation on Rabbits Free Flaps with Locally Applying Iodine Tincture

HOU Dong, CHENG Shao-hua, SONG Yang, SONG Peng-fei, WANG Lei, XUE Hui-chao

2019, 39(1):  46-51.  DOI: 10.3969/j.issn.1674-5817.2019.01.009

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Objective To investigate the impact of local 2% iodine tincture application on healing wounding flap and reducing seroma formation following a model created in rabbits. Methods Thirty-two New Zealand rabbits were randomly divided into groups A, B, C and D, group A and group B as control groups, group C and group D as treatment groups. The flaps of treatment groups were treated with 2% iodine tincture, and the control groups with normal saline. group A and group C were sacrificed on the third day after surgery, while group B and group D on the fifth day. We analyzed the results of weight, leukocyte count, neutrophil percentage, seroma volume of all groups and we evaluated tissue samples using histopathological examinations. Results Seroma formation was significantly decreased in groups C, D compared with groups A, B (P<0.05). The proportion of hyperplastic collagen fibrils and hyperplastic vessels number were both significantly higher in groups C, D than that of groups A, B (P<0.001). There is no significant difference among the four groups in terms of weight, leukocyte count and neutrophil percentage. Conclusion Two percent iodine tincture promoted angiogenesis and collagen synthesis of free flap, reduced seroma formation and accelerated wound healing of New Zealand rabbits.



Experimental Evaluation of Cornu Cervi Pantotrichum Intervention in Ovariectomized Rats with Osteoporosis

QU Lei-ming, GONG Wei

2019, 39(1):  52-55.  DOI: 10.3969/j.issn.1674-5817.2019.01.010

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Objective Traditional Chinese medicine show that kidney stores the essence, generates the marrow and nourishes the bones. Bone growing development are closely related to kidney essence, brain and bone marrow. This study come to revealed the regulation mechanism of Cornu Cervi Pantotrichum. Method Experimental rat models were established by removing ovaries. Rats randomly divided into five groups: the normal group, model group, Xianlinggubao group, Bujiale group, Cornu Cervi Pantotrichum group. After 12 weeks treatment, femoral bone mineral density (BMD) was measured by dual energy X-ray absorbinmetry. The activities of transforming growth factor-β1 (TGF-β1) and TGF-β induced early growth response 1 (TIEG1) in the bone, kidneys and hypothalamus was measured through ELISA. Results After 12 weeks treatment, femoral BMD in model group was significantly decreased (P<0.05)compared with normal group, the femoral BMD in Cornu Cervi Pantotrichum group was much higher than the model group (P<0.05), the activities of TGF-β1, TIEG1 in the bone, kidney and hypothalamus of model group rat was significantly decreased (P<0.05) compared with the normal group, the activities of TGF-β1、TIEG1 in the treatment groups were much higher than the model group, the femoral BMD in Cornu Cervi Pantotrichum group was much higher than the model group (P<0.05). Conclusion One of the main pathological mechanisms in osteoporosis induced by ovarian removal is considered to be the abnormal activities of TGF-β1, TIEG1, possibly involving the feedback regulation mechanism of the hypothalamus-kidney-bone. Cornu Cervi Pantotrichum may control the disease by regulating the hypothalamus-kidney-bone feedback mechanism.

Establishment and Quality Control of SPF Golden Hamster Colonies

ZHANG Hua-qiong, XIA Shuang, WU Yan-ru, HE Fan

2019, 39(1):  56-60.  DOI: 10.3969/j.issn.1674-5817.2019.01.011

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Objective To establish high-quality SPF golden hamster colonies in accordance with WHO pre-certification of live attenuated Japanese Encephalitis Vaccine. Methods A new SPF golden hamster colony was established by caesarean based on the established mating method. The colonies were under general quality control with regular sampling tests. Results The foundation stocks and production stocks of SPF golden hamsters were established, which were negative in 19 virological items、6 parasitological items and 17 bacteriological items. Conclusion A new SPF golden hamster colony is successfully established, the quality meets the requirements of WHO TRS 980.



The Current Situation and Recommendations in Laboratory Animal of Shanxi Province

PANG Wen-biao, Xu Guo-qiang, CHEN Zhao-yang, SONG Guo-hua

2019, 39(1):  61-64.  DOI: 10.3969/j.issn.1674-5817.2019.01.012

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This paper makes a detailed investigation on current situation of laboratory animal in Shanxi province. The article further analyzed the problems existing in the laboratory animal in our province and put forward corresponding countermeasures and suggestions to promote healthy and sustainable development of the laboratory animal industry in Shanxi Province.

Research Progress of Animal Model Establishment and Gene Editing on Plasmodium falciparum

KUANG De-xuan, WANG Wen-guang, SUN Xiao-mei, DAI Jie-jie

2019, 39(1):  65-71.  DOI: 10.3969/j.issn.1674-5817.2019.01.013

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Plasmodium falciparum is the pathogen of human malaria. The animal model of plasmodium falciparum is an important experimental tool to study the infection process of plasmodium falciparum, the mechanism of chronic pathogenesis, the evaluation of drugs and the vaccine on plasmodium falciparum. Up to now, the animal model research of plasmodium falciparum mainly involves non-human primates, immuno-deficient mice, transgenic technology and CRISPRR/Cas9 system. In this paper, the animal model of plasmodium falciparum in vitro/vivo infection and gene editing were summarized and analyzed to provide reference for the establishment of animal model and gene editing of plasmodium falciparum in the future.

Research Advances in Rat Model of Endometriosis

JI Lian, MA Tie, LIU Dong-yan

2019, 39(1):  72-76.  DOI: 10.3969/j.issn.1674-5817.2019.01.014

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In order to establish reliable rat model to study the etiology, pathogenesis and treatment of endometriosis, this article reviewed the literature on the rat model of endometriosis, including the pathogenesis, modeling method, the refinement, evaluation criterion and the application of rat endometriosis model. This review may offer an important reference for choosing animal models to study endometriosis in the future.

The Research Progress of Biological Significance of Birds’ Call

YANG Li-qiong, XIE Jun, LIU Fang-fang, CHEN Jian, XU Fan

2019, 39(1):  77-82.  DOI: 10.3969/j.issn.1674-5817.2019.01.015

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Sound exists extensively in nature, it exists not only in various natural phenomena, but also in the communication between various creatures. There are not only the vibration of the vocal organs in birds’ call, but also the abundant neurobiological information. This article reviews the four types of bird calls and their biological significance, includmg begging call, song, alarm call and flight call.