Table of Content

25 February 2015, Volume 35 Issue 1

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Establishment and Preliminary Evaluation of Hepatitis B Virus Transfection Mouse Model by Using Hydrodynamic Injection

PENG Xiu-hua, CHEN Li-xiang, REN Xiao-nan, SHI Bi-sheng, XU Chun-hua, ZHOU Wen-jiang, ZHOU Xiao-hui

2015, 35(1):  1-5.  DOI: 10.3969/j.issn.1674-5817.2015.01.001

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Objective To construct a hepatitrs B Virus (HBV) transfection model in C57BL/6 mice with hydrodynamic injection and to further confirm the effectiveness of this mouse model for anti-HBV drug evaluation. Methods C57BL/6 mice were injected hydrodynamically via the tail vein with 10 μg of pAAV-HBV1.2 plasmid in 2 mL PBS. Results The serum level of HBsAg and HBV DNA level were monitored every week. Two weeks after the hydrodynamically injection (HI), all the mice were HBsAg positive while 22.2 % of the mice were still positive five weeks after HI. HBV DNA levels in the serum persisted higher than 10³ copies/ml for four weeks after HI. Histopathological analysis showed inflammatory infiltration and high expression of HBcAg and HBsAg in the transfected mouse liver tissue. In addition, Lamividine were applied for the evaluating the potential of this mouse model for anti-HBV drug evaluation, and data showed significant down regulation of HBV DNA level in the Lamividine treated mice. Conclusion This HBV transfection mouse model maybe effective in screening efficient anti-virus drugs targeting HBV polymerase and in evaluating HBV therapeutic vaccines, and thus promoting the development of novel treatment strategies against chronic hepatitis B infection.



Development of a Double Antibody Sandwich ELISA for Detection of Mouse Hepatitis Virus

ZHOU Jie, GAO Cheng, HU Jian-hua

2015, 35(1):  6-9.  DOI: 10.3969/j.issn.1674-5817.2015.01.002

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Objective To develop a sandwich ELISA for detection of mouse hepatitis virus (MHV). Method The ELISA was standardized by pair-matching experiment, and the working concentrations of the mAb 3D4H9 and HRP-conjugate mAb 4G9F2 were 12.8 ng/mL and 70.84 ng/mL, respectively, and judging with P/N>2 and absorbance_450nm ≥0.248 as positive criteria. Results The DAS-ELISA was specific for detection of MHV, but no cross-reaction with minute virus of mouse, reovirus type 3, sendai virus, pneumonia virus of mice. The intra-assay and inter-assay coefficient of variability were within 10%. In addition, a total 358 samples were tested by the DAS-ELISA, and indirect ELISA, there was no significant difference between results. Conclusion The DAS-ELISA was sensitive and specific which provided a useful tool for diagnosis of MHV.



Comparative Study on Formation of Atherosclerosis and Phospholipase A2 Expression in ZDF and SD Rats

SI Xu-wei, XI Sai-fei, XU Xiao-ping, ZHUGE Fu-yuan, CHEN Xiao-zhen, CAI Yue-qin, CHEN Min-li

2015, 35(1):  10-16.  DOI: 10.3969/j.issn.1674-5817.2015.01.003

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Objective To observe the formation of Atherosclerosis(AS) and expression of cPLA2, sPLA2, LP-PLA2 in AS tissue of ZDF rats and SD rats. Methods 6 ZDF (fa/fa) rats and 6 SD rats, with high-fat feeding and small dose, multiple times, intraperitoneal injection of 7×10⁵ U/kg vitamin D3,to build ZDF rats AS model group and SD rats AS model group, 6 ZDF (fa/+) rats and 6 SD rats, respectively,as ZDF rats control group and SD rats control group, fed with normal diet. GLU, TC, TG, HDL-C, LDL-C were measured before and after the establishment of the model, blood Ca²⁺ was measured after the establishment of the model, HE staining and Real-time PCR to detect the expression of cPLA2, sPLA2, LP-PLA2 mRNA in the abdominal aorta. Results After the establishment of the model, fasting blood glucose, blood lipid and blood Ca²⁺ were significantly higher (P<0.01), typical AS and obvious calcification were appeared in abdominal aorta of ZDF rats AS model group and SD rats AS model group, fasting blood glucose, blood lipid of ZDF rats AS model group were significantly higher than SD rats AS model group, and AS lesion was relatively serious, in ZDF rats AS model group, the expression of cPLA2, sPLA2 were significantly increased (P<0.01), while the expression of LP-PLA2 was significantly decreased (P<0.01); in SD rats AS model group, the expression of cPLA2, sPLA2, LP-PLA2 were significantly increased (P<0.01). Conclusions With high-fat feeding and small dose, multiple times, injection of vitamin D3 can establish the AS model in ZDF rats and SD rats, and AS lesion is relatively serious in ZDF rats, the expression of PLA2s are different in ZDF rats and SD rats, and this may be related to molecular pathological mechanism of the formation of AS in ZDF rats and SD rats.



The Influence of Colla Corii Asini on Growth and Quality of Hair in Mice Treated by Cyclophosphamide

LI Hao, JI Guo-xia, WANG Ke-zhou, JI Chuan-liang, LI Lan-bo, ZHANG Yan, FENG Mo-zhu, LI Jian-mei, LIU Tao

2015, 35(1):  17-22.  DOI: 10.3969/j.issn.1674-5817.2015.01.004

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Objective To explore the effects of colla corii asini on the growth and quality improvement of hair in mice of anemia caused by cyclophosphamide. Methods The mice model of anemia was established by ip cyclophosphamide. C57BL/6 mice were divided into five groups (the high dose group, the medium group and the low dose group, the model control group and the blank control group), and the different dose of colla corii asini or physiological saline were respectively administrated for 20 days by gavage. The number of hair follicles, the various content of blood cell, the length and the amino acid content of the hair in mice were determined, and the microstructure of hair were observed by the electron microscope at the 0 day, 12 days and 20 days after the first treatment. Results The contents of white blood cells, lymphocytes and monocytes in all colla corii asini groups and the blank control group, were higher than that of the model control group. The amino acids in the new hair of medium and high dose group mice were higher than that of the model control group. At the 20 days after the first treatment, the length and thickness of new hair in mice of all colla corii asini groups (the high, medium and low dose group) was more than that of the model control group, and was less than that of the blank control group, but there was no significant difference among the hair length (P>0.05). Scanning electron microscope (SEM) images showed there were significant differences in new hair diameter among different groups, the diameter of all colla corii asini groups was more than that of the model control group, and was less than that of the blank control group. And the diameter of the high dose group was more than that of the medium and the low dose group. The number of hair follicle of all colla corii asini groups was more than that of the model control group, and was less than that of the blank control group. Conclusion The colla corii asini could increase the number of white blood cells in C57BL/6 mice of anemia caused by cyclophosphamide, promote the formation of new hair follicles in C57BL/6 mice, promote hair growth, increase the content of amino acid and improve the quality of new grown hair.



Effect of Extractive from Glaucescent Fissistigma Root on Inhibiting Human Gastric Cancer and Ovarian Cancer in Nude Mice

CHU Fang, LI Yu-yun, FEI Jian, LI Yan-shu

2015, 35(1):  23-26.  DOI: 10.3969/j.issn.1674-5817.2015.01.005

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Objective To observe the effect of the extractive of Glaucescent Fissistigma Root on inhibiting human gastric cancer and ovarian cancer in nude mice. Methods The nude mice human gastric cancer model was established by hypodermic inoculation with solid tumors which inoculated with MKN45 cells. The nude mice human ovarian cancer model was established by hypodermic inoculation with solid ovarian tumors from human. Mouse in experimental or control groups were respectively treated by extractive of Glaucescent Fissistigma Root or drinking water for 30 days and 0.5 mL per day. Body weight and the size of tumor was measured twice a week. At 30 days, the inhibitive rate of tumor treated by the extractive of Glaucescent FissistigmaRoot was observed to estimate its effect. Results The inhibitive rate in human gastric cancer and ovarian cancer groups were both inhibited by the extractive of Glaucescent Fissistigma Root compared to control, but it significantly inhibited in human gastric cancer group. Conclusion Human gastric cancer and ovarian cancer groups were both inhibited by Glaucescent FissistigmaRoot, and the inhibitive effect was significantly to human gastric cancer group.



Zebrafish Thrombosis Model Induced by Phenyl Hydrazine

ZHANG Yong, ZHU Xiao-yu, GUO Sheng-ya, LI Chun-qi

2015, 35(1):  27-31.  DOI: 10.3969/j.issn.1674-5817.2015.01.006

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Objective To develop an in vivo thrombosis model in live zebrafish. Methods AB strain zebrafish at 2 days post fertilization (dpf) were treated with various concentrations of phenyl hydrazine (0.5 μmol/L, 1 μmol/L, 2 μmol/L, 4 μmol/L and 8 μmol/L) for 24 hours and the venous blood speed and thrombosis in zebrafish were qualitatively assessed under a dissecting microscope. Subsequently, venous thrombus was quantified through measuring red blood cells stained with o-Dianisidine staining in zebrafish heart. Untreated zebrafish and zebrafish treated with 0.1% ethanol (vehicle) were used as controls. Results Reduced venous blood speed and thrombosis were observed in all the groups of zebrafish treated with phenyl hydrazine at concentrations from 0.5 μmol/L from 8 μmol/L. Thrombosis in zebrafish treated with 0.5 μmol/L, 1 μmol/L, 2 μmol/L, 4 μmol/L and 8 μmol/L of phenyl hydrazine were(19.79±1.55)%, (43.28±1.82)%,(69.85±1.12)%, (70.56±4.60)% and (72.25±2.11)%, respectively. Conclusion Phenyl hydrazine can induce thrombosis in zebrafish with an optimum concentration at 2 μmol/L and this thrombosis model may be useful for rapidly in vivo drug screening in live zebrafish.



Isolation and Purification of Mouse Spermatogonia

LI Yu-hua, DUAN Fei, ZHOU Xiao-yu, Li Yang, LI Run-sheng

2015, 35(1):  42-45.  DOI: 10.3969/j.issn.1674-5817.2015.01.009

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Objective To establish a simple and efficient method for separation and purification of mouse spermatogonia. Methods Spermatogonia were isolated and purified from neonatal mouse testis using collagenase IV/DNase I/dispase combination enzymatic digestion and differential velocity of cell adherence method. Isolated spermatogonia were analyzed by morphology and stra8 immunochemistry. Results The isolated spermatogonia were stra8 positive and cell purity was as high as 86%. Conclusion This method is efficient for separation and purification of mouse spermatogonia .



Application of Different Type Vascular Ligating Clips on Laparoscopic Cholecystectomy in Rabbits

CHEN Xian-hua, PAN Yong-ming, BAO Yi-hong, HU Fei, PANG Xiu-bing, CHEN Liang, ZHU Ke-yan, XU Jian-qin, CHEN Min-li

2015, 35(1):  46-51.  DOI: 10.3969/j.issn.1674-5817.2015.01.010

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Objective To provide an experimental basis for clinic application of vascular ligating clips on ligation effects, inflammation and its performance stability in vivo after cholecystectomy. Methods Thirty-six rabbits were divided into 3 groups, negative control group, experimental (Jiazhu) group and positive control (Hem-o-lok) group, 12 rabbits in each group. The gallbladder duct and gallbladder artery were closed by Jiazhu vascular ligating clips and Hem-o-lok clips in experimental group and positive control group respectively. Animals were euthanized at 6 and 12 months postoperation, the ligation effects and the gross lesion in ligation sites were observed, the serum GGT, TNF-α, CK levels and the vascular ligating clips weight as well as its intrinsic viscosity in vivo were also detected. Results The ligating clips were ligated firmly and no bleeding or bile leakage were found, and the pathological morphology of liver has also no abnormal in experimental group and positive control group. Compared with negtive control group, the serum GGT level in experimental group and Hem-o-lok group were significantly increased at 3 days and 14 days postoperation and CK level were also significantly increased at 3 days postoperation (P<0.05, P<0.01). Furthermore, the TNF-α level was significantly decreased at 3 days and 2 months postoperation (P<0.05), and was also significantly decreased at 6 months postoperation in Hem-o-lok group (P<0.05). The serum GGT, TNF-α and CK levels were no significant differences between experimental group and Hem-o-lok group (P>0.05). Compared with original samples, the weight of ligating clips and its intrinsic viscosity in experimental group and Hem-o-lok group had no obvious changes after 6 and 12 months implanted (P>0.05). Conclusion It is effective for the ligation of gallbladder duct and gallbladder artery by Jiazhu vascular ligating clips, besides, its performance is stable in vivo. The Jiazhu vascular ligating clips are similar validity and security with Hem-o-lok clips.



Comparative Study on Three Kinds of Osteoporosis Model in Rats

FENG Jia, XIANG Yang, XIA Yan, CHEN An-ping, YANG Nian-an, GONG Shu-shi, YUAN Lin

2015, 35(1):  52-55.  DOI: 10.3969/j.issn.1674-5817.2015.01.011

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Objective To observe and compare the effects of different modeling methods of osteoporosis on rats. Methods Female SD rats at age of six months were divided into normal group, ovariectomized group, glucocorticoid group, and retinoic acid group. After 6 weeks, the changes of serum calcium, phosphorus, alkaline phosphatase and bone density, bone histomorphometry parameters were compared. Results Compared with normal group, the serum Ca in ovariectomized rats was decreased, serum P and ALP were increased, bone mineral density, trabecular thickness and trabecular number were decreased, and trabecular spacing was increased.The Glucocorticoid group compared with normal group, there was no significant difference in each parameters. Compared with the normal group, retinoic acid rat’s serum Ca was decreased, serum P and ALP were increased,bone mineral density, trabecular thickness and trabecular number were decreased, and trabecular spacing was increased. Conclusion The osteoporotic model was successful.With the short-term intermittent use of glucocorticoid, effects on the cancellous bone in rats may not be obvious, large dose of retinoic acid can reduce bone mass of rats in short-term and change the bone micro structural, but it had obvious side effects.



Nephrotoxicity of EDTA Utilized to Treat Chronic Cadmium Poisoning in Rats and Mice

LI Guang-xian, ZHONG Zhi-yong, CHEN Jia-xing, YU Ri-an, TANG Xiao-jiang

2015, 35(1):  56-60.  DOI: 10.3969/j.issn.1674-5817.2015.01.012

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Objective To investigate the side effects of (EDTA) utilized to treat chronic Cadmium poisoning in animals. Methods Male SD rats and NIH mice were randomly divided to the control group and model group respectively. The model group animals were daily given CdCl₂ + 2-Mercaptoethanol(ME, ip) for 5 days. 50 days or 30 days later, the rats or mice were randomly divided into model control group and EDTA treatment group respectively. The control group and model control group were injected with saline, and the EDTA group animals were treated with EDTA solution .At the end of the experiment, the urine, blood, kidneys and testis were collected for detecting the Cd concentration and pathologic changes. Results Compared with the model control group, in the EDTA treated groups (both in rats and mice), the Cd levels of kidney, urine and blood all increased significantly (P<0.05); protein precipitation could be detected in kidney tubules. Conclusion The EDTA could promote the accumulation of cadmium in the kidney. EDTA is not recommended to treat chronic cadmium poisoning patients.



Molecular Characteristics and Pathogenicity of Newcastle Disease Virus from Domestic Ducks in China

WU Wei, LIU Huai-ran, LIU Sheng-wang, CHEN Hong-yan

2015, 35(1):  65-70.  DOI: 10.3969/j.issn.1674-5817.2015.01.014

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Objective To study the molecular characteristics of Newcastle disease virus from domestic ducks, and then compare the pathogenicity in SPF chicken and ducks. Methods By cloning and sequencing the complete genomic sequence of Newcastle disease virus (NDV) strain Du/CH/LGD/358/2011, and then conducted the pathogenicity test in SPF chicken and ducks. Results The genome was 15 198 nucleotides (nt) in length, followed the “rule of six” that is required for replication of NDV strains, and encoded six structural polypeptides. The cleavage motif of F protein was 112 ERQER/L117, which was the characteristic of lentogenic strain. Phylogenetic analysis based on complete genomic sequences and partial sequences of F gene revealed that Du/CH/LGD/358/2011 belonged to class I genotype 3b, an independent clade closely related to Hong Kong LBM viruses. In addition, the pathogenic results between SPF chickens and SPF ducks was hardly varied. Conclusion Du/CH/LGD/358/2011 belonged to lentogenic strain, both SPF chicken and ducks could estimating be used fpr the pathogenicity. This study provided the theoretical basis for mornitoring on SPF ducks.



The Effect of Puppy Raisers on Training Success Rates in Guide Dogs

ZHOU Zi-juan, ZHANG Ya-li, LIANG Jia, YU Jian-xiong, HAN Fang, WANG Liang, WANG Ai-guo, WANG Fu-jin, WANG Jing-yu

2015, 35(1):  71-75.  DOI: 10.3969/j.issn.1674-5817.2015.01.015

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Objective To explore the effect of the factors of puppy raisers on training success rates in guide dogs. Method By sorting out and analyzing the investigation materials of 69 puppy raisers from Chinese Guide Dogs Training Centre of Dalian, the evaluation forms including the quality of puppy raisers, parenting skills, the social environment, the time puppy raisers spent on interacting with their dogs were filled out according to the actual situation of puppy raisers, and then the puppy raisers were scored based on grading standard. Result According to the analysis of these four evaluation factors, it was found that the training success rates of dogs on 3~5 score raiser group is significantly higher than that of 1~2 score raiser group, respectively, 26/52 (50.0%) vs 3/17 (17.6%), P=0.019; 26/50 (52.0%) vs 3/19 (15.8%), P=0.006; 27/53 (50.9% ) vs 2/16 (12.5%), P=0.006; 28/58 (48.3%) vs 1/11 (9.1%), P=0.016. The correlation analysis of four factors above showed that except that there is no significant correlation between the quality of puppy raisers and the social environment (P>0.086, r=0.208), and it showed positive correlations in statistics between each other three factors (P<0.05), but the correlation coefficients were low (r<0.5). Conclusion The quality of puppy raisers, parenting skills, social environment, and the time puppy raisers spent on interacting with their dogs has a significant impact on the training success rate of guide dogs.



Liquichip Technology and Its Application in Detection of Protein and Nucleic Acid

LIU Chun-mei, WEI Xiao-feng, GAO Cheng

2015, 35(1):  76-81.  DOI: 10.3969/j.issn.1674-5817.2015.01.016

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Liquichip is a new type of detection technology developed in the 1990s, which is integrated with flow cytometry, laser technology and digital signal processing. Compared with the traditional clinical detection methods, the main advantages are the high-throughput, high-sensitivity, high reproducibility, wide detection range, fast response, and only small amount of testing sample wanted. Luminex has two core technologies: xMAP^® technology and xTAG^® Technology. xMAP^® technology utilizing an antigen-antibody reaction principle, can be used for the detection of proteins and nucleic acids. xTAG^® technology can be used for a number of genomic assay formats such as gene expression analysis, microRNA analysis, single nucleotide polymorphism (SNP)analysis, specific sequence detection and other applications.