Table of Content

25 April 2014, Volume 34 Issue 2

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Establishment of Different Malignant Potential Prostate Cancer Animal Model from Same Origination

ZHANG Zhen-hua, SHEN Zeng-li, HOU Chuan-ling, SONG Chun-jiao, SUN Ai-jing

2014, 34(2):  83-88.  DOI: 10.3969/j.issn.1674-5817.2014.02.001

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Objective Supplying ideal animal model for investigating the mechanism of tumor progress and hormone resistant in prostate cancer. Methods Prostate cancer tissue was obtain via prostatectomy and cut into specimens which orthotopic transplanted into castrate group and sham-castration group nude mice .T hen tumor was harvest and transplant to next generation. until emergence of lymph node metastasis in two groups.. The cell growth curve of cell lines is used to evaluate their growth condition. Boyden chamber is used to evaluate metastasis ability of cell lines. The rate of tumor formation, weight of tumor and range of tumor infiltration of three cell lines was confirmed by subcutaneous transplant. Result The tumorgenic in sham-castration group is 30% without lymph node metastasis in first generation, after three passages is 50%, lymph node metastasis in pelvic is 40%. In castrate group, no tumor formation and lymph node metastasis in first generation. One pelvic lymph node metastasis is find after the forth passages which tumor origin from the lymph node metastasis of sham-castration group.. There are significant differences of cell proliferation transfer ability and tumor rate in vitro and vivo between three cell lines. (p<0.05). Conclusion It's feasible that applying surgical specimens of prostate cancer to establishment .nude mice orthotopic graft model. It is possible to screen heterotransplant tumors and metastasis tumors without hormone dependency by graft rat's lymph node metastasis tumor into castrate nude mice.

Effect of Body Building and Spirit Fostering Exercise Training on Cognitive Function and Blood Pressure in SHR/SP Rat with Vascular Dementia

ZENG Gui-gang, ZHANG Shen, LI Jun, PENG Hai-dong, JIANG Hong-xu, WEI Pin-kang

2014, 34(2):  89-92.  DOI: 10.3969/j.issn.1674-5817.2014.02.002

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Objective To investigate the effect of body building and spirit fostering exercise training on the cognitive function and blood pressure in the Stroke Prone spontaneously hypertensive rat(SHR/SP) with vascular dementia (VD). Method The VD model was established by a permanent ligation of bilateral carotid artery (2-VO) in male SHR/SP rats. The VD model rats were randomly divided into model group(M group), body building group(B group), spirit fostering group(S group ) and body building & spirit fostering group(BS group) (n=10) respectively. The B group was trained by using treadmill training, S group was trained by using enriched environment, BS group was trained by using treadmill training and enriched environment. After 8 weeks, Morris maze tests were conducted for valuating the learning- memory abilities, the blood pressure was measured using a tail cuff method. Result In place navigation training, the latency of BS group was less than other groups, the latency of B group and S group were less than that of rats in model group, there was not different between B group and S group (P<0.05); In spatial probe ability test, the frequency of crossing platform and the ratio of time spent in the target quadran of rats in BS group were significantly higher than other groups, the frequency of crossing platform and The ratio of time spent in the target quadran of rats in B group and S group were higher than M group (P<0.05), there was not different between B group and S group (P<0.05); the blood pressure in BS group was lower than other groups, the blood pressure in B group was lower than S groups and M group, there was not different between S group and M group (P<0.05). Conclusion Combination of the spirit and body exercise training is one of the important thoughts of the basic TCM theory, the training under guidance of the theory can improve the cognitive function and blood pressure in SHR/SP rat VD model.



Chronic Restraint Stress Induced Anxiety- and Depression-like Behaviors in Spontaneously Hypertensive Rats

CAO Shuang, DING Jian-wei, LI Bing, YUAN Li-fen, DU Qin

2014, 34(2):  93-97.  DOI: 10.3969/j.issn.1674-5817.2014.02.003

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Objective To investigate the effects of chronic restraint stress (CRS) on anxiety- and depression-like behaviors in Spontaneously Hypertensive Rats(SHR). Methods SHR were randomly divided into control(CON) group and CRS group respectively. The changes of behaviors were detected by open field test (OFT),forced swimming test (FST) and elevated plus maze(EPM) testŁ¬the changes of blood pressure and levels of adrenocorticotropic hormone(ACTH) in plasma in different groups were also measured and compared. Results SHR in CRS group showed significant reductions in rearing incidence in OFT(PŁĽ0.05),the time spent in open arms during the EPM(PŁĽ0.05), and the immobility during the FST was increased significantly (PŁĽ0.05). Furthermore, the blood pressure and concentration of plasma ACTH increased significantly in CRS group compared to CON group. Conclusions CRS can induce anxiety- and depression-like behaviors in SHR.

Effect of Compound Glycyrrhizin on Expressions of Colonic NF-κB p65 and COX-2 in Ulcerative Colitis Rats

LU Guang-xin, LV Fei, DU Yong, YANG Yang-guo, ZHOU Chun-fang, JIN ling-li, YANG Jian-ye

2014, 34(2):  98-101.  DOI: 10.3969/j.issn.1674-5817.2014.02.004

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Objective To investigate the experimental therapeutic effect of the Compound Glycyrrhizin on the expression of NF-κB p65 and COX-2 in rats with ulcerative colitis(UC) induced by trinitrobenzene sulfonic acid/ethanol (TNBS/ethanol). Methods Forty adult Sprague-Dawley rats were randomly divided into 4 groups(n=10), Normal control group received no treatment, while the rest groups used trinitrobenzene sulfonic acid/ethanol enema to generate ulcerative colitis models. UC model group received saline water enema and decapitated three days later, Compound glycyrrhizin glycosides treatment group daily received glycyrrhizin glycosides solution enema, 40 mg·kg^-1·d^-1, Continuous enema 7 days and decapitated,, Sulfasalazine (SASP) treatment group daily received SASP solution enema, 0.5 g·kg^-1·d^-1, Continuous enema 7 days and decapitated, Each group received different treatment as described above, NF-κB p65 and COX-2 were detected by immunohistochemistry. Results Compared with Normal control group, the level of NF-κB p65 was significantly increased in UC model group、SASP treatment group and Compound glycyrrhizin glycosides treatment group [0.25±0.11 vs 5.58±0.85 vs 3.36±0.66 vs 3.21±0.58, P<0.01], and the level of COX-2 was significantly increased [0.21±0.13 vs 5.54±0.40 vs 3.23±1.05 vs 3.16±0.94, P<0.01]. Compared with Model group, the level of NF-ęB p65 was significantly decreased in SASP treatment group and Compound glycyrrhizin glycosides treatment group [5.58±0.85 vs 3.36±0.66 vs 3.21±0.58, P<0.01], and the level of COX-2 was significantly decreased [5.54±0.40 vs 3.23±1.05 vs 3.16±0.94, P<0.01]. Conclusion Compound Glycyrrhizin has therapeutic effect on ulcerative colitis in rat. The mechanism is associated with the reduced levels of NF-κB p65 and COX-2.

The Characteristics of Glucolipid Metabolism and Insulin Resistance in ZDF(fa/fa) Rats

XI Sai-fei, SI Xu-wei, ZHU Ke-yan, CHEN Xiao-zhen, YANG Tao-tao, CHEN Min-li

2014, 34(2):  102-106.  DOI: 10.3969/j.issn.1674-5817.2014.02.005

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Objective To observe the characteristics of glucolipid metabolism and insulin resistance in ZDF rats after feeding with high fat diet. Methods Six ZDF (fa/fa) rats in model group were fed with high fat diet, 6 ZDF (fa/+) rats in control group, fed with normal diet, lasted for 6 weeks. The body weight was measured every week. Glucose, cholesterol, triglycerides, high-density lipoprotein, low density lipoprotein were measured every two weeks, TC/HDL-C and AI were calculated. In 6^th week, the fasting insulin, OGTT and ITT were measured. HOMA-IR, HOMA-β were calculated. Results After feeding with high fat diet, the blood glucose and lipid levels in ZDF model group were rapidly increased (P<0.01), TC/HDL-C and AI were significantly increased (P<0.01), after 4 weeks, they remained stable.In 6^th week, fasting insulin and HOMA-IR were significantly increased (P<0.01), but HOMA-β was significantly decreased (P<0.01). Conclusion ZDF rats induced by high fat diet, exhibits hyperglycemia with hyperlipidemia, abnormal glucose tolerance and hyperinsulinemia, and also exhibits increased insulin tolerance and decreased insulin sensitivity. ZDF rats are suitable to study tyjpe 2 diabetes mellitus and metabolic syndrome which caused by insulin resistance.



Effect of HSP70 on Fibrosis of Nonalcoholic Fatty Liver Disease in Rabbits

DENG Li-li, ZANG Jia-na, YING Hua-zhong, YU Chen-huan, YU Bing

2014, 34(2):  107-112.  DOI: 10.3969/j.issn.1674-5817.2014.02.006

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Objective To investigate the change of HSP70 in nonalcoholic fatty (NAFLD) liver fibrosis of rabbit. Methods Forty male Japanese white rabbits were randomly assigned to control group and model group. The model group was divided into three subgroups, as 4-week, 6-week, 8-week groups, with 10 rabbits in each, and treated with peanut oil (1.2 ml/kg) subcutaneously (s.c.), twice a week for 4 weeks, 6 weeks and 8 weeks respectively. The rabbits of all groups were sacrificed at the designed time point. Samples of serum were collected for detecting the serum ALT, AST, TC, TG, FFA, FBG, HSP47, HSP70, HA, PC?, C˘ô, LN ,and the HOMA-IR were calculated. The liver tissues were collected for histopathological and immunohistochemical observation. Results The model groups had hepatic injury and high level of insulin at the beginning of 4 weeks. With the process of NAFLD liver fibrosis, HSP70 had gradually increased. Conclusion The expression of HSP70 was positively correlated with NAFLD liver fibrosis, and it might have close relation with the development of NAFLD liver fibrosis.

Expression, Activity and Pharmacokinetic Study of A Novel Long-Acting Recombinant Human Chorionic Gonadotropin

HUANG Hu, YANG Fan, WU Mao-bo, ZHANG Yu-jie, ZHONG Ling, HOU Yong-min

2014, 34(2):  113-119.  DOI: 10.3969/j.issn.1674-5817.2014.02.007

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Objective To make a novel long-acting hCG-Fc fusion protein that could prolong hCG's serum half-life and improve the patients' compliance. Methods The expression vector was constructed by the gene engineering that contains the gene encoding hCG-Fc which Fc fragment was fused into the 3' end of hCG gene. The expression vector was transfected into CHO cells and stable clones were selected to express hCG-Fc fusion protein specifically. The serum free culture medium of cells grown was collected for protein purification using affinity based chromatography. The analysis of purified protein was performed as follow: molecular weight by SDS-PAGE, protein identity by Western blotting, activity in vitro by ELISA, activity in vivo by seminal vesicle weight gain assay, and serum half-life by single-subcutaneous injection into rats. Results The molecular weight of hCG-Fc fusion protein is similar to the theoretical size. The ELISA activity of hCG-Fc protein is 2 965 IU/mg and in vivo activity is 2 288 IU/mg. The serum half-life in rats is 174 h. Conclusions hCG-Fc fusion protein is active and functional based on its in vitro activity and in vivo activity. Pharmacokinetic study shows that its serum half-life is almost six times of the recombinant hCG's. This novel long-acting fusion protein can significantly reduce the times of injection and improve the patients' compliance of medicine, and also could have huge social and economic benefits.



Effects of Excess Fluoride on Secretion of GAG and II Type Collagen in vitro Chondrocytes of Rats

WANG Ning, FAN Yu-lan, SHI Zhi-chong, ZHENG Wei-dong, LU Wei, XIAO Ping

2014, 34(2):  120-125.  DOI: 10.3969/j.issn.1674-5817.2014.02.008

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Objective To observe the effect of secretion of GAG and II type collagen of excess fluoride on in vitro chondrocytes of rats. Methods Chondrocytes of rats was separated from the 2-month old female rats, and cultured in the cell bottles. After identification, six groups were designed in the research including control group and NaF group (6.25 µmol/ml, 12.50 µmol/ml, 25.00 µmol/ml, 50.00 µmol/ml and 100.00 µmol/ml). After 6 days of administration, GAG contents were measured. Alcian Blue dye and II type collagen immunohistochemistry dye were also performed on the in vitro chondrocytes of rats. Results After 6 days of treatment, there were no significant difference of GAG contents of cell culture medium between NaF groups and control group (P>0.05). Alcian Blue dye showed that secretion of GAG content of all groups did not change after administered by NaF. But II type collagen immunohistochemistry dye showed that scores of II type collagen immunohistochemistry dye increased according to dosage of NaF leveled up (P<0.05). Conclusions NaF may not affect the content of GAG secreted by the chondrocytes of rats in vitro, but can affect the secretion of II type collagen.



Effects of High Fat Diet on Arterial Ring's Tension in SD Rats

ZHANG Yi, ZHAN Wei-wei, WU Yong-ji, ZHOU Wu-gang, CHEN Dong-rui, JIANG Wei-min, ZHENG Lin

2014, 34(2):  126-130.  DOI: 10.3969/j.issn.1674-5817.2014.02.009

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Objective To observe the changes of arterial ring's tension in SD rats with hypercholesterolemia and atherosclerosis. Methods In vitro isometric tention experiment was used to measure arterial ring's maximum contractile tension, arterial ring'maximum relaxation pencentage of the aorta in cholesterol-fed SD Rats (group T1, n=10, for 4 weeks; group T2, n=10, for 12 weeks) and normal control rats(group C1, n=10; group C2, n =10). All parameters and blood biochemical markers(total cholesterol, triglycerides, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol) among groups were analyzed with ANOVE factor analysis.Correlation was analyzed with Pearson analysis.Light microscopic evaluation was used to demonstrate atherosclerotic changes in the aorta. Result The maximum contractile tension value to KCl and phenephrine and the maximum relaxation pencentage of the aorta in group T1 were significantly lower than those of group C1 controls (P<0.05). The maximum relaxation pencentage of the aorta in group T2 rats were significantly lower than those of group C2 controls (P<0.05). The maximum contractile tension value to KCl and phenephrine in group T2 were significantly higher than those of group T1(P=0.001, P<0.05). However, no significances were found with the maximum relaxation percentage in group T1 and T2 (P>0.05). Correlation analysis shows: the maximum contractile tension value of the aorta was positively correlated with triglycerides (r=0.435, P<0.05). The maximum relaxation pencentage of the aorta was negatively correlated with systolic pressure, total cholesterol and low-density lipoprotein cholesterol (r=-0.318, P=0.046, r=-0.449, P=0.004; r=-0.411, P=0.008), also it was negatively correlated with high-density lipoprotein cholesterol(r=0.324, P=0.041). Light microscopy confirmed morphologic typical changes of aortic atherosclerosis in group T1 and T2. Conclution High fat diet will lower the ability of rats's arterial contraction and relaxation in earlier state. The maximum relaxation percentage was much lower associated with the increasement of total cholesterol and low density lipoprotein.

Genetic Analysis of Rhesus Macaques(Macaca mulatta) Using Microsatellite Markers

YANG Na, LIU Xiao-shuai, ZHOU Liang, ZENG Dai-wen

2014, 34(2):  131-135.  DOI: 10.3969/j.issn.1674-5817.2014.02.010

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Objective Inbreeding is the big problem in captive colonies of rhesus macaques.To preserve genetic variability, the exact pedigree must established by genetic markers. Methods Fourteen autosomal short tandem repeat (STR) loci with high information content were used to 64 rhesus macaques by fluorescently-labeled primer polymerae chain reaction (PCR) and capillary electrophoresis typing. Then, these loci were evaluated by unique genetic characterization, parentage determination, and estimation of parameters. Results Seven of the 14 markers have high gene diversity (H_e) and polymorphic information content (PIC)(>0.8), the combined probability of genetic identity (CPI) at all loci is 1.21E-18, and the combined probability of no-parent exclusion (CPE1) or single parent exclusion (CPE2) at all loci can reach 0.9985 and 0.9999 respectively. Conclusion These markers are reliable and highly informative genetic markers, can provide for powerful parentage determination, unique genetic characterization, and accurate estimation of parameters, which are all useful for genetic management of rhesus macaques.

Application of PCR Methods for Detection of Cilia-Associated Respiratory Bacillus in Mouse

FENG Yu-fang, XING Jin, YUE Bing-fei

2014, 34(2):  136-139.  DOI: 10.3969/j.issn.1674-5817.2014.02.011

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Objective To establish an effective PCR assay for detection of cilia-associated respiratory bacillus, and applicate the method for detection in mouse. Methods Sequence of CAR bacillus was obtained from NCBI genbank. A plasmid was structured by selecting the appropriate sequence. And primers were designed based on the sequence; the positive amplified fragments were sequenced to verify the reliability of the method. Results The PCR method for detection of CAR bacillus was successfully established. Conclusion The establishment of the method will help the detection of experimental animals for CAR bacillus.



Application of in Vitro Fertilization and Embryo Transfer in Breeding of APP/PSEN1 Double Transgenic Mice

LÜ Xiang-chuan, ZHANG E-nuo, ZHOU Sheng-lai, YU Yang, WANG Wei, ZHANG Mei-ying, ZHENG Zhi-hong

2014, 34(2):  140-144.  DOI: 10.3969/j.issn.1674-5817.2014.02.012

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Objective To study the techniques of in vitro fertilization (IVF) and embryo transfer applied for breeding APP/PS1 double transgenic mice. Methods The fertilization rates and birth rates were analyzed in both natural mating group and IVF group for breeding APP/PS1 double transgenic mice. The female C57BL/6 mice were given intraperitoneal injection of appropriate dose of PMSG-hCG. The super ovulation oocytes were collected for IVF with sperms which were taken from 12 weeks, 24 weeks, 36 weeks, 52 weeks and 72 weeks old APP/PS1 double transgenic male mice respectively. The 2-cells embryos were than transfer to the fallopian of pseudopregnant mice. The genotypes of newborn mice were identified by PCR. Results The fertilization rates were 73.3% in group of natural mating, but 95% in group of IVF. The statistic differentiation was significant in newborn mice number (P<0.05) between natural crossing group to IVF group, but was not marked in genotype positive rate (P>0.05). The fertilization rates, birth rates and genotype positive rate were evaluated in 12 weeks, 24 weeks, and 36 weeks old APP/PS1 double transgenic male mice by statistic analysis which showed that there were no significant difference among them (P>0.05 ). The IVF rates were 85.6%, 84.1% in 52 weeks, and 72 weeks old APP/PS1 double transgenic male mice those were so aging enough that could not breed the filial generation by usual method. Conclusion The application the techniques of IVF-ET can enlarge rapidly the breeding population of APP/PS1 double transgenic mice.



A Modified Delivery of Mouse Cauda Epididymides at Room Temperature

WANG Mei-shan, KONG Peng-cheng, ZHU Yan, ZHU Lian, JIANG Man-xi, CHEN Xue-jin, LI He-ping

2014, 34(2):  145-148.  DOI: 10.3969/j.issn.1674-5817.2014.02.013

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Objective To explore the optimal conditions for Cauda epididymis transport under room temperature and eliminate the effect of medium on the transport of cauda epididymides at room temperature. Methods The transport methods of cauda epididymides were modified in this study and the sperm motilities, fertility and developmental abilities of IVF embryos after transportation at room temperatures or 4~8℃ in 20 μl of medium M2 or medium-free tube were compared. Results Under room temperatures, delivery of cauda epididymides without medium was significantly superior to with medium, but was inferior to 20 μl M2 medium under 4~8℃. Conclusion The modified medium-free system was beneficial to room temperature transportation of cauda epididymides and maybe eliminate the side effects of medium on spermatozoa, and the delivery of cauda epididymides under room temperature was useful for transporting some valuable strains between mouse banks and research facilities.



Loop-mediated Isothermal Amplification Technique and Its Application

ZHAO Lijuan, ZHOU Jie, GAO Cheng

2014, 34(2):  162-166.  DOI: 10.3969/j.issn.1674-5817.2014.02.018

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Loop-mediated isothermal amplification(LAMP) is a novel nucleic acid amplification method. At a certain temperature, LAMP performed only one step to terminate the process. Due to its high efficiency, simplicity, rapidly, high specificity and the result of LAMP is easily detected by naked eyes, LAMP has been widely applied in the qualitative and quantitative detection of viruses, bacteria and clinical diagnosis of disease. In this paper, we introduced the principle of LAMP and its applicability in molecular detection and diagnosis.

Situation on Research of Murine Norovirus

LIU Qin, WEI Xiao-feng, GAO Cheng

2014, 34(2):  167-170.  DOI: 10.3969/j.issn.1674-5817.2014.02.019

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Norwalk virus, also known as norovirus, is the major pathogen to cause human nonbacterial gastroenteritis, in addition, Norwalk virus is also found in cattle, pigs and mice . Norwalk virus can be divided into five genotypes (GI-V), and the murine norovirus (MNV) belongs to GV. MNV is a newly discovered virus infection in laboratory mice, and is considered to be the most popular form of mouse virus. Now, MNV is the only one that can be replicated efficiently in cells of the Norwalk virus, so MNV is regarded as a research model for the molecular mechanism of Norwalk virus. Moreover, MNV and human Norovirus (HuNV) have many similar characteristics of molecular biology, and also is considered to be the ideal virus model for study of human norovirus.