Table of Content

25 April 2012, Volume 32 Issue 2

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Detection of Microorganisms Carried by Experimental Heliothis armigera

AN Xue-fang, XIAO Yu-zhou, XIA Ke-xiang, ZHANG Fan, ZHU You-ling

2012, 32(2):  89-93.  DOI: 10.3969/j.issn.1674-5817.2012.02.001

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Objective Microbial isolation, culture, and identification in vitro and in vivo were carried out on Heliothis armigera of different seasons and different ages that were bred indoor in the institute to investigate the carriage of microorganisms. Methods Common culture medium and fungi culture medium were used for the in vivo and in vitro microbial isolation, culture and purification of heliothis armigera, respectively. The preliminary identification was performed through colony morphological observation, Gram's staining and microscopic observation. Then 16SrRNA and 18SrRNA analysis technology was applied for further identification of isolated bacteria and fungi. Meanwhile, PCR was used for amplification detection of highly pathogenic HaNPV for Heliothis armigera. Results Four strains of Bacillus were isolated, which belonged to Bacillus subtilis Cohn, Bacillus licheniformis, Bacillus cereus and Bacillus, respectively. Four strains of fungi were isolated, including 2 strains of Yeasts, one strain of Aenicillium Link and one strain of Aspergillys Micheli. The result of HaNPV detection was negative.



The Safety Evaluation of Zhengqingfengtongning injection

CHEN Zi-yuan, LI Zi-shan, TIAN Hong

2012, 32(2):  94-97.  DOI: 10.3969/j.issn.1674-5817.2012.02.002

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Objective To investigate the safety of Zhengqingfengtongning injection and evaluate the rationality of security project in the quality standards. Methods The safety of the Zhengqingfengtongning injection was studied with active systemic anaphylaxis(ASA), passive cutaneous anaphylaxis(PCA) in guinea pig, the depressor substance test in cat and muscle stimulation test in rabbit. Results ASA and PCA reaction didn't occur at the dose of 12.5 mg/ml; maximum concentration without blood pressure reduction was 0.125 mg/ml in cat; In rabbit muscle stimulation test,the response was less than level 2. Conclusion The results showed that Zhengqingfengtongning injection was safe for clinical use, and the safety project in quality standard was reasonable.

Observation on Function of Compound Momordica Charantia L. Capsule for Reducing Blood Glucose

HUANG Chao-pei, MA Yu-yan, YAO Si-yu, QIN Hui-yan, ZHAO Peng, YANG Jun-feng

2012, 32(2):  98-100.  DOI: 10.3969/j.issn.1674-5817.2012.02.003

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Objective To investigate the function of Compound Momordica Charantia L. capsule for reducing blood glucose. Methods Mice were injected with alloxan to build hyperglycemia animal model and then randomly divided into three test groups (225, 450, 900mg/kg) and model control group. The mice were administered with the compound for 30 days, then the fasting blood-glucose and sugar tolerance between test groups and control group were compared. Normal mice were chosen to carry out the test of reducing fasting blood - glucose at the same time. Results The fasting blood - glucose was obvious lower than that of model control group (P<0.01), and the blood-glucose decline rate was greater than that of control group (P<0.01) in the hyperglycemia model mice of 900 and 450 mg/kg group. There were not significant difference between the sugar tolerance of test groups and model control group in hyperglycemia model mice and between the fasting blood - glucose of test group and model control group in normal mice. Conclusion Compound momordica charantia L. capsule had the function for reducing blood-glucose.

Isolation and Culture of Bone Marrow Mesenchymal Stem Cells (MSCs) of Tibetan miniature pig, and Lentivirus-mediated EGFP Gene Transfer into MSCs

TANG Hua, BI Wen-hao, AI Wei, LI De-sheng, PAN Guang-hui, SHI Jun-wen, LIU Wei, HUANG Wei, XIAO Dong, GU Wei-wang

2012, 32(2):  105-110.  DOI: 10.3969/j.issn.1674-5817.2012.02.005

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Objective To establish a simple and effective method used to isolate and purify bone marrow mesenchymal stem cells (BMSCs) of Tibetan miniature pigs, and further set up the lentivirus-mediated in vitro gene delivery system for BMSCs of Tibetan miniature pigs. Methods ①MSCs of Tibetan miniature pigs were initially isolated by density gradient centrifugation, followed by purifying MSCs using differential attachment method, and further passage 3 BMSCs of Tibetan miniature pigs were characterized by CD45, CD90 and CD105. ②According to the standard protocol from Invitrogen, lentiviruses carrying EGFP gene were produced, and then infected MSCs, followed by EGFP assay under fluorescent stereo microscope and fluorescence activated cell sorting (FACS) for determining the infecting efficiency. Results BMSCs of Tibetan miniature pigs obtained by the combined use of density gradient centrifugation and differential attachment method were shown to enjoy the characteristics of MSCs. Additionally, the BMSCs of Tibetan miniature pigs were successfully and efficiently infected by lentivirus supernatant, and the infecting efficiency was 84.1%. Conclusion High purified BMSCs of Tibetan miniature pigs can be obtained by the combined use of density gradient centrifugation and differential attachment method, and lentivirus-mediated in vitro gene delivery systems for BMSCs of Tibetan miniature pigs were successfully established.



Construction of Expression Vector pcDNA3.1/myc-His-DJ-1^M26I and Expression in NIH3T3 Cells

ZHANG Mei-ying, XU Ying-qi, WANG Wei, ZHAO Yue, YANG Wei, YU Meng, GUO Xiao-chong, QIN Ying, ZHENG Zhi-hong

2012, 32(2):  111-115.  DOI: 10.3969/j.issn.1674-5817.2012.02.006

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Objective To construct the expression vector pcDNA3.1/myc-his-DJ-1 and pcDNA3.1/myc-His-DJ-1^M26I to lay a foundation of further study on function of DJ-1^M26I and establish transgenic animals. Methods The 26^th amino acid of DJ-1 was mutated by gene mutation kit, then pcDNA3.1/myc-his-DJ-1 and pcDNA3.1/myc-His-DJ-1^M26I were constructed. The two plasmids were transfected into NIH3T3 cells respectively using liposome method, then the cells were selected with G418 and detected the DJ-1 gene expression at DNA, transcription and protein levels. Results 1 clone of pcDNA3.1/myc-his-DJ-1 and 3 clones of pcDNA3.1/myc-his-DJ-1^M26I were obtained after G418 selection by PCR analysis. The expression of DJ-1-His were detected by RT-PCR and Western blot in pcDNA3.1/myc-his-DJ-1 and pcDNA3.1/myc-his-DJ-1^M26I transfected cells. The profieration rate of DJ-1^M26I transfected cells was lower than normal NIH3T3 cells (P<0.05) by MTT essay. Conclusion The expression vectors pcDNA3.1/myc-his-DJ-1 and pcDNA3.1/myc-His-DJ-1^M26I were constructed successfully.



Effects of Foreign DNA Length on Integration Efficiency in Preparation of Transgenic Mouse

CHI Jun, KUANG Ying, SHI Ji-jing, GONG Hui, MAO Ji-fang, FEI Jian, WANG Zhu-gang

2012, 32(2):  116-119.  DOI: 10.3969/j.issn.1674-5817.2012.02.007

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Objective To analysis the relevance of foreign DNA length to integration efficiency on the randomly gathering data in preparation of transgenic mouse. Methods The statistics analysis was performed on 29 randomly gathered data of generation of transgenic mouse. Result According the regression equation of statistics analysis in this paper, the integration efficiency of foreign DNA was increased with the shorter length of foreign DNA (P<0.001). The foreign DNA fragments integrated into mouse chromosome DNA with 10% efficiency when the length of foreign DNA were less than 6 000 bp. Conclusion There is a negative correlationbetween integration efficiency and foreign DNA length in microinjection.



Effect of XUEBIJING on Multiple Organ Dysfunction Syndrome Induced by Lipopolysaccharide in Sheep

SHI Zhi-yong, LUO Xiao-hong, WAN Dong-jun, LU Lu, WANG Hong-yi, NIU Ting-xian

2012, 32(2):  120-124.  DOI: 10.3969/j.issn.1674-5817.2012.02.008

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Objective To investigate the prevention and cure efficacy of XUEBIJING on multiple organ dysfunction syndrome (MODS) induced by lipopolysaccharide (LPS) in sheep. Methods Fourty sheep were randomly divided into 4 groups: the control (Group A), model (Group B), precaution (Group C) and cure group (Group D), 10 sheep each group . After anesthesia, the MODS model was established by infusing LPS at 6 μg/kg within 30 minutes intravenously. Then the sheep were infused with XUEBIJING (2 ml/kg) by intravenous drip before and 1 hour after injecting LPS in Group C and Group D respectively; animals in Group D received XUNBEJING (2 ml/kg) by intravenous drip once daily for 3 days. Meanwhile, the sheep were infused with isotonic Na chloride at the same dose in Group A. The indexes of blood gas, blood routine and blood biochemistry were determined at the different time points. Results After intravenous injecting with LPS, the physical sign was alleviative to some extent in Group C and Group D than that in Group B. The death rates of sheep were 0%, 60%, 40% and 50% in Group A , Group B, Group C and Group D respectively. Compared to the values of oxygenation indexes prior to injection with LPS, it decreased obviously (P<0.05 or P<0.01) within 3 hours in Group B, Group C and Group D, but there were not significant statistical differences observed between three groups (P>0.05). At the different time points, the level of aspartic transaminase (AST), alanine aminotransferase (ALT), total bilirubin (TBIL) and creatinine (CRE) were obviously increased compared to that prior to injection (P<0.05 or P<0.01) . But the number of white blood cell (WBC) was obviously decreased at 3 h~6 h, then it began to increase at 12 h and increased significantly at 24 h (P< 0.01). Conclusion XUEBIJING could improve physical sign appearance, degrade the death rate of MODS induced by LPS and it had interventional effect on damage in multiple organ .



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

WANG Zhi-yuan, LIU Yue-huan, YU Chen-huan, YING Hua-zhong, CAI yue-qin, Zhang li-zong

2012, 32(2):  125-128.  DOI: 10.3969/j.issn.1674-5817.2012.02.009

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Objective To investigate differences on immunological function of small bowel between WHBE rabbit and Japanese white rabbit. Methods Sixteen WHBE rabbit and Sixteen Japanese white rabbit were used and each group at different stage was 8. Levels of secretory IgA (SIgA), Substance P (SP), IL-4, IFN-γ, somatostatin (SS) and Vasoactive Intestinal Peptide (VIP) in serum and small bowel were determined by ELISA. Results The levels of sIgA, SS、SP and the ratio of IFN-γ/IL-4 in serum and small bowel of grown WHBE rabbit were much lower than those of weanling WHBE rabbit. In particular, the levels of sIgA, SS, IFN-γ and IL-4 in small bowel of grown WHBE rabbit lower than those of grown JW rabbit. Conclusions Immunological function of WHBE Rabbit was much weaker than that of Japanese Rabbit. It may warrant further evaluation as a possible animal for the research of irritable bowel syndrome.

Comparison on Animal Models of Hyperlipidemia

SHEN Long-hai, AN Yong-tong, ZHANG Zhen, YAN Yong-gao

2012, 32(2):  129-132.  DOI: 10.3969/j.issn.1674-5817.2012.02.010

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Objective To summarize various animal models of hyperlipidemia applying in hypolipidemic experiments in our laboratory. Methods The animal models varied in some respects: the animals explored (mouse, SD rat and quail), the prescriptions of gastric perfusion intralipid and high fat diet, the experimental periods. Result According to the serum biochemical detection, the hyperlipidemia models of rat and mouse were more suitable for screening the drugs of derucing high cholesterol high low-density lipoprotein. Quail model formed faster and more obvious, which was more suitable for lipid-lowering drug preliminary screening, but the individual variation was very significant. Each model has its own advantages and disadvantages. Conclusion The models with different animals, prescriptions and experimental periods have their own characteristics, which would be chosen according to the experimental purposes.

The Inhibiting Effect of Coix Seed Oil Injection on Xenograft Model of Human Liver Cancer Cell SMMC-7721

YIN Bei-pei, LU Hong-qi, SHEN Long-hai, LI Bing-sheng

2012, 32(2):  133-136.  DOI: 10.3969/j.issn.1674-5817.2012.02.011

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Objective To study the inhibiting effect of coix seed oil injection on human live cancer SMMC-7721 grafted in nude mice. Methods The model of human live cancer SMMC-7721 grafted in nude mice was established, the tumor size and body weight of mice bearing tumors were observed and measured, then relative tumor growth rates and tumor inhibition rates calculated. After the last treatment, tumors and various organs were harvested for immunohistochemistry and HE staining. Result The relative tumor growth rates of three dose groups of coix seed oil injection were 33.02%, 35.78% and 39.41%, and the inhibition rates were 49.54%, 46.86% and 41.71% respectively. The positive Ki-67 staining rate of tumors in nude mice treated with coix seed oil injection was significantly lower than that in the control group. Main organs showed normal state according to hematoxylin and eosin staining. Conclusion Coix seed oil injection has certain inhibition efficacy on the xenograft model of human liver cancer cell SMMC-7721.



Establishment of Minipig Abdominal Wall Zipper Model and Its Application in Surgical Teaching

TANG Qiu, LIU Zhixue, YU Chenlin, CUI Shufang

2012, 32(2):  137-141.  DOI: 10.3969/j.issn.1674-5817.2012.02.012

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Objective To build a minipig model of abdominal wall zipper, the teaching practice test was carried out to discuss the application of minipig in teaching. And the research data was collected, which lay the foundation for the late period application of minipig model of abdominal wall zipper. Method Twenty healthy, juvenile minipigs were selected and divided randomly into two groups, with 10 as control for conventional surgery, 10 for the model of abdominal wall zipper. The minipigs were undergone intestinal anastomosis and intestinal fistula surgery. The intra and postoperative recovery period was dynamically observed. The time course of the experiments, quantity of anesthetic, minipig's mental status and diet consumption were recorded. Result The minipig model of abdominal wall zipper has strong practicality in the surgical teaching because it's a mature and stable model, In intestinal anastomosis surgery, the index was better than the group under conventional surgery such as time course of experiments, quantity of anesthetic, minipig's mental status and so on. Moreover, structural features of the model can also facilitate the dynamic observation after surgery for the researchers. Conclusion The minipig model of abdominal wall zipper was established successfully. Application of the model in the surgical teaching appeared effectively. It may be provided a brand-new animal model for future related research.

A Comprehensive Method of Strain Conservation Mediated by Spermatozoa in Mice

LI Dun-Gao, JIANG Man-Xi, CHEN Xue-Jin, LI He-Ping

2012, 32(2):  142-145.  DOI: 10.3969/j.issn.1674-5817.2012.02.013

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Objective To establish a comprehensive system for mouse strain conservation through natural mating, sperm cryopreservation and assisted reproductive technology. Methods Mouse fresh epididymal sperm were collected by vasectomy of unilateral testis and the vasectomized mice were naturally mated for breeding. If no satisfactory results obtained from natural breeding, then the cryopreserved sperm were thawed for In Vitro Fertilization (IVF). If no results obtained, Intracytoplasmic Sperm Injection (ICSI) was used to reconstructed embryos and which were finally transferred into pseudopregnant recipient female mouse. Results The results show that parts of mice vasectomized unilateral testis such as BDF1 and C57BL/6J mice could be used for natural breeding. Among the remaining sterile males, only ICR mice could be preserved through IVF while ICSI technology was the last approach to save GFP mice with C57BL/6J background. Conclusion The genetic background of mouse sperm cryopreservation was a key factor in IVF and setup a comprehensive system, which could achieve the purpose of strain conservation while mouse strains could be utilized at maximum extent.



Effect of YIGUTANG Containing Serum on Osteoblast Proliferation and IL-6 Secretion

CHEN Fang, ZHU Yi-liang, YANG Li, TANG Ying

2012, 32(2):  146-148.  DOI: 10.3969/j.issn.1674-5817.2012.02.014

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Objective To research the effect of YIGUTANG containing serum on osteoblast proliferation which was from the skull of new born Wistar rat in vitro, and IL-6 secretion. Methods Iliac bone specimens were obtained from the newborn rat calvaria, after that the bone pieces were digested by collagenase-trypsin. The proliferation of osteoblast was observed using MTT reduction assay. The IL-6 in the supernatant was detected by ELISA. Results The proliferation of osteoblast could be stimulated by the serum with different dosages of absorbed YIGUTANG (group A 、B 、C). Meanwhile, the levels of IL-6 secretion in group A, B and C could be significantly decreased compared with the control group (P<0.05). Conclusion The proliferation of osteoblast can be stimulated by the serum with absorbed YIGUTANG and the capability of producing IL-6 in osteoblast also can be restrained by the serum.

Replication and Evaluation of Irritable Bowel Syndrome Animal Models

LIU Yan-xia, LIU Lei, LIU Jun-kang

2012, 32(2):  165-169.  DOI: 10.3969/j.issn.1674-5817.2012.02.019

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The article summarized the replication methods and evaluation indicators of irritable bowel syndrome animal models. Combined with peripheral sensitization, relatively mild stress stimulus method was a relatively better choice which was to enhance the success rate of molding and consider animal welfare. Defecation frequency and character was the most intuitive and economic indicator in evaluation, but its specificity was poor. Intestinal sensitivity was the most specific indicator which depended on the detection of stress and correlated with clinical research.

Advances in Research on Relationship between 5-LO/FLAP and Coronary Artery Disease

LIU Zhi-hua, CHEN Min-li

2012, 32(2):  170-174.  DOI: 10.3969/j.issn.1674-5817.2012.02.020

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Coronary artery disease(CAD) is one of the cardiovascular disease that harmful to human health in the world nowadays. The pathogenesis of CAD is very complicated and until now it has not been completely clarified, the concept that CAD is a chronic inflammatory disease of the coronary artery, is now well accepted. Leukotrienes(LTs) are important inflammatory mediators, which play a strong role in promoting inflammatory reaction. 5-lipoxygenase (5-LO) is the key enzyme in the forming process of leukotrienes(LTs), while the 5-lipoxygenase activating protein(FLAP) is necessary if the 5-LO needs to be activated. Recent studies have found that 5-LO/FLAP play important roles in the formation and development of CAD. In this review, mainly focused on the relationship between 5-LO/FLAP and CAD.