Table of Content

25 June 2013, Volume 33 Issue 3

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Sequence Analysis on Complete Mitochondrial Genome and Phylogeny of Microtus fortis fortis

GAO Jun, NI Li-ju, SUN Feng-ping, WANG Jin-xiang, HU Jian-hua, GAO Cheng, LI Kai, XIAO Jun-hua, ZHOU Yu-xun

2013, 33(3):  167-173.  DOI: 10.3969/j.issn.1674-5817.2013.03.001

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Objective To obtain the nucleotide sequence of complete mitochondrial genome sequence of Microtus fortis fortis to provide molecular data for the genetic structure and phylogeny research of Microtus.Fortis. Methods By means of conventional and long distance PCR and sequence with the “primer walking” strategy to obtain the complete mitochondrial genome of M.f.fortis (Genbank: JF261174).The phylogenetic tree was constructed based on Cyt b gene to investigate the phylogenetic position of M.f.fortis. Results The length of mitochondrial genome of M.f.fortis is 16312bp, include 13 protein coding genes, 2 ribosomal RNAs, 22 transfer RNAs and one major noncoding region (CR region). The extended termination associated sequences (ETAS-1 and ETAS-2), conserved sequence block 1 (CSB-1) and a Poly(C)₁₀ section were found in the CR region. The putative origin of replication for the light strand (O_L) of M.f.fortis showed high conservation in stem and adjacent sequences, but the difference existed in the loop region among different species and subspecies. Phylogenetic analysis results based on the cytochrome b gene showed the closest phylogenetic relationship with Microtus middendorffi in the genus Microtus. Conclusion The mitochondrial genome sequence of M.f.fortis showed a typical vertebrate pattern. This study can provide useful molecular data for the further study about phylogenic relationships and subspecies taxonomy of M.fortis in the future



Prokaryotic Expression, Purification and Identification of Estrogen Receptor β in Beagle Dogs

XU Qin, ZHAO Yan-bin, REN Xiu-mei, SUN Zhao-zeng, YE Hua-hu, LIU Yi, LI Wei, BAI Jie-ying, ZENG Lin, HU Zhong-ming

2013, 33(3):  174-179.  DOI: 10.3969/j.issn.1674-5817.2013.03.002

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Objective To obtain the estrogen receptor β (ERβ) and its splicing isoforms for elucidating its functional roles in the reproductive manipulation of Beagle dogs, the prokaryotic expressed canine pMAL-p5x/ERβ480 was established and optimized. Methods The cDNA specifically encoding ERβwas amplified from the total RNA of the Beagle dog hypothalamus, sequenced and blasted against other ERβcDNA sequences in the GeneBank. The amplified cDNA fragment was then cloned into a prokaryotic expression vector, pMAL-p5x, to produce recombinant plasmid pMAL-p5x/ERβ480. The constructed recombinant plasmid was transformed into E.coli DH α and E.coli BL21(DE3). The MBP-ERβ fusion protein was induced after the addition of IPTG into the growth media. The expressed product was purified by Amylom Resin affinity chromatography, and identified by SDS-PAGE. Results Sequence analysis indicated that the coding region of the cDNA fragment was about a length of 480 bp. The fusion protein was expressed comparatively well under the following conditions : in 0.2% glucose, 100 µg/ml Ampcillin, 0.1mmol/L IPTG at 37℃ for 5h or in 0.2% glucose, 50 µg/mL Ampcillin, 0.2 mmol/L IPTG at 37℃ for 3h. The SDS-PAGE results showed that the molecular weight of recombinant ERβ was of about 60 000, just as expected. Conclusion The pMAL-p5x/ERβ prokaryotic recombinant plasmid was developed successfully, and the beagle dogs MBP-ERβ fusion protein was obtained and biochemical characterized to some extent. It was anticipated that the fusion protein would be useful for the production of the dog species-specific ERâ polyclonal antibody and for the further functional analysis.



Preliminary Research on Mouse Model of Influenza Virus by Aerosol Infection

YANG Yu-qin, ZHU Zhao-qin, XU Chun-hua, HE Jing, PENG Xiu-hua, ZHOU Xiao-hui, HU Yun-wen, ZHOU Wen-jiang

2013, 33(3):  180-184.  DOI: 10.3969/j.issn.1674-5817.2013.03.003

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Objective Preliminary study on the model of influenza which was established by simulation of human natural infection in mouse. Methods ICR mice were infected with H1N1 FM1 and H1N1 PR8 virus by aerosol inhalation. The symptoms and body weight of mice were observed everyday. At 5, 12, 21 day after infection,the mice were sacrificed. The lungs of mice were weighed, then were done virus assay and pathological observation. Results The clinical symptoms of infection mice were obvious, the body weight lost seriously, lung index increased and pathological changes significantly in the early mid-term, for example the typical pulmonary edema, pulmonary interstitial disease, inflammation and pulmonary congestion appeared in the lung of mice. The influenza virus also can be detected in the early stage. The symptoms of mice getting better, the virus were not detected in the late. Conclusions The established infection mouse model of the influenza virus aerosol by aerosol inhalation is feasible.



Viral Particle Titer and Transgene Length Affect Integration Rate of Lentiviral Vector Mediated Transgenic Mice

GUO Xin-bing, GONG Xiu-li, CHEN Yan-wen, FANG Yu-dan, ZHANG Jing-zhi

2013, 33(3):  185-189.  DOI: 10.3969/j.issn.1674-5817.2013.03.004

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Objective To study the effeet of viral particle titer and the transgene length on the integration rate of transgenic mice mediated by lentiviral vector particles. Methods Eighteen kinds of lentiviral vectors particles, which carried various lengths of inserts, were used for subzonal injection of mouse fertilized eggs, followed with transplanting those eggs into the oviduct of pseudopregnanting mice. Neonates were then identified by PCR analysis. Results For short inserts (<2 kb), only 2 × 10⁸ of virus titer was needed to achieve a high integration rate. While the length of inserts were increase (4-5kb), 1 × 10⁹ viral particles were required for having a reasonable integration rate. However, transgenic mice was not able to be achieved when the inserts were longer than 6kb, in our hands. Conclusion Integration rate of transgenic mice was largely affected by both the viral titer and the length of inserts. It increases with the particle titer, while decreases with the length of inserts

Physiopathological Changes in Rabbit Model of Cardiac Arrest Resuscitation

ZHANG Dong, LI Nan, LI Hong-xiang, WANG Yu-shan

2013, 33(3):  190-194.  DOI: 10.3969/j.issn.1674-5817.2013.03.005

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Objective To discuss physiopathological changes in a rabbit model of asphyxia induced cardiac arrest and advantages of the model applied to the study of post-cardiopulmonary resuscitation. Methods Fifteen clean-grade adult rabbits were included in this study. Cardiac arrest was induced by asphyxia, the duration of which was 7 minutes. The changes of blood pressure and electrocardiogram were observed before and after cardiac arrest. And the time of cardiac arrestˇ˘the time to restoration of spontaneous circulation(ROSC)、the time to restoration of spontaneous breath, and the electrocardiogram when cardiac arrest occurred were recorded. The rate of restoration of spontaneous circulation and survival rate in 6 h, 12 h, 24 h, and 48 h were calculated. The arterial blood gas analysis was tested before and after cardiac arrest. Results ①MAP before closing trachea is 116.18±8.89 mmHg, at the moment of restoration of spontaneous circulation, was 121.03±16.75 mmHg, at 15 minutes since that, was 90.92±13.68 mmHg, and remained at that level thereafter. According to the electrocardiogram recorded at the moment of cardiac arrest, we found 2 cases of ventricular fibrillation, 11 cases of electromechanical dissociation, and 2 cases of asystole with a straight line. The electrocardiogram showed the progression of ST segment elevation and recovery from the restoration of spontaneous circulation to 30 minutes. ②The rate of restoration of spontaneous circulation from cardiac arrest was 100%(15/15), and the time was 147.60±22.09 s. The time to restoration of spontaneous breath was 403.33±130.18 s. The survival rate in 6h, 12h, 24h, and 48h were 93.33%(14/15), 73.33%(11/15), 53.33%(8/15) and 26.67%(4/15) separately. ③At the moment of restoration of spontaneous circulation, pH of the arterial blood gas analysis obviously decreased (P<0.05), and PaO₂、PaCO₂ and lactic acid obviously increased(P<0.05), compared with those before asphyxia. Conclusion The rabbit model of asphyxia induced cardiac arrest can simulate the characteristics of the pathophysiological changes in clinical cardiopulmonary resuscitation. The main adventages include the research needs of laboratory measurements and hemodynamic monitoring, the experimental operation is easy to grasp and without thoracotomy.



Atrial Fibrillation Model By Pacing Double Surperior Pulmonary Veins

CHEN Shi-quan, JIANG Chen-yang, SHENG Xia, HUANG Song-qun, GONG Xin-cheng

2013, 33(3):  195-199.  DOI: 10.3969/j.issn.1674-5817.2013.03.006

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Objective To evaluate the feasibility of double-superior-pulmonary veins (PVs) pacing in building atrial fibrillation(AF) model in canine. Methods After open-chest surgery, two decapolar catheter were implanted outside left- and right- superior PVs in 10 healthy Beagle dogs, rapid pacing (1 200 beat/min) were simultaneously performed for 6 hours. AF inducibility, effective refractory period (ERP) and window of vulnerability (WOV) were measured. Results AF model was successfully built in 8 canines. AF induction rate was 80%. After rapid pacing, a significant decrease and then increase could be found in ERP recording (145.±15.1 vs 130±16.3^* vs 136±11.4^**, ^*P=0.004. ^**P=0.494). After five hours AF inducing, the total atrial fibrillation induce window (? WOV) were increased, as compared with the baseline, but without significant difference (26.7±65.3 vs 20±52.9, P>0.05). While in AF model induced by acetylcholine (0,1,3,6 hours), the time of atrial fibrillation induced were first rising then declining, and this trend and the correlation between the change trend of ERP. Conclusion Double superior PV pacing model can be used in pathogenesis of atrial fibrillation, electrophysiology reconstruction, myocardial reconstruction, molecular biology change clinical research, also can be used for tachycardia sex and part of heart failure cardiomyopathy.



Evaluation of Left Ventricular Diastolic Function by Multi-parameter Echocardiography in Rabbits

WANG Fang, YANG Jun-hua

2013, 33(3):  200-203.  DOI: 10.3969/j.issn.1674-5817.2013.03.007

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Objective To evaluate the left ventricular diastolic function in healthy New Zealand White rabbits by multi-parameter echocardiography. To obtained normal values and analysis their correlation. Methods Left ventricular diastolic function can be measured by Pulsed Doppler, Tissue Doppler imaging and color M-mode Doppler. twenty-five rabbits were used in this study. Valves of mitral flow spectrum (E,A) and E/A were detected by Pulsed Doppler. Early and late diastolic peak velocity of mitral annulus (e’,a’) and e’/a’, E/e’ were measured by Tissue Doppler imaging (DTI). Isovolumic relaxation time (IVRT) was measured by Pulsed Doppler. Early diastolic flow propagation velocity (Vp) was measured by color M-mode Doppler. And we obtainednormal values of left ventricular diastolic function. Results The peak of E and A ,the peak e’ and a’ mixed together in three cases of all. Heart rate range was 180-324 beats/min. The normal values of left ventricular diastolic function were: E=0.44~1.05 m/s,A=0.23~0.47m/s,E/A=1.47~2.67.e’=6.96~19.92cm/s, a’=3.82~12.77cm/s, e’/a’=0.88~2.58, E/e’=2.70~8.80. IVRT=22.90~58.06 ms, Vp=38.90~81.52 respectively. There were significant correlation between E/A and e’/a’. No statistically significant among E/A, e’/a’, IVRT, Vp. Conclusions Left ventricular function of rabbits in cardiovascular disease can be evaluated by Pulsed Doppler and Tissue Doppler imaging.



Establishment and Preliminary Application of ELISA for Detecting Antibody to Mouse Hepatitis Virus in Mongolian Gerbil

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

2013, 33(3):  204-209.  DOI: 10.3969/j.issn.1674-5817.2013.03.008

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Objective To develop a ELISA method for determination of Mouse Hepatitis Virus antibody in Mongolian gerbils. Methods The cultured DBT cell were vaccinated with MHV-V1、MHV- V3、MHV-JHM virus for preparation the normal DBT antigen and special MHV antigen, the best working density of enzyme union and the normal or special antigen were titrated and the experiments of accuracy, sensitivity, stability and specificity were done. Result The best working density of normal, special antigen and the enzyme union is 0.4 µg/ml, 5ěg/ml and 1∶5000 respectively; The inter-assay coefficient of variation of normal antigen and special antigen is 7.9% and 6.8% respectively, the intra-assay average coefficient of variation is 12.7% and 9.7% respectively; The detection sensitivity is 1:1280; There is no cross-reactivity with Sendai virus (SV) and Lymphocytic Choriomeningitis virus (LCMV). The stability test shows the relative deviation is below 25%. Conclusion The ELISA method is good in duplication, stability, specificity, and sensitivity, as a result of which ELISA may be used in detecting the antibody to MHV in Mongolian gerbil .

The Foetus of Beagles Obtained in vivo and Fetal Fibroblasts Cultured

ZHANG Dun-wei, WANG Xiao-min, LIAN Xue-ke, LIU Yun-zhong, GAO Xiang, NI Qing-chun

2013, 33(3):  210-214.  DOI: 10.3969/j.issn.1674-5817.2013.03.009

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Objective To explore some methods of obtaining fetuses of 30 days by surgical operation and the ways of the fetal fibroblasts culture. Methods Identified the estrus of female Beagles for timed mating, then observed development of the fetues to determine the time of pregnancy by B-ultrasonic examination. When the female beagle was 30 days pregnant, removed the fetuses from its one side of the uterus by surgical operation, and then separated fetal fibroblasts with trypsin digestion after removing its head, limbs and internal organs. The cells were cultured and passaged in vitro. Meanwhile, the remaining fetuses of the other side of the uterus were retained, and the female Beagle was nursed carefully to insure development of the fetuses. Results Successfully isolated and cultured the canine fetal fibroblasts, and kept the remaining fetuses developing to full term and survived. Conclusion This experiment reserved the female dog which can be used as receptor for embryo transfer; the remaining pups can be used as the control group of the full-sib relationship for cloning Beagles in the future.



Comparative Study on Immunological Functions of Intestina Parva Between WHBE Rabbit and Japanese White Rabbit

HU Hui-ying, WANG Zhi-yuan, KE Xiang-fu, YU Chen-huan, TU Jue, ZHANG Li-zhong, YING Hua-zhong

2013, 33(3):  215-217.  DOI: 10.3969/j.issn.1674-5817.2013.03.010

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Objective To investigate differences on immunological function of small bowel between white hair black eye (WHBE) rabbit and Japanese white (JW) rabbit. Methods The weanling and 2 months old male WHBE rabbits and JW rabbits were used with 8 in each group. Two kinds of cell separation mediumsŁ¬ficoll and percoll ,were adopted for the isolation and purification. MTT test was applied to detect the lymphocyte proliferation induced by ConA and LPS. Results The levels of lymphocyte proliferation in small bowel of grown WHBE rabbits were much higher than those of grown JW rabbit. Conclusions Immunological function of lymphocytes in small bowel of grown WHBE Rabbit was much weaker than that of Japanese Rabbits. It may warrant further evaluation as a possible animal for the research of irritable bowel syndrome.

Application of Caesarean and in vitro Fertilization for Bio-cleansing in Mice

ZHU Lian, KONG Peng-Cheng, WANG Mei-Shan, CHEN Xue-Jin, JIANG Man-Xi, CUI Li

2013, 33(3):  218-221.  DOI: 10.3969/j.issn.1674-5817.2013.03.011

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Objective Caesarean and in vitro fertilization were used to rederive the contaminated SPF mouse colonies. Methods The contaminated mice with normal reproductive capacity were hysterectomized according to the vaginal plugs and abdomen palpatiion after natural mating. The fetus was removed from the uterus when labor and the pups were suckled by foster mothers. The foster nursing and the survival rates of the pups were observed. The in vitro fertilization and embryos transfer was used to the mouse strains whose reproductive capacity was poor or breeding failure. Both sperm and the eggs were incubated in the HTF medium, and 3.5 days embryos were transferred to the uterus of SPF recipient mice. Results All the re-derived mice through both methods reached the standard of SPF animals. Conclusions For the mice which did not meet the SPF standard but pathogens on them could not vertically transmit; the two methods can effectively improve their microbial level.

Comparison on Anesthetic Effect of Intravenous Pentobarbital Sodium or Isoflurane Anesthesia in Mini-pig

CAI Li-ping, YU Chen-lin, ZHAO Shan-min, TANG Qiu

2013, 33(3):  222-224.  DOI: 10.3969/j.issn.1674-5817.2013.03.012

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Objective To observe the anesthetic effect of pentobarbital sodium iv or isoflurane anesthesia on mini-pig. Methods A total of 10 healthy Bama mini-pigs were equally randomized into two groups. One group was administered with pentobarbital sodium intravenously,and the other group was administered with isoflurane . Changes in narcotic indicators were observed. Results There was no significant difference in the number, body weight, anesthesia performing hours, the adverse reaction rate, SPO₂ and pH value between the two groups (P>0.05), but both groups had significant difference in the onset time of anesthesia, postoperative recovery time, the number of cases of intraoperative restlessness, MAP and HR (P<0.05) between the two groups. Conclusion Anesthetic dose of sodium pentobarbital needs to be added during the experiment, while isoflurane anesthesia has better postoperative analgesic effect and less side effects, being a safe and effective analgesic option for different experiments.

The Research and Application of Mammary Gland Epithelial Cells

YAN Ya-bin, HUANG Ying

2013, 33(3):  237-242.  DOI: 10.3969/j.issn.1674-5817.2013.03.016

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Transgenic animal mammary gland bioreactor has been developed for the drugs production as one of the most promising bio-pharmaceutical methods in recent decades. The key technique in the process of the mammary gland bioreactors generation relies on the construction of exogenous gene vectors. It is crucial to establish a simple and quick method to test the accuracy and expression level of constructed gene vectors. As the growth progress and biophysiological lactation functions of mammary glands could be representatively investigated in mammary epithelial cells in vitro, mammary epithelial cells are becoming ideal cell models for detecting the efficacy of constructed-vectors in transgenic animals generations. In this review, it briefly introduced the structure of mammary gland tissue and characteristics of proliferation, differentiation, induction and maintenance of mammary epithelial cells. Additionally, it was summarized the culture approach of mammary epithelial cells and its application in biological and medical researches.

Progress on Immune Mechanism of Microtus fortis in Protecting against Schistosoma japonicum Infection

SHAO Guo-yan, XIE Jian-yun, GAO Cheng

2013, 33(3):  243-246.  DOI: 10.3969/j.issn.1674-5817.2013.03.017

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Microtus fortis (Mf) can be infected with Schistosoma japonicum but without any pathogenic clinical symptom. Mf has a congenital, hereditary resistance mechanism against the infection of Schistosoma japonicum. In recent years, since Mf is acclimated into an experimental animal, the resistance mechanism was deeply investigated by many researchers, this review summarize relative study progress from the aspect of specific and non-specific immunity of Mf.