Table of Content

25 February 2012, Volume 32 Issue 1

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Cloning and Identifying Expression Vectors of α-1,3 Galactosyltransferase Gene from Bama Minipig in Guangxi

GUO Xiao-ping, CHEN Shi-jin, LAN Gan-qiu, JIANG Qin-yang, GUO Ya-fen

2012, 32(1):  1-7.  DOI: 10.3969/j.issn.1674-5817.2012.01.001

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Objective For the purpose of producing GGTA1 gene silencing bama-mini pig, cloned and analyzed the function of α-1,3 galactosyltransferase gene of bama-mini pig then constructed two expression vectors. Methods The GGTA1 gene was amplified from liver tissue by RT-PCR using a specific pair of primers which were designed and synthesized according to the GGTA1gene sequence released in GenBank, the amplified cDNA fragment was ligated into pMD18-T vector after purification. The recombinant was verified by the method of PCR and sequencing, then the enzyme digested products was ligated into pEGFP-N₁ to construct the expression vectors. identified by PCR and digesting reaction. Results The α-1,3 galactosyltransferase gene of bama-mini pig exist the absence of 5-exon(81-116) which containing 1080 bp, and existence of 5-exon which containing 1116 bp. Conclusions There exit the absence of 5-exon in the bama-mini pig, Construction of the both expression vectors (pEGFP-GGTA1-1 and pEGFP-GGTA1-2) can be transfected into PK-15 cells to identify their functional expression, also can be used in the experiment of gene silencing pig α-1,3 galactosyltransferase gene.



Construction of Reference Standard Plasmids of Real-time Quantitative PCR for Detecting Theiler's Murine Encephalomyelitis Virus UTR Segment

YUAN Wen, ZHANG Yu, WANG Jin, LIU Xiang-mei, HUANG Ren

2012, 32(1):  8-13.  DOI: 10.3969/j.issn.1674-5817.2012.01.002

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Objective To construct a recombinant plasmid as the standard for Theiler's murine encephalomyelitis virus (TMEV)detection by real-time quantitative PCR. Methods A specific TMEV untranslated region (UTR) fragment in size of 1014 bp was amplified by RT-PCR, The PCR product was ligated with pMD18-T vector and transformed into E.coli DH5α. Recombinant plasmid was identified by PCR and sequencing. The concentration of recombinant plasmid was analyzed through its absorption at 260 nm. Standard curve was constructed by real- time quantitative PCR with ten-fold serial dilutions of recombinant plasmid. Results The fragment of UTR was successfully cloned into pMD18-T vector. The quantitative standard curve of recombinant standard plasmids was obtained, statistics analysis indicated that there was a good linear correlation between the Ct value and the concentration gradient of standard plasmids. The regression value was over 0.99. Conclusion The standard plasmid for detecting TMEV with real-time PCR has been constructed successfully.



Effects of Chronic Lead Poisoning on Caspase Familial in Cranial Nerve Cells of Rats

DIAO Bo, HU Dai, ZHOU Fang, ZHANG Yi

2012, 32(1):  14-16.  DOI: 10.3969/j.issn.1674-5817.2012.01.003

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Objective To research the effects of lead acetate with different concentration on caspase familial in cranial nerve cells of rats, and to investigate the possible mechanism of apoptosis in cranial nerve cells induced by lead poisoning. Method Fourty Wistar rats were divided into 4 groups randomly: normal group (distilled water, A group), high concentration group (2 g/L, B group), middle concentration group (1 g/L, C group), and low concentration group (0.5 g/L,C group), each group drunk lead acetate aqueous solution with different concentration for 2 months; the content of caspase-3, caspase-8, and caspase-9 proteins in brain tissue homogenate were detected by spectrophotometric method. Result Compared with A group, the content of caspase-3、caspase-8、caspase-9 protein in brain tissue of B, C, D groups increase significantly (P<0.001, P<0.01, P<0.05) with concentration dependent. Conclusion Lead poisoning could increase the expression of caspase familial, and significantly induce apoptosis in cranial nerve cells.

Comparison on Three Kind Models of Acute Gastric Ulcer in Mice

WANG Hui, ZHOU Xing, ZHANG Yi-yun, ZHUO Shu-hong

2012, 32(1):  17-19.  DOI: 10.3969/j.issn.1674-5817.2012.01.004

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Objective To compare three models of acute gastric ulcer in mouse, and describe the modeling method and some related notes. Method Three kinds of mice ulcer models induced by binding and water immersion-restraint stress, ethanol and reserpine respectively under different conditions; the extent of gastric mucosal injury was evaluated grossly and histologically. Results Three models all are able to form a stress ulcer, which are mainly appeared superficial. The water immersion-restraint stress method in 21±1℃ with 15 h and the ethanol method in 0.2 ml or 0.1 ml per mouse both have effects, while both have a low modeling death rate. Conclusion The Ethanol method is the best for modeling of acute gastric ulcer in mouse.



Effect of HP-β-CD on Haematology and Serum Biochemical Parameters in SD Rats

ZHU Kui-cheng, XUE Jing-li, WANG Chun-yao

2012, 32(1):  20-22.  DOI: 10.3969/j.issn.1674-5817.2012.01.005

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Objective To investigate the effects of HP-β-CD on clinical symptom, hematological parameters and blood biochemical parameters in rats. Methods Fourty SD rats were randomly divided into 4 groups. 6-week rats (SD) were administrated with HP-β-CD at doses of 500, 1000 and 2000 mg/kg/day by continuous intravenous infusion. Results There was no effect on survival and outer appearance and behavior comparing with control group. At both 1 000 and 2 000 mg/kg/day groups, there was a decrease in the number of RBC, content of Hb and HCT, while plasma BUN level increased (P<0.05 or P<0.01).It showed increasing in plasma CRE level at 2 000 mg/kg/day. Conclusion HP-β-CD could induce hemolysis and renal function damaged in rats at both 1 000 and 2 000 mg/kg/day dose.

Epigenetic Influences of Bisphenol A on Male Reproductive Toxicity in Zebrafish

QIAO Kun, LIU Yuan-yuan, LI Yu-hua, WANG Yu-zhu, DING Xun-cheng, LI Wei-hua, LI Runsheng

2012, 32(1):  30-37.  DOI: 10.3969/j.issn.1674-5817.2012.01.009

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Objective To investigate the epigenetic modifications of zebrafish sperm maturation and embryo development by bisphenol A (BPA). Methods 20 dpf juvenile zebrafish were exposed to ethanol solution (control groups), BPA solution of 0.5mg/Land 1.5 mg/L (treatment groups) until 100 dph. As for the male selected from each group, the gonadal Dnmts mRNA expression levels were tested by quantitative PCR, the gonadal genome methylation levels were measured by immunohistochemistry. In addition, the levels of histone methylation including H3K9me3, H3K4me3, H3K27me3 and H3K36me3 were detected using Western-blot and immunohistochemistry. Males of each group were mated with untreated female fish to observe and record the indicators relating to embryonic stage. Results The expression levels of Dnmt1、dnmt6、dnmt7、dnmt8 mRNA in BPA-treated groups increased significantly compared to the control group (P<0.05). Levels of genome DNA methylation and H3K9me3 in mature sperm of BPA 1.5 mg/L-exposed group increased significantly, while the number of eggs of female fish which mated with this group is reduced to 63%±2.49% of these fish with control group, the 12h embryonic mortality increased to 202.53%±6.21% and the 72 embryonic malformations increased to 399.08%±12.16%, these differences are of statistical significance (P<0.05). Conclusion BPA exposure selectively raised levels of DNA methylation and histone H3K9me3 in mature sperm of zebrafish, these changes may be an important foundation of the increased developmental abnormalities of F1 offspring.



A New Mouse Model of Acute Hepatitis B Virus Infection

TANG Hong-bin, WU Kai-lang, CHENG La-ying, JIANG Ming-sen

2012, 32(1):  38-42.  DOI: 10.3969/j.issn.1674-5817.2012.01.010

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Objective To establish an acute HBV mouse model by tail vein injection of infectious sera. Methods Mice sera at 3 days after hydrodynamics-based transfection was collected and injected into the tail vein of BALB/c mice with volume of 0.1ml and 0.2ml respectively. The serum was slowly administrated within 10-12s. DNA levels of HBV e antigen (HBeAg), HBV surface antigen (HBsAg), HBV core antigen (HBcAg) and HBV were determined and compared before and after infectious sera injection. Results The levels of serum HBsAg, HBeAg, HBV DNA and liver HBcAg in mice injected with 0.1ml viral sera were higher than that injected with 0.2ml and hydrodynamic injected pBule-HBV. Conclusions The method of intravenous injection with low dose of infectious sera could be used to establish a mouse model of acute HBV infection.



Effects of Carbenoxolone on Morphine-induced Conditioned Place Preference in Mice

CHEN Zhao-qin, JIANG Zheng-lin, GONG Yong-sheng

2012, 32(1):  43-46.  DOI: 10.3969/j.issn.1674-5817.2012.01.011

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Objective To investigate the effect of gap juction inhibitor carbenoxolone (CBX) on morphine addiction in mice. Methods Conditioned place preference (CPP) was established in ICR mice through prolonged morphine exposure. The influence of CBX on the acquisition or expression of CPP were observed through behavior test. Changes of the astrocytes were examined by immunofluorescence of glial fibrillary acidic protein (GFAP). Results ①Contrast to the control group, CPP was induced in the morphine-dependent group (P<0.05). ②CPP was inhibited significantly in the CBX-pretreated group(P<0.05), but not significantly in the CBX-treated group (P>0.05). The morphine may lead to increase of GFAP immunostaining in ventral tegmental area (VTA). ③The GFAP immuno-reactive cells in CBX-pretreated group were fewer than those in morphine group. Conclusion CBX can suppress the acquisition of CPP induced by morphine, but the effect of CBX on the express of CPP was weak. CBX maybe play directly or by indirect effect of GFAP.



A Novel Mouse Model for Ulcerative Colitis-associated Carcinogenesis Induced by Azoxymethane and Dextran Sulfate Sodium

ZHENG Hai-ming, ZHAO Hang, ZHENG Ping

2012, 32(1):  47-50.  DOI: 10.3969/j.issn.1674-5817.2012.01.012

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Objective To develope an ulcerative colitis-associated carcinogenesis model in mice. Methods Female BALB/c mice were randomly divided into 4 groups: the control group, the azoxymethane (AOM) group, the dextran sulfate sodium (DSS) group and the AOM + DSS group. AOM was intraperitoneally injected. DSS was fed for 1 week then with ordinary water for 2 weeks, such 3 weeks formed a cycle and 9 weeks in all. The body weight was detected weekly and the mental status, stool character, hairs glossy and mortality were daily observed and recorded. Levels of TNF-α was tested by ELISA. Inflammation and pathology were observed by HE staining. Results ①After drinking DSS,the mice showed diarrhea, bloody stool, weight loss, depressed and hairs messy. Post-oral tumor were seen anal prolapse. Non-DSS groups had no more performance. AOM + DSS group appeared one death. The body weight was significantly decreased at 4,7,8,9 week after treatment compared with the control group (P <0.05). ②The TNF-α was significantly increased in DSS groups (P<0.05). ③HE staining: DSS groups showed infiltration of inflammatory cells and (or) the formation of lymphoid follicles. The tumors were induced in all of the AOM + DSS mice (9/9), which were most located in distal colon and the pathology were showed high atypical hyperplasia and (or) carcinoma in situ. Dysplasia or cancer was not observed in the other three groups. Conclusion Ulcerative colitis-associated carcinogenesis model in mice can prepared with a single intraperitoneal injection of azoxymethane prior to three repetitive oral administrations of dextran sulfate sodium. This method have the advantages with shorter time, high formation of tumor and stability of histological type.



Analysis on Genetic Quality of Five Production Colonies of C57BL/6J Mouse

ZUO Bao-fen, DU Xiao-yan, HUO Xue-yun, LI Zhen-kun, CHEN Zhen-wen

2012, 32(1):  51-55.  DOI: 10.3969/j.issn.1674-5817.2012.01.013

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Objective To learn and analyze the genetic quality of C57BL/6J mouse so as to provide some reference for laboratory animal genetic quality control and management for production units and for the utilization of C57BL/6J. Methods Forty two microsatellite loci which were optimized previously and distribute 17 autosomes and X chromosome were selected in present study. PCR and STR scanning by these 42 loci were performed to detect the genetic quality of 5 main domestic C57BL/6J mouse populations from Beijing, Shanghai and Nanjing. Results The results of STR scanning showed that 42 microsatellite loci demonstrated monomorphism in C57BL/6J populations from Shanghai and Nanjing. However, populations from Beijing displayed polymorphism. Thereinto, Animal number 3 from Jing 1 population presented polymorphism at locus D15Mit105 compared to other 9 mice and other 4 populations. Meanwhile all the mice in Jing1 population at locus D10Mit3 show polymorphism compared with other 4 populations. Animal number 8 at locus D14Nds1 and animal number 6 at locus D2Nds3 showed polymorphism within Jing 2 population. Animal number 1 from Jing 3 population also showed polymorphism within population. Conclusions C57BL/6J mouse from Shanghai and Nanjing showed genetic consistency. However, genetic differences existed in 3 production populations in Beijing. These results should be paid much more attention by producers and users.



The Perioperative Pain Control in Canine with Tibial Fracture and Internal Fixation

WANG Shou-li, ZHU-Jun, ZHU Yun-li, FU Pei-liang, WU Yu-li, WU Hai-shan, ZHAO Hui

2012, 32(1):  56-59.  DOI: 10.3969/j.issn.1674-5817.2012.01.014

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Objective To observe the influence on dog from different analgesia regimes during perioperative periods and to explore the suitable regime for dogs. Methods All the animals were randomly divided into four groups receiving different analgesia regime. All the dogs experienced the same surgery of tibial fracture and internal fixation. The pain was evaluated according to the selected pain score system, the results were analyzed. Result The physical status and behavior of dogs who received multimodal analgesic regime changed significantly less than that of regular regime. Conclusion Multimodal analgesic regime for surgery animal can make animals suffer less during research period and contribute to the mortal spirit, help to elevate the welfare of experimental animals.

Morphologic Observation on Left Bundle Branch and It's Purkinje Fiber Networks of Bovine Heart

ZHOU Nan-qian, LI Zhan-sheng, SHEN Biao, WU Man-yang

2012, 32(1):  60-63.  DOI: 10.3969/j.issn.1674-5817.2012.01.015

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Objective To collect data for comparative anatomy on the structure and distribution of the left bundle branch and Purkinje fiber networks in bovine heart. Methods The morphosis of left bundle branches and Purkinje fiber networks were observed after perfusing Chinese ink into 22 bovine heart gross specimens. The left -angle coordinate was built by the anterior and the posterior papillary muscles line as x-axis, and the main trunk of the left bundle branch serves as y-axis.Then the right ventricle was divided into four quadrants. The density of Purkinje fiber networks was measured by naked eyes and expressed with the number of signal“+”. Results The main trunk of the left bundle branch, which showed the flat band shape, ran along the sub-endocardium of the left part of inter-ventricular septum.It's length was 27.07±5.96 mm and it's width was 5.64±2.12 mm; There were three types in the first branch: shape of two branches(31.82%), shape of three branches (63.64%), and networks(4.55%); The density of Purkinje fiber networks of sub-endocardium in left ventricle varies in different quadrants .The types Ⅲ and Ⅳ quadrants mainly indicated “++”, 17 (77.27%) and 15 (68.18%)cases in them respectively; The typesⅠand Ⅱ quadrants mainly indicated “ +++”, and there were 12 (54.55%) and 9 (40.91%) cases in them respectively. Conclusion The structure and distribution of left bundle branch and it's Purkinje fiber networks of bovine heart is similar with human heart but there is some specificity for bovin heart.



Animal Models of Alzheimer's Disease

LOK Keng-hoe, ZHAO Wen-juan, YING Ming

2012, 32(1):  80-88.  DOI: 10.3969/j.issn.1674-5817.2012.01.020

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Alzheimer's disease (Alzheimer's disease, AD) is a neurodegenerative disease, its learning ability, behavioral and expression impairments exacerbate with age. AD animal models than simulate the AD disease's pathology are an essential tool to study this disease. A number of transgenic animal models had successfully established, including APPPS1, PS1/PS1, Tau protein transgenic mice and rapid aging-SAMP8 mice. Suitable animal model play an important role in the AD pathogenesis and drug development research. This article reviews the most common animal model of AD and its role in drug development.