Table of Content

30 April 2009, Volume 29 Issue 4

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Establishment of Human/Goat Stem Cell Chimerism Model via an in Utero Transplantation Under Sonographic Guidance

XU Miao1，HUANG Wen-ying1，HU Wei1，XIONG Can1，SHENG Min2，ZENG Fan-yi3，HUANG Shu-zhen1

2009, 29(4):  211-214. 

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Objective To establish a non-suigical method for in utero transplantation of stem cells into goats under sonographic guidance. Methods Human hematopoitic stem cells from cord blood were delivered into the peritoneal cavities of the fetal goats at 45-55 days of gestation under sonographic guidance (displaying transect of the peritoneal cavities) by injection using a #7 piercing needle . The fetal development was observed and miscarriage rate was noted. The level of chimerism obtained in the fetal goats was studied at set intervals. Results Human hematopoitic stem cells were injected into 49 goat recipients under sonographic guidance, obtaining 36 live bom goats with miscarriage rate (26.7%, 13/49) similar to those from the uninjected control group, but significantly lower than the laparatomy group (63%). Conclusions The method described herein is efficient and safe, with much lowered miscarriage risk comparing to that using surgical interventions for delivering stem cells into fetal goats, and is suitable for producing large numbers of chimeras with many useful applications including serving as animal models for studying stem cell biology in a live organism.

Expression of VP7 Gene of Lapine Rotavirus in Recombinant Baculovirus

ZHOU Jie1，WANG Yu2，HU Jian-hua1，GAO Cheng1

2009, 29(4):  215-219. 

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Objective To express the LaRV VP7 protein gene with Bac-to-Bac baculovirus expression system. Method According to LaRV VP7 gene sequence in Genbank, a pair of specific primers was designed and synthesized. The VP7 ORF gene was amplified by RT-PCR., and then inserted into the transfer vehicle pFastbacHTa to obtain the recombinant transfer vectors pFastBHa-VP7, which was used to transform into E. coli DHlOBac, producing specific transposition with Bacmid to yield the recombinant shuttle vehicles, Bacmid-VP7. Then the Bacmid-VP7 was further used to transfect Sf9 insect cells by lipofectin to generate recombinant baculovirus expressing VP7 recombination protein. Results The recombinant baculovirus and Bacmid-VP7 was obtained. The IFA and Western blotting detection confirmed that the gene had been expressed in sf9 cells, and the expression product could specially react to the sera of LaRV. Conclusion The LaRV VP7protein is expressed successfully by Bac-to-Bac baeulovirus expression system, which may lay foundation for development of diagnostic technique.

Cloning and Eukaryotic Expression of NP Gene of Rabies Virus

LI Min，YUAN Ju-fang，DING Lu-jing，YANG Li-ping，ZHANG Guang-zhou，SHAO Jun-shi，MA Lan-zhi，ZENG Lin

2009, 29(4):  220-222. 

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Objective To detect the immunogenicity of NP gene of RV. Method NP gene of RV was amplified by RT-PCR and then cloned into plasmid pVAXl to construct recombinant plasmid pVAXl-NP.The Vero cells were transfected with plasmid pVAXl-NP. Expression of the NP protein was detected by SDS-PAGE and Westem-blot assay. The mice were immuned with plasmid pVAXl-NP. Result The plasmid PVAXl-NP was obtained and the NP gene was expressed in Vero cells .The expression products had immunogenic ity and the antibody level of immuned mice was higher. Conclusion The NP protein of RV had immunogenic ity.

The Nitric Oxide Synthesis Inhibitor NW-Nitro-L-Arginie Methyl Ester(L-NAME) Causes Cognitive Ability Impairment in Offspring Rats

ZHU Wei-min1，WANG Chun-fang1，LI Xiao-tian1，ZHOU Guang-xin2，REN Yun-yun1，PAN Hua2

2009, 29(4):  223-227. 

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Objective To explore the relationship between exposure to the nitric oxide synthesis inhibitor N①-nitro-L-arginine methyl ester (L-NAME) and the impairment of cognitive ability in offspring rats. Methods Maternal rats are randomly divided into 3 groups: (1) high dose group: maternal rats are subcutaneously injected with 125 mg/kg L-NAME in the 13th gestational day, and persisting in 7 days;(2) low dose group: maternal rats are subcutaneously injected with 75mg/kg L-NAME in the 13th gestational day，and persisting in 7 days;(3) control group: maternal rats are subcutaneously injected with 1 ml of 0.9% NaCl in the 13th gestational day, and persisting in 7 days. Afler the maternal rats is spontaneous delivery, the offspring rats are fed till they are 8 weeks old. The offspring rats were tested for visual-spatial learning and memory and spatial localization abilities in Morris water maze. Results In the animal model of preeclampsia, in the 1st day of place navigation test, the escape latent period of high dose group and low dose group is significant longer than that of control group (P＜0.05). In the 4th day, the escape latent period of high dose group is significant longer than that of control group (P＜0.05). In spatial exploration test, the percent of IV quadrant distance of low dose group is significant lower than that of control group(P＜0.05). Conclusion Exposure to the L-NAME results in the impairment of intelligence of the offspring rats. The study pattern has not significant difference. And the memory and space direction ability of offspring rats is impaired.

Establishment and Evaluation of Myocardial Ischemia Reperfusion Injury Model in Rats

ZHOU Xue-wu，LIAO Zi-fu，CHEN Jing，LIU Liang-ming

2009, 29(4):  228-232. 

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Objective To establish and evaluate myocardial ischemia reperfusion injury of rats by using left coronary artery ligation. Methods The anesthetized rats of twenty-one were chest-opened. The left coronary artery was ligated with silk thread to produce ischemia of 45 minutes and was then relaxed to induce repeifusion of 180 minutes. The standard II lead of electrocardiogram was recorded. The mean arterial pressure and myocardial contractile function were measured. The average areas of myocardial ischemia/necrosis and survival rate of rats were calculated. Results The width and height of QRS wave were increased (P＜0.01) after ischemia and were then decreased (P＜0.01) significantly after reperfusion. The ST segment and T wave were up-shifted (P＜0.01) afler ischemia and were then lowered (P＜0.01) obviously after reperfusion. MAP, HR, LVSP, + dp/dtmax and -dp/dtmax were significantly reduced (P＜0.01) and LVEDP was remarkably increased (P＜0.01) afler ischemia. The tendency of the above indices after reperfusion was the same as that of ischemia. At 180 minutes, the average area ratio of ischemic zone/left ventricle and necrotic zone/ischemic zone were 34.3% and 49.6% respectively, and the survival rate of rats was 49.6%. Conclusions It was an available method to establish myocardial ischemia reperfusion injury model in rats by using left coronary artery ligation. The changes of electrocardiogram,MAP and myocardial contractile function,as well as myocardial ischemia/necrosis were obvious. The successful rate was achieved.

Sleeve Gastrectomy Model in Rat

WANG Yi-long1，CAI Jing-li2，XU Lu-bai2，WANG Ji-sheng2，XU Jia-rong2，KE Zhong-wei3，ZHENG Cheng-zhu3

2009, 29(4):  233-236. 

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Objiective To establish an animal model for sleeve gastrectomy in SD rat. Methods Twenty male SD rat were operated with sleeve gastrectomy. Ten male SD rat were operated with laparotomy. The weight of the rats were determined before operation, 1st day post operation and on Fortnightly basis for 16 weeks. Results All rats remained alive in the control group, 13 rats remained alive in the SG group. In the control group，weight gain was achieved obviously after operation， whereas in the SG group, weight gain was achieved until the 2th week. In the SG group, weight gain was significantly lower than in the control group. Gastric dilatation were observed obviously in the SG group (2.02 士 0.20 w 3.02 土 0.52，P＜0.01; 0.91 土 0.10 w 1.58 土 0.35，P＜0.01). Conclusion The SG experimental model established on SD rat was reliable ,and it may be used for study the mechanisms of sleeve gastrectomy.

Comparative Research on the Sequence of 5' End of MtDNA D-loop Region of Four Breeds of Mini-pigs

LI Hong-tao1，WU Qing-hong2，XIAO Dong2，YUAN Jin2，WANG Wan-shan2，ZHANG Jia-ning2，GU Wei-wang2

2009, 29(4):  237-240. 

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Objectives To analyze the sequence of 5' end of mtDNA D-loop region of Tibet minipigs, Bama miniature pigs and Guizhou xiang pigs and WZS pigs，to investigate genetic differentiation and the phylogenetic relationship between mini-pigs and several other breeds of Chinese domesticated pigs. Methods The fragment of 5' end of D-loop region in four breed mini-pigs were amplified and sequenced, followed by being multiple alignment compared with each other，then variation sites and haplotype were made. Genetic relationship tree between mini-pigs and other China domestic pigs were established. Results Compared with Rongchang pig，704bp sequence at 5' end of the Tibet mini-pigs D-loop had 20 polymorphic sites which deduced 26 haplotypes. There were two mainly haplotypes which have three transform sites at 305，500 and 691，one is t，a，a，respectively; another is c, g, g , respectively. Bama miniature pigs, Guizhou xiang pigs and WZS pigs had 4，4 and 3 haplotypes, respectively. Tibet mini-pigs had close relation with domestic pigs in southwest China by using phylogenetic tree. Conclusions There were genetic differentiation in Tibet mini-pigs colony and had a closely genetic relationships with southwest Chinese domestic pigs. Bama miniature pig and Guizhou xiang pig and WZS pig had less genetic differentiation that may be the results of artificial selection.

Induction of T Lymphocytes by Dendritic Cells Transfected with IL-2 Gone for Therapy of Nude Mice Transplanted with Pancreas Cancer Cell Line PC3

QIAO Wei-wei1，GU Jian-zhong2，DU Wei-dong3

2009, 29(4):  241-243. 

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Objective To study the antitumor effect of cytotoxic T lymphocytes (CTL) induced by dendritic cells (DCs) transfected with IL-2 gene. Methods DCs collected from peripheral blood monocytes were transfected with IL-2 gene and CTL was induced by it. The proliferation of the CTL and its cytotoxic activity to PC3 was assessed. Nude mice with PC3 transplanted tumor were treated with CTL. The kinetics growth was recorded，RT-PCR-ELISA was used to determine the telomerase of transplanted tumor. Result CTL induced by DCs transfected with IL-2 gene can kill PC3 effectively, and it suppress the growth of experimental PC3 tumor and its telomerase. Conclusion DCs transfected with IL-2 gene activate the anti-tumor immune effect of CTL T lymphocytes and it may be a good immune therapy to malignant tumor.

The Cytotoxic Effects of Acetone Extractions from Three Kind Beddings on Mouse Tumour Cell Lines

MENG Jin-ping，SUN Shu-hua，YANG Xu，WANG Yan-rong，LIU Yun-bo

2009, 29(4):  244-247. 

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Objective To investigate the cytotoxic al effects of beddings on growth activity of mouse cell lines. Methods The acetone extraction of three kind beddings(com cob, pine wood shaving, poplar wood shaving ) were respectively added in mouse hepatoma cell line Hepa 1 -6(Hepa 1 -6), the Lewis Lung carcinoma (LLC)and the Melanoma B16(B16) with different concentration. MTT method was used to detect the cell grown activity and compare the cytotoxical effects of different beddings(pine wood shaving, poplar wood shaving and corn cob). Results The acetone extraction of three kinds of bedding provided with inhibition effects on three cell lines. The coefficients on Hepal-6 in linear regression models are: corn cob -0.875，pine wood shaving -0.747，poplar wood shaving -0.826. The coefficients oil LLC are: com cob -0.527，pine wood shaving -0.812, poplar wood shaving -0.865. The coefficients on B16 are: corn cob -0.727，pine wood shaving -0.860，poplar wood shaving -0.888. The 50% concentration of inhibition (IC50) of beddings to Hepal-6 are: com cob 8.86 mg/ml, pine wood shaving 19.10 mg/ml, poplar wood shaving 91.37mg/ml. The IC50 of beddings to LLC are: corn cob 4.42 mg/ml, pine wood shaving 7.72 mg/ml, poplar wood shaving 24.5 mg/ml. The IC50 of beddings to B16 are: corn cob 8.86 mg/ml, pine wood shaving 19.10 mg/ml, poplar wood shaving 91.37 mg/ml. Conclusion There are evident negative relativity between the three beddings and Hepal-6、LLC、B16 cell grown activity (p<0.001). Cytotoxic role in cell growth inhibition is pine wood shaving＞poplar wood shaving＞ corn cob.

Comparison on Different Breeding System and Identification of C57BL/6J-HBV Transgenic Mice

SHEN Yan1，XU Chun-hua2，LI Xiang-rui3，FEI jian4，ZHOU Wen-jiang5

2009, 29(4):  248-253. 

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Objective To explore the breeding rule of C57BL/6J-HBV transgenic mice and to conduct a comprehensive study on biological characteristics of the transgenic mice from the DNA, RNA, proteins and virological level. Method C57BL/6J-HBV transgenic mice were breeded in two different ways of mating and breeding, PCR was used to detect the positive rate of transgenic in each generation. RT-PCR and real-time PCR were used to semi-qualitatively or quantitatively detect the expression of HBV DNA in transgenic mice and to explore the transcription characteristics of mRNA coded by HBV. ELISA and Western Blotting were used to HBV virus antigen expression and secretion. Results There were significant differences (P＜0.05) of both litter size and weaning between two mating mode. The positive rate of reciprocal cross progeny was higher than the rate of backcross progeny. Four founder HBV positive transgenic mice were crossed with normal C57 BL/6J mice, and real-time PCR was performed to detect the HBV genome of the offspring during every passage. Analysis showed that the positive rate was always around 40-50%, indicating the HBV genome has integrated into the C57BL/6J-Tg mice’s genome, expressed stably and can transmit into the next generation reliably. Viral antigen can be stablely expressed in the transgenic mice liver tissue and secreted into the blood. Conclusion The transgenic mouse model can be of high expression model of HBV viral antigen with long-term stability and be applicted as the animal model for hepatitis B virus-related research.

Weight of Main Organs and Organ Coefficient in WHBE Rabbits

XU Jian-qin，CHEN Min-li，ZHANG Li-zong，CHEN Liang，PAN Yong-ming，XU Xiao-ping

2009, 29(4):  266-268. 

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Objective To measure the organ weight and organ coefficient of WHBE rabbits. Methods The weight of body and main organs of adult WHBE rabbits were measured and calculated their organ coefficient, and compared between male and female in this experiment. Results Between male and female, the thyroid gland weight and coefficient showed significantly differences (P＜0.01)，but no difference was seen in the other organ coefficient (p>0.05). Conclusion The above data could be useful in the biomedical researches.

An Analysis of Blood Gas and Electrolytes in WHBE Rabbits among Different Ages

ZHOU Wei-min，XU Jian-qin，CHEN Min-li，PAN Yong-ming，WANG De-jun，ZHANG Li-zong

2009, 29(4):  269-272. 

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Objective To observe the changes of blood gas，electrolytes and energy metabolism in WHBE rabbits among different ages. Methods One hundred and thirty-three WHBE rabbits were divided into 3 groups, infant，young or senile groups respectively, the blood sample were taken from center artery in ear, and the blood gas and electrolytes indexes were detected by NOVA blood gas and electrolytes analyzers. Results (1) Effect of gender，the Glu index was lower in male infant rabbits than that of female ones(P＜0.05)，and SO₂，Hb, K+ indexes were higher in male young rabbits than those of female ones(P＜0.05)，no sexual difference was shown in the blood gas of senile group(p>0.05). (2) Effect of age, the PCO₂，BE-ECF，BE-B，SBC，HCCV，TCO₂，a/A indexes were higher in young group than those of infant group (P＜0.05，P＜0.01)，while PO₂ SO₂, O₂Ct，A, PO₂/FI indexes were lower in young group than those of infant group (P＜0.05，P＜0.01)，pH, PCO₂，BE-ECF, BE-B, SBC, HCCV, TC02, a/A, Osm indexes were higher in senile group than those of infant and young groups (P＜0.05，P＜0.01)，while PO₂, SO₂，Hct，Hb，O₂Ct，A，PO₂/FI indexes were lower in senile group than those of infant and young groups (P＜0.05，P＜0.01). Conclusion Gender has effect on Glu in infant group and on SO₂, Hb, K+ in young group. The blood gas, electrolytes and energy metabolism in WHBE rabbits were changed with increasing age, and those related with its physiological function.

Study on Anti-oxidation Activity in WHBE Rabbit with Irritable Bowel Syndrome of Spleen Deficiency Type

ZHANG Li-zhong，XU Jian-qin，PAN Yong-ming，CHEN Liang，WANG De-jun，PENG Ding-guo，YING Hua-zhong，CHEN Min-li

2009, 29(4):  273-276. 

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Objective To observe the change of anti-oxidation activity in WHBE rabbit with irritable bowel syndrome of spleen deficiency type. Methods Twelve WHBE (white hair and black ear) rabbits and 12 Japanese white rabbits were randomly divided into two groups respectively, with 6 rabbits in normal control group and 6 rabbits in model group. The irritable bowel syndrome of spleen deficiency type rabbits model was made by using the methods of moist heat, stress and stimulation adding with taking senna decoction，and each model group rabbits were recovered naturally for 10 days after the successful establishment. The levels of superoxide dismutase (SOD), the content of malondialdehyde (MDA), the activities of glutathione peroxidase (GSH-Px), nitric oxide (NO) and nitric oxide synthase (NOS) were determined. Results Compared with normal control groups, the activities of the serum SOD and GSH-Px were significantly decreased in WHBE rabbits and Japanese white rabbits after the successful establishment of the model (P＜0.05，P＜0.01)，the content of MDA，the activities of NO and NOS were significantly increased (P＜0.05，P＜0.01). After 10 days nature recover, the activity of GSH-Px in the natural recovery groups was still lower, while the content of MDA and the activity of NOS in the WHBE rabbits were all significantly higher than those in the normal rabbits (P＜0.05). All the anti-oxidation indexes in Japanese white rabbits returned to the normal level, except for the GSH-Px activity was still higher than normal control group (P＜0.05). Conclusion The anti-oxidation activity of WHBE rabbit of irritable bowel syndrome of spleen deficiency type were significantly decreased similar to clinic in human, and recovered slowly, so the WHBE rabbits may be suitable for the study of irritable bowel syndrome of spleen deficiency type in huaman.

The Current Situation of Asthma Animal Model

CUI Fei-fei，XIN De-li

2009, 29(4):  277-277. 

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As morbidity of asthma was increasing in recent years, it was necessary to investigate the etiology, pathology, treatment of bronchus asthma. But there were many limitations to study in human, so that the animal model of asthma played a Irreplaceable role in asthma investigation. In recent years, more and more animal asthma models were applied to further elucidate the pathology, physiology, and the machanisms of immunity. And study in animal also gave us many results which can not achieved in human body. Even in the aspects of development of new drugs, animal model gave us the experimental therapy that can not applied in human. Here, the established animal asthma models and interrelated sensibilization substances were reviewed.