Table of Content

25 August 2016, Volume 36 Issue 4

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Preparation and Determination of CD4 Polyclonal Antibodies of Tree Shrew

LU Kong-jie, XU Jing-wen, ZHANG Xue-mei, WU Zhong-xiang, REN Fang-fang, MA Na, GONG Wei, YAN Li-wei, ZHU Wen-bing, DONG Shao-zhong

2016, 36(4):  243-249.  DOI: 10.3969/j.issn.1674-5817.2016.04.001

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Objective To express tree shrew (Tupaia belangeri) CD4 protein in prokaryotic cells, purify the expressed product and prepare its polyclonal antibodies,and determinate CD4 protein in vivo. Methods CD4 gene was cloned and inserted into express vectors. And then recombinant plasmids pET30a (+)-CD4 and pGEX-5X-1-CD4 were constructed. The expressed recombinant proteins His-CD4 and GST-CD4 were expressed and purified. The polyclonal antibodies of CD4 protein were prepared with three different adjuvants including Al (OH)₃, MF59 and Freund’s. The serum antibody specificity was determined by Western blot. Results The expressed recombinant proteins His-CD4 and GST-CD4, with relative molecular masses of about 53.5×10³ and 70.0×10³ respectively. And the concentrations of proteins His-CD4 and GST-CD4 were 600 μg/ml and 1mg/ml respectively. Three groups of adjuvants all can induce antibodies. The comparison results of Geometric Mean Titer (GMT) is that the Freund’s adjuvant group (GMT: 97 420) showed the highest geometric mean of antiserum, followed by Al (OH)₃ adjuvant group (GMT: 67 202) and MF59 adjuvant group (GMT: 55128). The Freund’s adjuvant group was significantly higher than the original protein group (P<0.001). The Al(OH)₃ adjuvant group was significantly higher than the original protein group (P<0.01). The MF59 adjuvant group was higher than the original protein group (P<0.05). Conclusions Mouse antiserum with high specificity was prepared,and it could bind to CD4 protein of tree shrew in vivo, which laid a foundation of further study on the preparation of monoclonal antibody against CD4 and the research of CD4 function and immune related experiments of tree shrews as viral infection models.



Establishment of Orthotopic Transplant Model of Human Lung Cancer and Observation of In vivo Biofluorescent Imaging in Nude Mice

LIU Qian, LIU Song, Han Feng-feng, GUO Xue-jun

2016, 36(4):  250-256.  DOI: 10.3969/j.issn.1674-5817.2016.04.002

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Objective To establish orthotopic transplant models of human lung cancer and study the in vivo biological characteristics of human lung cancer with high and low metastatic potential cells in nude mice. Methods The low-metastatic human lung cancer cell line SPC-A-1 and high-metastatic human lung cancer cell line SPC-A-1sci were tranfected with green fluorescent protein/Luciferase (GFP/Luc) gene by lentiviral vector, they were orthotopically transplanted into the lung of nude mice. The tumor growth and metastasis were observed by using in vivo biofluorescent imaging system and anatomical methods. Results The human lung cancer cell lines with stable high-level GFP/Luc expression and orthotopic transplant animal models were successfully established. In vivo biofluorescent images showed that the luciferase expression rates of SPC-A-1-GFP-Luc and SPC-A-1sci-GFP-Luc cells were 50.0% (12/24) and 62.5% (20/32), respectively. The expression intensity and area of luciferase expression in SPC-A-1sci-GFP-Luc cells were higher than that in SPC-A-1-GFP-Luc cells. Anatomical examination showed that tumor formation rates of SPC-A-1 cells and SPC-A-1sci cells were 50.0% (12/24) and 62.5% (20/32), respectively. Meanwhile, the spontaneous metastasis rate was 25.0% (3/12) and 40.0% (8/20) in vivo. Conclusions Lung orthotopic transplant animal model could reproduce the biological characteristics of tumor growth and metastasis. In vivo biofluorescent imaging system was suitable for the objective evaluation of the biological characteristics.



Screening of Mouse Strain Susceptive to Chronic Stress Depression and Preliminary Research on Its Mechanism

XU Long-jin, WANG Kou-zhou

2016, 36(4):  257-262.  DOI: 10.3969/j.issn.1674-5817.2016.04.003

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Objective To screen mouse strain that susceptive to chronic unpredicted mild stress(CUMS) and preliminarily research its susceptive mechanism. Methods KM, ICR, BABL/c and C57BL/6 mice were used to establish CUMS depression model. Body weight, sucrose preference test(SPT), open field test (OFT) were used to evaluate the sensitivity of mice to CUMS. Hippocampal levels of glucocorticoid reccptor (GR), 5-hydroxytryptamine(5-HT), kynurenine(KYN) were measured and used for susceptive mechanism research. Results Body weight, SPT, horizontal movement score and vertical movement score of C57BL/6 mice were found to susceptive to CUMS. SPT, horizontal movement score and vertical movement score of BABL/c showed similar tendency. Hippocampal levels of GR and KYN of normal C57BL/6 mice were significantly lower or higher than that of normal KM, ICR and BABL/c mice. Conclusions The C57BL/6 mouse is most susceptive to CUMS among 4 strains tested, and hippocampal levels of GR and KYN may be the main mechanism responsible for its susceptibility.

Generation of Rag1 and Rag2 Deficient Mice and Their Application in Tumor Xenograft

SHEN Ru-ling, SHI Jia-hao, SHENG Zhe-jin, FENG Xiao-long, WU Wen-ting, FEI Jian

2016, 36(4):  263-269.  DOI: 10.3969/j.issn.1674-5817.2016.04.004

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Objective To establish recombination-activating genes, Rag1, Rag2 deficiency mouse models and study the possibility as tumor xenograft recipients. Methods The traditional embryonic stem (ES) cell targeting and blastocyst microinjection were performed to develop the Rag1 and Rag2 deficiency mouse models. The flow cytometry assay was conducted to analyze the T/B lymphocyte contents in peripheral blood. The tumorigenicity on different immunodeficiency mouse strains was assessed by exogenous tumor cell transplantation experiment. Results Rag1 and Rag2 deficiency mouse models were established. The T/B lymphocytes of peripheral blood in two strains of homozygous mutant mouse models were significantly reduced compared with those of wild type mice. A549 tumor cells had stronger tumorigenicity in Rag1, Rag2 mutant mice than that in BALB/c nude mice and NOD-SCID mice. Conclusion Rag1, Rag2 deficiency mouse models were successfully developed. Two mouse models display serious immune system defects, which could be used as exogenous tumor transplant recipients to establish tumor model.



Production of Recombinant Simian Type-D Retrovirus p27 Gene and Evaluation of its Diagnostic Potential

ZHOU Jie, YANG Yan-fei, HU Jian-hua, TAO Ling-yun, GAO Cheng

2016, 36(4):  270-275.  DOI: 10.3969/j.issn.1674-5817.2016.04.005

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Objective To Clone and express p27 gene of simian type-D retrovirus and evaluate its diagnostic potential. Method The p27 gene was amplified by RT-PCR and cloned into the prokaryotic expressive vector pET-28b(+) after sequencing. Recombinant plasmid was transformed into E.coli Rosetta DE3) and recombinant protein was analysed by SDS-PAGE and Western blot. The protein was purified affinity chromatography with Ni-NTA, and its diagnostic value was evaluated by ELISA. Results The highest soluble expression level of recombinant protein was obtained after being induced at 37℃ for 4 h, with the concentration of Isopropyl-β-D-thiogalactoside (IPTG) was 0.5 mmol/L, and the recombinant protein had immunological activity. After purified, the concentration of protein was 2.043 g/L, and the purity was 93.3%. The indirect ELISA was developed using the purified recombinant protein, detection of 3 standard positive sera and 22 negative sera showed the detection rate were 100%. Conclusion Recombinant p27 protein from prokaryotic expression system had perfect antigenicity, which would can be used as the candidate antigen of simian type-D retrovirus.



Effect of Acupoint Sticking Therapy on Eosinophils and Ultrastructure of Lung Tissue in Asthmatic Rats

QIAO Ming, YANG Zheng-ming, HUANG Qian, LIU Lan-ying

2016, 36(4):  276-279.  DOI: 10.3969/j.issn.1674-5817.2016.04.006

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Objective To observe the effect of acupoint sticking therapy on eosinophils infiltration and the lung tissue ultrastructure of asthmatic rats. Methods Using rat model of asthma which is established following Palmans’ method, we observed the effect of acupoint sticking therapy on the whole blood leukocyte count, eosinophils and ultrastructural changes in lung tissue. Results The whole blood leukocytes and eosinophils counts were significantly increased in asthma model group compared with the sham group. After treatment with acupoint sticking therapy, the whole blood leukocytes and eosinophils counts were significantly decreased and the ultrastructure of alveolar epithelial cells was also obviously improved in asthma rats. Conclusion Acupoint sticking therapy can reduce the whole blood leukocytes and eosinophils counts and alleviate inflammatory cell infiltration and alveolar epithelial cell necrosis in the lung of asthmatic rats to treat asthma.



Isolation and Primary Culture of Fibroblast-like Synoviocytes Using Modified Explant Culture Method from Rabbit with Aduvant Arthritis

CHEN Fang, CHEN Li-feng, LIU Qin, WANG Li-ping, ZHANG Yi

2016, 36(4):  280-284.  DOI: 10.3969/j.issn.1674-5817.2016.04.007

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Experimental Study on SV25T4 AcrySof IQ ReSTOR Multifocal Toric Intraocular Lens in Rabbits

FANG Li, ZHAO Jing-chuan, LI Xue-lai, ZHOU Sen-ting, ZHOU Qi-meng, FENG Qin, CHEN Xian-hua

2016, 36(4):  285-289.  DOI: 10.3969/j.issn.1674-5817.2016.04.008

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Objective To evaluate the intraocular biocompatibility by observing inflammatory reaction after implantation of SV25T4 AcrySof IQ ReSTOR multifocal toric intraocular lens (IOLs) in rabbits. Methods Left eyes were performed phacoemulsification with SV25T4 AcrySof IQ ReSTOR multifocal toric intraocular lens implantation and right eyes were performed phacoemulsification with SN6AD AcrySof IQ ReSTOR multifocal intraocular lens implantation in 24 rabbits. All of the eyes were examined with slitlamp microscope and measured the intraocular pressure at the 1st, 7th days and 1st,3rd,6th months postoperatively. The explanted IOLs were assessed for optical properties. The enucleated eyes were performed histopathological evaluations. Results There were mild corneal edema and anterior chamber flare in both eyes at 1stday postoperatively, obviously improved at 1st week postoperatively, there is no significant difference between two groups during the observation period (P>0.05). Optical testing items of the explanted IOLs were in accord with the requirement of YY0290.2-2009. Histopathological observation showed mild hyperemia of ciliary body and few lymphocytes infiltration. Conclusion The SV25T4 AcrySof IQ ReSTOR multifocal toric intraocular lens has good biocompatibility, meeting for the requirement of safety and validity as material of ocular implants in cataract surgery.



Observation on Growth and Development of Meriones meridianus jie in Indoor Breeding

LIAO Li-fu, XU Yi-mei, LI Wei, SHI Shen

2016, 36(4):  290-294.  DOI: 10.3969/j.issn.1674-5817.2016.04.009

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Objective To observe the growth and behavior of Meriones meridianus jie at 4th generation in domestication. Methods Body weight, body length, tail length, hind foot length and self thermoregulation ability were measured, and changes of the morphological characteristics and behavior were observed from birth to 150 days of M.meridianus. Results The changes of appearance morphology showed from glabrous to hair at the age of 0-5 days old. The hair growth and activity were increased gradually at 6-13 days old. The eyes began to open at 14 days old.The animal began to feed baits at 17 days old with increasing bait and decreasing milk gradually. The thermoregulatory mechanisms was not established before the age of 25 days old. The growth of body weight began to differentiation between female and male at 30 days old with the male slightly greater than the female.The female and male were grew slowly thereafter, and took part in breeding after 70 days old. Conclusion According to the behavior and growth characteristics of M. meridianus, growth and development of the animals is divided into four stages: suckling stage between the birth to 24 days old, juvenile stage between 25~40 days old, subadult stage between 40 to 70 days old, and adult stage over 70 days old.



Breeding of Inbred Dwarf Rats and its Main Biological Characteristics

YAO Ju-fang, HUA Zheng-yu, WANG Bin, SUN Zhi-kai, DAI Hui-li

2016, 36(4):  295-300.  DOI: 10.3969/j.issn.1674-5817.2016.04.010

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Objective To breed the inbred strain of dwarf rat and determine the main biological characteristics. Methods The spontaneous mutant litters looks like dwarfism from the Wistar conlony was bred and maintained by brother×sister (B×S) mating system for 22-23 progenies and nominatured as spontaneous dwarfism rat (SDWR) according the standard. As reference of strain, indexes of growth and development, body measurements, reproductive performance, organ weight, serum biochemical (12 items) and hematology (12 items) about dwarf rat at different age stages were measured, and be compared with background Wistar rat. Results Up to February 2016, the dwarf rat was inbreded continuously for 22-23 generations, and named as SDWR temporarily. The weight of weanling, youth and adult individuals in SDWR was respectively only about 45.7%, 37.5%, 36.2%(♂) and 49.7%, 41.7%, 40.3%(♀) of the same age Wistar rats (P<0.01). The body and tail length of youth and adult individuals in SDWR is respectively 77.5%, 72.3% (♂) and 73.8%, 68.9%(♀) of Wistar rats (P<0.01). The weights of major organs (heart, liver, spleen, lung, kidney, testis or oviduct tubes) in youth and adult SDWR are significantly lower than that of the same ages Wistar rats (P<0.01). Conclusions Through 22-23 progenies inbreeding, the established inbreeding strain of SDWR exhibited obvious dwarfism symptoms in growth, body measurements, reproductive performance and organs weight, and has stable heredity. The rat may be useful as animal models for the study of inherited dwarfism in human.



Effects of Different Drugs and Disinfectants on Sporulation of Coccidian Oocysts in Rabbit

WEN Fu-li, ZHANG Jia, ZHENG He-ping, MA Lei, ZHANG Shi-lan, WANG Shou-kun

2016, 36(4):  301-306.  DOI: 10.3969/j.issn.1674-5817.2016.04.011

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Objective To study inhibitory effect of different drugs and disinfectants on coccidian oocyst sporulated in rabbit, and to provide reference for the prevention and treatment of rabbit coccidiosis. Methods Selected 5 kinds of drugs and disinfectant for gradient test, respectively. To observe their effects on sporulation process of rabbit coccidia oocysts in vitro. Results (1) Inhibition test was performed under 5 kinds of anticoccidial drugs.The drugs of inhibition rate for coccidian oocyst sporulated were shown in turns: Toltrazuril Solution > Decoquinate Premix > Dinitolmide Premix > Diclazuril Premix > Amprolium Hydrochloride and sulfaquioxaline Sodium. The median effect concentration (EC₅₀) were shown as follows: Amprolium Hydrochloride and sulfaquioxaline Sodium > Diclazuril Premix > Dinitolmide Premix > Decoquinate Premix > Toltrazuril Solution. Take 15 mL/L Toltrazuril Solution effect of 8h has the strongest inhibitory on the sporulation of rabbit coccidian oocysts. (2) The rate of 5 kinds of disinfectants which inhibit rabbit coccidian oocyst from being sporulated was shown as follows:Carbon disulfide > Phenol > Ammonia > Symclosene > Tincture of iodine. The EC₅₀ were shown as follows:Tincture of iodine > Symclosene > Ammonia > Phenol > Carbon disulfide. The hours for 50% of maximal effect (EH₅₀) were shown as follows:Carbon disulfide > Phenol > Ammonia > Symclosene > Tincture of iodine. (3) The drug resistance of rabbit coccidiosis oocysts with strong toxicity were shown as follows: E.magna > E.stieda > E.irresidu > E.intestinal. Conclusions The 15 mL/L toltrazuril solution with effect of 8 h has the strongest inhibitory on the sporulation of rabbit coccidian oocysts. Carbon disulfide at concentration of 6% with effect of 4 h can completely inhibit rabbit coccidian oocysts from being sporulated. Drug resistance to rabbit coccidia oocysts was associated with body size of the parasite, and the larger body size with high sensitivity.

Progress on Methods for Establishing Animal Model of Vaginitis

LI Dong-dong, PANG Qin-xia, ZHU Min, KANG Le

2016, 36(4):  317-322.  DOI: 10.3969/j.issn.1674-5817.2016.04.015

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Vaginitis is the common infectious diseases of the women caused by invasive pathogens, which is often happened after vaginal mucosa or cervical injury (such as childbirth, abortion, surgery, etc.). However, the etiology and pathogenesis of vaginitis is still not entirely clear yet. Using animal models of human diseases to research human diseases is an important mean to clarify the pathogenesis of the disease or drug efficacy. Establishing a stable and reliable animal model of vaginitis is an important method for reseaching the pathogenesis and pathophysiology changes of human vaginitis and have an important role in promoting the development of new drugs and vaccines. There are three types of animal models of vaginitis: bacterial vaginosis, vulvovaginal candidiasis and trichomonas vaginitis according to the type of infection. The animal models of bacterial vaginosis are mainly infected by Gardnerella vaginalis, Neisseria gonorrhoeae, Escherichia coli or Staphylococcus aureus. The animal models of vulvovaginal candidiasis are mainly infected by Candida albicans or Candida glabrata. The animal models of trichomonas vaginitis are mainly infected by Trichomonas vaginalis or Tritrichomonas foetus. Mice, rats, rabbits and non-human primates are mainly selected to prepare animal models of vaginosis. In addition, pig vaginal mucosa and human epithelial cells are also commonly used in the preparation of animal models of vaginosis.

The Effect of Size and Hardness of Feed on Feed Utilization in Laboratory Animals

KOU Hong-yan, YAN Li-xin, LIU Yue-chang, MIAO Yu-tao

2016, 36(4):  323-326.  DOI: 10.3969/j.issn.1674-5817.2016.04.016

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This paper briefly reviews the effects of size and hardness on feed production and physiological and biochemical indexes of laboratry animals. The optimum size of feed may be useful for control the cost of production, and the suitable size and hardness of the feed may improve the feed utilization in laboratory animals.