Table of Content

25 August 2013, Volume 33 Issue 4

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Construction of Recombinant Fowl Adenovirus Serotype 1 Exprsessing Exotic Green Fluorescence Protein

ZHOU Jie, ZHAO Li, HU Jian-hua, GAO Cheng

2013, 33(4):  249-255.  DOI: 10.3969/j.issn.1674-5817.2013.04.001

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Objective To construct a recombinant fowl adenovirus expressing exotic green fluorescence protein(eGFP). Methods The flanking fragments of non-essential region(nt40 065~nt43 684) in fowl adenovirus genome were cloned into a transfer plasmid as well as eGFP. Through homologous recombination between the transfer plasmid and wild fowl adenovirus in chicken hepatocellular carcinoma cell line(LMH), the non-essential region between the flanking fragments was replaced by the complete eGFP expression cassette. In order to improve the occurrence probability of recombination, the transfection conditions such as logarithmic growth phase of LMH, the ratio of plasmid and lipofectamine, cells seeding density were explored. The optimum transfection condition was determined as follows: Seed 3×10⁵/ml LMH cells into 6 cell culture plate, 36 h later, cells were transfected with 8 μl lipofectamine 2 000 and 3.0 μg plasmid pUC-LR-eGFP. Recombinant virus was screening by plaque purification. Results recombinant adenovirus serotype 1 (rFAdV-1) expressing eGFP was obtained. Conclusion These results provided favorable references for fowl live vaccine development and related fundamental research.



Impact of Different Method in Sperm Membrane Treatment on Exogenous Plasmid Transgenic Efficiency by Sperm

JIA Xiao-feng, XU Yan, LIU Miao, WANG Liang, SHI Hui-juan

2013, 33(4):  256-260.  DOI: 10.3969/j.issn.1674-5817.2013.04.002

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Objective To explore the effect of three methods in removing mouse sperm membrane including ultrasound, NaOH and lysolecithin on the efficiency of transgenic by intracytoplasmic sperm injection (ICSI). Methods The mouse sperms were treated with the three methods above mentioned. Both of sperm membrane and DNA integrity of the sperm cells were evaluated by flow cytometry after died with Transgreen and propidium iodide (PI). The treated sperms were incubated with GFP-carrying plasmid and then conducted ICSI. The effect of these methods on efficiency of transgenic was evaluated by the results of fertilization cleavage and blastocyst with fluorescence. Results The rate of removing sperm membrane by lysolecithin method was higher than that of ultrasound and NaOH methods (99.2% vs. 94.7%, 90.9%)(P<0.05), but the rates of sperm DNA damage, the development of embryos from ICSI and blastocyst with fluorescence were similar among these methods (P>0.05). Conclusion The three methods of mouse membrane treatment could be used to improve mouse transgenic efficiency by ICSI.



Injury Effects of Different Dosage of Ionizing Radiation on Testicles of Male Mice

LIU Li-na, ZHOU Xi, CHEN Ya-lin, SUN Huan, TANG Ying

2013, 33(4):  261-264.  DOI: 10.3969/j.issn.1674-5817.2013.04.003

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Objective To study the injury effects of different dosage of ionizing radiation on the testicles of mice. Methods One hundred and twenty male KM mice were randomily divided into five groups at the radiation dosage of the control(0 Gy), 2.5 Gy, 3.5 Gy, 4.5 Gy and 5.5 Gy respectively. On day 3, day 7 and day 14 after ⁶⁰Coγ exposure, the epididymis index, testicle index, comparative sperm count and sperm abnormality rate were determined. The pathological changes of the testes were observed by hematoxylin and eosin stain. Results On day 3 after exposure, the epididymis index was increased, but it was decreased subsequently. The epididymis index of 4.5 Gy and 5.5 Gy groups was presented significant differences on day 14 after exposure, compared with that of the control group and day 3 group (P<0.01or P<0.05). The testicle index in each group was decreased obviously on day 7 and 14 after exposure (P<0.01or P<0.05). As to 5.5 Gy group, the testicle index was decreased significantly compared with that on day 3 after exposure (P<0.01). The sperm abnormality rate of all groups, especially in 5.5 Gy group increased significantly on day 3, 7 and 14 after exposure (P<0.01or P<0.05). The comparative sperm count of 5.5 Gy group was decreased evidently (P<0.01) and the myoid cells and basal membrane of seminiferous tubules were appeared blurry. All the index of 2.5 Gy group had no significant difference compared with those of the control group. Conclusion The injury degree of testicles in mice is positively correlated with the radiation dosage. The 3.5 Gy and 4.5 Gy are the suitable doses for establishing the model of pathological injuries in the testicles of mice.



Experimental Study on SPECT/CT Bone Fusion Imaging in Diagnosis of Stress Fracture in Rabbit

CAI Liang, ZENG Gui-gang, QIAO Wei-wei, SHI Hong-cheng, ZHANG Shen, CHEN Shu-guang, HU Peng-cheng, LI Pei-lei

2013, 33(4):  265-270.  DOI: 10.3969/j.issn.1674-5817.2013.04.004

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Objective To discuss the gain value of SPECT/CT bone fusion imaging and bone planar scintigraphy in the diagnosis of stress fracture. Methods Twelve male New Zealand White rabbits were randomly divided into two groups, including 8 in the experimental group and 4 in the control. In the experimental group, 8 rabbits were given electronic stimulation regularly to let it jump in a special device for five weeks. Meanwhile, the control group was allowed for free activities. All of them had undergone both ⁹⁹Tc^m-MDP bone planar imaging and SPECT/CT bone fusion imaging. All of bone planar images, bone SPECT images and SPECT/CT fusion images were diagnosis by 2 nuclear medicine physicians in double-blind method. All results were confirmed by pathological examination. Results The sensitivity, specificity and accuracy of bone planar imaging diagnosis of stress fracture were 31.25％(5/16)、87.50％(7/8) and 50.00％(12/24), respectively; the sensitivity, specificity and accuracy of SPECT/CT bone fusion imaging in the diagnosis of stress fracture were 75.00％(12/16)、87.50％(7/8) and 79.17％(19/24). Conclusions SPECT/CT bone fusion imaging has a higher sensitivity and specificity in the diagnosis of stress fracture, and it can further improve the diagnostic accuracy.



Vaginal Cytologic Examination on Estrous Cycle in Mini-pig

TIAN Fang, XIA Min-jie, WANG Yu-zhu, ZHOU Xin-chu, LI Wei-hua, DING Xun-cheng

2013, 33(4):  271-274.  DOI: 10.3969/j.issn.1674-5817.2013.04.005

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Objective To examine the vaginal cytology of estrous cycle in Guangxi Bama mini-pig. Methods Twelve mini-pig were selected and divided into different groups-diestrus group, proestrus group and estrus group. Vaginal smears of the pigs were obtained and stained with hematologic stain and Papanicolaou stain respectively .Epithelial cell morphology was observed and counted under the optical microscope. Results Leukocytes, para basal cell, small intermediate cell, large intermediate cell and superficial cell were found in vaginal smears. Leukocyte percentage was significantly higher during diestrus than that during estrus or proestrus (P<0.01). Para basal cell plus small intermediate cell counts were significantly higher during proestrus than that during estrus or diestrus (P<0.01). Superficial plus large intermediate cell counts were significantly higher during estrus than that during diestrus or proestrus (P<0.01). Conclusion Vaginal smears can be used to estimate different stage of estrous cycle in Guangxi Bama mini-pig.



Establishment and Application of ELISA for Detection of Anti-Toxoplasma Antibodies in Guinea Pig

LI Xiao-bo, FU Rui, GONG Wei, WANG Ji, WEI Li, WANG Shu-jing, YUE Bing-fei, HE Zheng-ming

2013, 33(4):  275-278.  DOI: 10.3969/j.issn.1674-5817.2013.04.006

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Objective To establish and apply a ELISA method for detecting the toxoplasma antibodies by recombinant antigen in guinea pig. Methods ELISA plates were coated using optimized recombinant toxoplasma antigen. we then determine optimal HRP-conjugated IgG concentration and test the assays specificity, sensitivity, stability and repeatability. Results The optimal HRP-IgG concentration is 1∶8000. There is no cross reaction when guinea pig sera with LCM, SV, PVM and Reo3 antibodies positive were added. The control positive serum could be detected at a maximum dilution of 1∶2560. Stability tests within six monthes show that the relative deviation of A₄₉₀ is less than 15%. The coefficient variation of the same batch is 5.7%-9.5%, of different batches is 1.9%-9.0%. The tox antibodies positive rate of 182 samples is 7.14%(13/182). The results coincidence rate between the assay and IFA is 100%, the positive coincidence is 92.3% and then negative is 100% compared to commercial kit. Conclusion The ELISA based on recombinant tox antigen can be used for routine test of guinea pig tox antibodies and be popularized.



Investigation on Gastrointestinal Parasites in Captive-bred Macaca mulatta and Macaca fascicularis in Guangxi

LI Jian, QUAN Chen-yu, SHI Wei, ZHOU Qing-an, ZHANG Hong-man, HUANG Wei-yi, HE Guo-sheng

2013, 33(4):  279-284.  DOI: 10.3969/j.issn.1674-5817.2013.04.007

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Objective To understand the infection status of gastrointestinal parasites in captive-bred non-human primates (Macaca mulatta and Macaca fascicularis) in Guangxi, and provide basis for prevention and control strategies. Methods The monkeys were divided into three groups, the growth group, the breeding group, and the quarantine group. Totally 784 fresh feces samples from M. mulatta and M.fascicularis were collected at six monkey shelters in Guangxi, during 2008~2012. The direct smear method (DS), saturation salt solution float method (SSF) and formaldehyde-ether precipitation method (FEP) were used for fecal parasite examination under the microscope. Results The total infection rate was 72.4% among 784 sample. Five protozoan species Ameoba, Trichomonas sp., Giardia sp., Balantidium coli and Coccidia), five species of nematodes(Trichuris sp., Streptopharagus sp., Physaloptera sp., Oesophagostmum sp. and Strongyloides sp.), two species of cestodas (Hymenolepis sp. and Bertiella studeri), one speice of trematoda and one speice of arthropod were identified. The gastrointestinal parasites infection of the growth group was possessed of the fewest among three groups, while the quarantine group was the largest. Relatively higher prevalence of protozoan was observed in both the growth monkey group and the breeding group, and the highest infection rate was found in both Amoeba and B.coli. Conclusions The results suggest that the protozoans with simple lifecycle and the soil-transmitted nematodes which are difficult to kill by antihelmintic products nowadays, have become the major parasitic species that does serious harm to the captive-bred monkeys for experiment used in Guangxi. Herein, the infestation status could provide reference for prevention and controling strategies on parasitic disease of monkeys. Some species identified from this study have been considered as the pathogens of zoonose, which will have important public sanitation significances of countermeasure on disease spread between humans and captive-bred monkeys.



The Protecting Machanism of Litchi Seed Aqueous Extracts on Learning and Memory Damage Induced by D-galactose in Mice

YE Hong-mei, ZHONG Chun-yan, Huang min-xian, Lu Jun-hua

2013, 33(4):  285-289.  DOI: 10.3969/j.issn.1674-5817.2013.04.008

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Objective To investigate the protecting machenism of Litchi Seed Aqueous Extracts on the learning and memory obstacles induced by D-galactose-induced in mice. Methods Fifty KM mice were randomly divided into normal group, model group, Huperzine A (0.4 mg·kg^-1·d¹) group and Litchi Seed Aqueous Extracts (15 g·kg^-1·d¹, 60 g·kg^-1·d¹) groups, 10 mice in each group. D-galactose (500 mg·kg^-1 ·d¹) were injected to all mice for 8 consecutive weeks by subcutaneous injection except normal group. At the beginning of 5 weeks, all drugs were given by gavage for 4 weeks. Water maze was used to detect the spatial learning and memory. The mitochondrial membrane potential were recorded with flow cytometer (FCM); the expressions of bcl-2 mRNA, bax mRNA and amyloid beta-protein precursor(β-APP mRNA) in the brain tissue were assayed by reverse transcription polymerase chain reaction. Result Litchi Seed Aqueous Extracts could obviously improve the condition of learning and memory obstacles in model mice. And it could significantly enhanced the mitochondrial membrane potential, reduced the expressions of bax mRNA and β-APP mRNA, increased the expression of bcl-2 mRNA. Conclusion Litchi Seed Aqueous Extracts possesses improving the learning and memory obstacles induced by D-galactose in mice which may be concerned with its enhancing the mitochondrial membrane potential, suppressing apoptosis and down regulation of the β-APP mRNA expression in the brain tissue.



Effect of β-carboline derivative on Subcutaneous Transplant Hepatocellular Carcinoma Growth in BALB/c Nude Mice

ZHANG Yan, FAN Wen-xi, XU Yi-mei, SHI Sheng, GUI You-jun, YAN Shun-sheng

2013, 33(4):  290-295.  DOI: 10.3969/j.issn.1674-5817.2013.04.009

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Objective To investigate the effect of â-carboline derivative HMD on subcutaneous transplant hepatocellular carcinoma in BALB/c nude mice. Methods A tumor model of human HCC was created by subcutaneous inoculation of 2×10⁷ (cells/ml,0.2ml) HepG2 cells into BALB/c male nude mice. The mice were randomized to low-(0.22 mg/kg), medium-(0.44 mg/kg), and high-dose HMD(0.87 mg/kg)group, saline group and 5-Fu group. The mice in each group received iv injection of the drug for 15 days. The effects of HMD on the growth of human hepatocellular carcinoma, the weight and pathological changes of nude miceŁ§S main organs were observed. Results Compared with saline group, the mice in HMD groups and 5-Fu group showed significant inhibition in the growth of the transplanted tumor (P<0.01).The inhibitory rate of HMD on the mouse weight. Compared to the saline group, the inhibitory effects of HMD(0.44, 0.87 mg/kg) were of statistic significance (P<0.01). Contrast to the negative control, there were no significant difference to the weight of kidney and kidney index in HMD groups and 5-Fu group, but there were significant differeces in the weight of liver and liver index (P<0.05). Conclusion HMD exhibit an inhibitory effect on the growth of transplanted HepG2 tumor, and the mice don’t appear toxicity symptom.



Preliminary Study on Breeding and Management of Naked Mole Rat in An Artificial Environment

SUN Wei, TANG Qiu, ZHAO Shan-min, LIN Li-fang, YUAN Zi-yan, ZHANG Lu, YU Chen-lin, CAI Li-ping, XU Chen, CUI Shu-fang

2013, 33(4):  296-300.  DOI: 10.3969/j.issn.1674-5817.2013.04.010

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Objective To explore the methods of breeding and feeding of naked mole rat. Methods The pregnancy rate, fetal interval, litter size, number of weaning and survival of naked mole rat with the generation of full-sib mating were studied under suitable feeding and management condition. Results Naked mole rat can be adapt in an artificial environment through seven pairs of naked mole rat breeding. Female naked mole rats were caged with males at 8-12 months of age. The rate of first pregnancy was 71.43%. The litter size of first-born of five female naked mole rats was 9.20±3.90. The weaning rate was 7.60±5.03 with survival rate 75.10%±42.49%. The second births have totally 5 litters with the birth interval 89.00±19.53 days and litter size 9.60±5.68. The weaning rate was 6.20±4.09 with survival rate 74.33%±34.19%. The third births have totally 5 litters with the birth interval 99.60±17.17 days and litter size 12.40±4.77. The weaning rate was 10.00±3.08 with survival rate 82.91%±16.70%. Conclusion It was possible to establish the conventional naked mole-rat in laboratory.



Comparative Study on Autophagy Regulation between Naked Mole Rat and C57BL/6 Mouse

LIN Li-fang, ZHAO Yi-ning, ZHAO Shan-min, XU Chen, CUI Shu-fang

2013, 33(4):  301-305.  DOI: 10.3969/j.issn.1674-5817.2013.04.011

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Objective A comparative study between naked mole rat and C57BL/6 mouse on autophagy regulation is conducted, providing a reference for the research of mechanism in high autophagy, anti-cancer, slow aging of NMR. Methods Electron microscopy and western blotting are use to detect the activtity of autophagy in the liver and lung tissue of newborn naked mole rat and C57BL/6. Reagents interfere the autophagy level of fibroblasts derived from naked mole rat and mouse. The cell number is tested after 24, 48, 72 hours by CCK-8 kit. Meanwhile, expression of Beclin 1 at each time point is detected by western blotting. Results Compared with mice, the naked mole rat tissue have significantly higher LC3B expression. The NMR cells have a certain amount of resistance from 3-MA. Rapamycin’s proliferation inhibition on NMR-S(fibroblasts derived from skin of naked mole rats)was significantly greater than that of mouse cells. Conclution NMR tissue possess a high autophagy, Cells from NMR have greater ability of self-regulation under extraneous autophagy inhibition, compared to C57BL/6.



A Report on Breeding of GFP Nude Mouse

ZHANG Li-bo, Liu Biao, TIAN Xiao-yun, WU Zheng-lin, ZHOU Sen-mei, YOU Jin-wei, FANG Tian, YUN Shi-feng

2013, 33(4):  306-309.  DOI: 10.3969/j.issn.1674-5817.2013.04.012

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Objective To breed a strain of BALB/c nude mice with green fluorescence and to observe the expression of green fluorescent protein in various organs. Method The nude mice expressing stable green fluorescence were developed by hybridizing, self-breeding and backcrossing BALB/c-nu/nu male mice with C57BL/6-TgN (GFP) female mice according to the principles of genetics. The genetic quality, thymus growth and green fluorescent protein expression in multiple organs were observed. Result The GFP nude mouse in line with BALB/c mouse was successfully bred. The mouse were presented a yellow-green appearance and characterized by athymism. The green fluorescence can be seen clearly in major organs such as liver, kidneys and pancreas under a fluorescent microscope. Conclusion A BALB/c GFP nude mouse model is developed successfully, its appearance is yellow-green and its whole-body expresses the green fluorescent protein stably.



Determination of Physiological Parameters in Juvenile Rhesus Monkeys

XING Feng-ying, YAN Guo-feng, YANG Wei-min, CHEN Xue-jin, LI Yao

2013, 33(4):  310-314.  DOI: 10.3969/j.issn.1674-5817.2013.04.013

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Objectives To detect the physiological parameters of juvenile rhesus monkeys. Method One hundred healthy 3 to 4-year-old juvenile rhesus monkeys (♂50, ♀50)were measured for a comprehensive examination about their hematological, cardiopulmonary function and other physiological parameters, including 30 routine blood tests, 22 blood biochemical indexes, 2 blood coagulation tests as well as blood pressure, electrocardiogram and respiratory rate. Result Mean corpuscular hemoglobin concentration (MCHC) (P<0.01) in the female rhesus monkeys was significantly lower than that of male monkeys; whereas platelet count (PLT), thrombocytocrit (PCT), eosinophil(EO), basophilic granulocyte (BASO), EO percentage (%), BASO percentage(%) (P<0.01) and white blood cell count (WBC), lymphocyte count (LYMPH) (P<0.05) were significantly higher in females juvenile rhesus monkeys than those of males. For the blood biochemical parameters,urea nitrogen (BUN), total bilirubin (TBIL), γ-glutamyl transpeptidase (γ-GTP), inorganic phosphorus (IP) (P<0.01) and glutamic-oxalacetic transaminease (AST), albumin (ALB), bilirubin (Total & Direct) (DBIL) (P<0.05) was significantly lower in the female than in male; while glucose (GLU),calcium (Ca) and triglycercide (TG) was significantly higher in females than in males (P<0.05). There were no significant differences in parameters considering blood coagulation tests,blood pressure and electrocardiogram between male and female juvenile rhesus monkeys. Respiratory rate was significantly reduced in female than in male (P<0.01). Conclusion The physiological biochemical parameters in experimental juvenile rhesus monkeys which might provide basis for reference values for animal experiments and disease diagnosis using juvenile rhesus monkeys.

Intracytoplasmic Sperm Injection and Transposon-mediated Transgenic Technology

KONG Peng-cheng, WANG Mei-shan, ZHU Lian, LI He-ping, JIANG Man-xi, CHEN Xue-jin

2013, 33(4):  319-323.  DOI: 10.3969/j.issn.1674-5817.2013.04.015

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Intracytoplasmic sperm injection (ICSI) has been widely used for generating offspring in human infertility clinics and in reproductive researched on mice. Many researchers engaged in animal transgenesis still consider it somewhat cumbersome. However, the greatest advantage of ICSI-mediated transgenesis is that it allows introduction of very large DNA transgenes (e.g., yeast artificial chromosomes), with relatively high efficiency into the genomes of hosts, as compared to pronuclear injection. Recently, researchers have developed an active form of intracytoplasmic sperm injection-mediated transgenesis (ICSI-Tr) with fresh sperm utilizing transposons. The transgenic efficiencies rival all transgenic techniques except that of lentiviral methods so that it is worthy of promotion and application in animal transgenic research.



Establishment and Assesment of Varicocele Model in Rat

CAO Huan, ZHOU Tie, HOU Jian-guo

2013, 33(4):  324-328.  DOI: 10.3969/j.issn.1674-5817.2013.04.016

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Varicocele is one of the most significant causes of infertility in male, Recently, varicocele has been given more and more attention and relative studies have gradually gone in depth. Successfully establishing animal model is crucial and critical to varicocele research. It has been 30 years in rat varicocele model development and the method is still improving. This review introduces relative progress on animal model development of varicocele.