Table of Content

25 October 2012, Volume 32 Issue 5

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Mutation Screening of Two Candidate Genes in B6-Co Mouse Affected with Cornea Opacity: Ankrd55 and Ddx4

LI Yao, WU Liu-cheng, LU Ze-yan, ZHENG Liang-feng, GE Liang-yu, SHAO Yi-xiang

2012, 32(5):  371-375.  DOI: 10.3969/j.issn.1674-5817.2012.05.001

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Objective To find mutational site of Ankrd55 and Ddx4 gene, clone and sequence these gene in B6-Co mutant strain mouse with cornea opacity phenotype. Methods The primers were designed according to mRNA. The target gene was amplified by PCR and RT-PCR in which the PCR templates were mRNA, and then recombinant vectors were transformed to competent cells after DNA fragments were connected to the T vector. The positive clones were selected and plasmid DNA extraction was detected by electrophoresis. The fragments by EcoRI digestion for identification of positive clones and at the end sequencing. Results The C base in sequence of Ddx4 chromosome 13 113 412 349 was replaced by A by sequence alignment with genome data base, with coding protein Pro replacement by Gln. Conclusion There is a single base mutation in Ddx4 gene, and the mutation may relate to the EOB phenotype of B6-Co mice. And it needs to be studied further.



Dynamic Observation on In vivo Bioluminescence Imaging of Experimental Metastatic Animal Models in Nude Mice

YAN Ming-xia, ZHU Miao-xin, LIU Lei, Li Jing, Lin He-chun, ZHAO Fang-yu, YAO Ming

2012, 32(5):  376-382.  DOI: 10.3969/j.issn.1674-5817.2012.05.002

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Objective To observe the tumor metastasis in deep organisms of the nude mice by in vivo bioluminescence imaging system. Methods The SMMC-7721-GFP/Luc cells with different concentrations were intravenously inoculated into the tail vein and spleen of the BALB/c-nu/nu mice, the distribution and expression of luciferase in nude mice were monitored by in vivo bioluminescence imaging system. Results The experimental metastatic animal models had been successfully established. The distribution and expression of luciferase ascended with cell concentration increased and decreased with the passage of time. Conclusion The in vivo bioluminescence imaging system may monitor the in vivo growth and metastasis of tumors and provide for studying the mechanisms of tumor metastasis and development of anticancer drug.



Reproductive Toxicity of Dibutyl Phthalate in Parent and Progeny Zebrafish

XU Hao, WANG Yu-zhu, WEN Cheng-li, CHENG Qian-qian, TIAN Fang, XIA Min-jie, LI Run-sheng, LI Wei-hua, DING Xun-cheng

2012, 32(5):  383-389.  DOI: 10.3969/j.issn.1674-5817.2012.05.003

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Objective To study the reproductive toxicity effects of dibutyl phthalate (DBP) on F₀ (parent) and F₁ (parent) zebrafish. Methods Fourty-eight pairs of adult zebrafish were randomly divided into 4 groups: blank control group, 0.01% acetone solvent control group, 625 µg/L DBP low-dose group, and 1250 µg/L DBP high-dose group. After 30-day exposure, F₀ zebrafish in each group were mated and the number of eggs, fertilization rate and 72-hours hatchability were recorded. After being kept for 180 days in clean water, F₀ and F₁ zebrafish in each group were again mated, and the replacement mating test were conducted. The Vetellogenin (VTG) mRNA of F₀ and F₁ zebrafish was measured by RT-PCT, and gonad histopathology was performed. Results The fertilization of F₀ and F₁ zebrafish in 1250 µg/L DBP group was significantly lower than that in blank control or in 625 µg/L DBP group; histological examination showed that DBP slightly inhibited ovarian development of the F₀ and F₁ zebrafish and slightly increased the cortical vesicular stage oocyte (Coc) and perinucleolar stage oocyte (Poc). In F₀ male zebrafish of 1250 µg/L DBP group, the sperm count in the testes was reduced, and the spermatocyte (sc) and Leydig cells were slightly increased. In F₁ male zebrafish of 1250 µg/L DBP group, the development of testes was inhibited significantly, manifested as the co-existence of abnormal seminiferous tubules, reduced sperm count and Leydig cell hyperplasia. Exposure to DBP could not induce the expression of VTG mRNA in either F₀ or F₁ male zebrafish, indicating that DBP has no estrogen-like effects on zebrafish. Conclusion Exposure to 1250 µg/L DBP can induce reproductive toxicity in F₀ and F₁ zebrafish. The number of eggs and egg hatchability were reduced. Abnormal seminiferous tubules, reduced sperm count as well as Leydig cell hyperplasia were found.



Establishment of Virtual System of Liver Three-dimensional Structure in Zebrafish

LIN Jin-xing, WEI Xiao-feng, CHU Xiao-hong, HU Jian-hua, GAO Cheng, CHEN Qiu-sheng

2012, 32(5):  390-395.  DOI: 10.3969/j.issn.1674-5817.2012.05.004

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Objective To establish the zebrafish liver three-dimensional structure of virtual system, for morphological basis of application research on zebrafish as model organism. Method The adult zebrafish liver tissue structure were observed by light and electron microscopy through the production of large amounts of tissue sections. After collecting of zebrafish liver morphology image, the software of zebrafish liver three-dimensional structure virtual system was established by use of computer image processing software on zebrafish liver shape reconstruction. Result The virtual system software of zebrafish liver three-dimensional structure was established. Conclusion The realization of zebrafish liver was preliminary reconstructed.



Comparison of SNP and STR Genotypes in Chromosome 1 Substitution Strains

ZHAO Li-ya, ZHAO Ying, CHAO Tian-zhu, LIU Lei, XING Zheng-hong, CHEN Guo-qiang, XIAO Jun-hua

2012, 32(5):  396-400.  DOI: 10.3969/j.issn.1674-5817.2012.05.005

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Objective To compare the genotyping results of STR and SNP and choose typing methods for three chromosome 1 substitution strains. Methods The three chromosome 1 substitution strains included 40 samples were typed respectively by 20 SNP and 10 STR. Results A total of 6 samples, 9 samples, 4 samples were identified respectively with SNP among 40 samples of C3H/He, FVB/N and AKR chromosome substitution strains. A total of 6 samples, 10 samples, 6 samples were identified respectively with STR among the 40 samples of C3H/He, FVB/N and AKR chromosome substitution strains. Conclusion For each strain with different gene background,. 3 groups of STR on chromosome 1 were chosen as genotyping methods of C3H/He chromosome 1 substitution strain. 3 groups of SNP were chosen as genotyping methods of FVB/N and AKR chromosome 1 substitution strains.



Observation on Oxidative Stress in Chronic Myocardial Ischemia in Mini-pig

XU Xiao-ping, PAN Yong-ming, CHEN Liang, XU Jian-qin, WANG De-jun, ZHU Ke-yan, YANG Yu-wei, CHEN Min-li

2012, 32(5):  401-406.  DOI: 10.3969/j.issn.1674-5817.2012.05.006

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Objective To observe the effects of oxidative stress on chronic myocardial ischemia in mini-pigs. Methods Fifty-five Bama mini-pigs were randomly divided into three groups: normal control (Ctr) group, high-fat (HF) group and chronic myocardial ischemia (CMI) group, five animals in each group. The chronic myocardial ischemia model was made induced by vitamin D₃, isoproterenol and high-fat diet for 24 weeks. The changes of phosphokinase isoenzyme (CK-MB), platelet aggregation rate (PAR) and the SOD, MDA, GSH-Px, NO, LD, ATPase in myocardial tissues were detected. Results Compared with normal control group, the serum CK-MB activity, PAR, the myocardial tissues of MDA and LD contents in CMI group were significant increased (P<0.05, P<0.01), the myocardial tissues of SOD, SOD/MDA ratio, GSH-Px, NO, Na⁺-K⁺-ATPase, Ca⁺⁺-ATPase and Mg⁺⁺-ATPase in CMI group were lowered (P<0.05, P<0.01). PAR and the myocardial tissues of SOD/MDA ratio and MDA content in HF group were significant increased (P<0.05, P<0.01), while the myocardial tissues of SOD, GSH-Px, Na⁺-K⁺-ATPase and Mg⁺⁺-ATPase were significant decreased (P<0.05). Compared with HF group, the serum CK-MB activity in CMI group was significant higher than that of HF group (P<0.01) and the myocardial tissues of Na⁺-K⁺-ATPase and Ca⁺⁺-ATPase were significant lower than those of HF group (P<0.05, P<0.01). Correlation analysis showed that there was a positive correlation between CK-MB and PAR, MDA in CMI group (P<0.05, P<0.01), and a significant negative correlation between CK-MB and SOD, SOD/MDA, GSH-Px, NO respectively(P<0.05, P<0.01). The platelet aggregation rate was positively correlated with CK-MB and MDA (P<0.05), and was negative relation with SOD, SOD/MDA and Na⁺-K⁺-ATPase (P<0.05, P<0.01). There was a significant positively correlation between LD and MDA in myocardial tissues (P<0.05), and LD was negatively correlated with SOD/MDA, Na⁺-K⁺-ATPase, Ca⁺⁺-ATPase and Mg⁺⁺-ATPase (P<0.05). NO level was positively correlated with SOD, SOD/MDA, GSH-Px in myocardial tissues(P<0.05, P<0.01), and was negatively related with CK-MB and MDA (P<0.05). Conclusion Oxidative stress injury was participated in the formation of chronic myocardial ischemia.



Effect of Telmisartan on Blood Pressure of Mice under Intermittent Hypoxia Condition

HE Hong-xing, ZHENG Yi-min, CHEN Xiao-yang, WANG Wan-yu

2012, 32(5):  407-410.  DOI: 10.3969/j.issn.1674-5817.2012.05.007

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Objective To observe the effect of chronic intermittent hypoxia in sleep apnea syndrome patients on renin angiotensin system and the protection of angiotensin receptors blocker on cardiovascular system, so as to explore the mechanism which sleep apnea syndrome act on cardiovascular system. Methods Thirty-two healthy male C57BL/6J mice were randomly divided into 4 groups: the CIH group, the telmisartan treatment group, the air control group, and the blank control group, with 8 mice in each group. The period of chronic intermittent hypoxia was lasted for 12 weeks. The blood pressure and blood flow of mouse tail were measured. Results (1) The level of systolic blood pressure in the CIH group was significantly higher than other groups and came down to the lowest after ARB treatment. (2) The level of diastolic blood pressure in ARB treatment group was significantly lower than other groups. (3) The tail blood flow of CIH group was significantly higher than those of the blank control group, the air control group, and the ARB treatment group. Conclusion The chronic intermittent hypoxia might induce high systolic blood pressure, and that ARB could decrease the hypertension caused by CIH. At the same time, CIH could increase the tail blood flow of mouse, and ARB could inverse this influence of CIH.

Effect of Lycopene on Angiotensin-converting Enzyme 2 Expression in Kidney Tissue of Rats with Diabetic Nephropathy

WU Bo, JIANG Hai-yan, WANG Yi-long, CHEN Tong-ke, ZHU Jia-yin, ZHAO Hui-ling

2012, 32(5):  421-425.  DOI: 10.3969/j.issn.1674-5817.2012.05.011

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Objective To observe the expression changes of angiotensin-converting enzyme 2(ACE2) in kidney tissue of rats with diabetic nephropathy, and explore the renal protection mechanism of lycopene. Methods The rats were divided into 5 groups: nomal control group, diabetic nephropathy (DM) model group, and Lycopene treated groups with high-dose (20 mg·kg^-1·d^-1), middle-dose (10 mg·kg^-1·d^-1), and low-dose (5 mg·kg^-1·d^-1) respectively. After 8 weeks, the blood and urine index was measured. The expression level of ACE2 protein in kidney tissue was detected respectively by immunohistochemistry and western blotting. Results The concentrations of blood glucose, serum insulin, creatinine, urea nitrogen, AngII and urine protein in lycopene and diabetes groups were all significantly higher than those of normal control group (P<0.05); After treated by lycopene, serum creatinine, urea nitrogen, AngII and urine protein were greatly improved (P<0.05). The expression of ACE2 protein in diabetes group was greatly decreased than that of normal control group (P<0.01), while ACE2 protein levels in the high- and middle-dose groups treated with lycopene were obviously up-regulated (P<0.05), especially in high-dose lycopene group (P<0.01). Conclusion Lycopene can decrease urine protein, improve the renal function, and up-regulate the expression of ACE2 protein in diabetic nephropathy.



Development of Hypoxic Experimental Apparatus for Nerve Cells and Its Evaluation

QIAN Long-hua, CHEN Hui-jin, ZHOU Ze-han, LI Wen-juan, MAO Feng-xia

2012, 32(5):  426-430.  DOI: 10.3969/j.issn.1674-5817.2012.05.012

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Objective To develop a hypoxic experimental apparatus for nerve cells with good function and easy to use, which can be used to explore the pathogenesis and management of neonatal brain injury by simulating a hypoxic-ischemia process on a cellular level. Methods A set of hypoxic experimental apparatus was designed and developed. Using this hypoxic experimental apparatus, the oxygen-glucose deprivation (OGD) model of nerve cells were respectively established with various OGD time of 30 min, 45 min, 60 min, 2 h and 3 h, respectively. The influence of different OGD time on the survival rates of nerve cells was assessed 24 hours after OGD by using Cell Counting Kit-8 (CCK-8) and Hoechst33342/PI double fluorescence dying method. Results The set of hypoxic experimental apparatus consisted of four parts including a hypoxic chamber, a commercial source of non-oxygen gas, a water-sealed bottle for draining air, and a CO₂ incubator. The hypoxic chamber developed was lucid with good closure property and easiness to open and carry. The results for evaluating the influence of different OGD time on the survival rates of nerve cells established by using the hypoxic experimental apparatus showed that the apoptotic and necrotic cells tended to increase significantly with the extension of OGD period gradually. The survival rates of nerve cells were from 85% with 30min OGD reduced to 22% with 3 h OGD. About 45~60 min OGD could cause 40~55% of nerve cells to die. Conlusions The set hypoxic experimental apparatus developed is with good function and easy to use, which can ensure the successful establishment of OGD model of nerve cells.



Observation on Mitomycin's Effect for Reducing Fibrous Tissue Hyperplasia after KTP Nd:YAG laser in Rabbit

LI Jin, YANG Wen-lei, DU Xiao-yan, ZHANG Lin

2012, 32(5):  431-435.  DOI: 10.3969/j.issn.1674-5817.2012.05.013

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Objective To observe the effect of Mitomycin(MMC) for reducing fibrous tissue hyperplasia after KTP Nd:YAG laser recanalisation in rabbit. Method The rabbit model of lacrimal duct obstruction was established, and the model rabbit was divided into two groups injecting with saline solution and MMC respectively. Result The appearance of lacrimal duct part obstruction and fibrous tissue hyperplasia was later in the MMC group, and the fibrous tissue hyperplasia is lighter. Conclusion MMC can reduce fibrous tissue hyperplasia after KTP Nd:YAG, and improve the success rate of KTP Nd:YAG laser recanalization.



Feasibility Analysis of Himalayana Marmot as an Alternative Model of Drug Mitochondrial Toxicity Evaluation

ZHANG Ping-hu, TAO Yuan-qing, JIANG Zhen-zhou, WANG Zhong-dong, FAN Wei, ZHANG Lu-yong

2012, 32(5):  436-440.  DOI: 10.3969/j.issn.1674-5817.2012.05.014

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Objective To explore whether Marmots himalayana could replace woodchuck as an alternative model for evaluating the toxicity of nucleotide analogue drugs, the phylogenetic relation of Marmots himalayana with other Marmots were analyzed based on a molecular marker, mitochondrial cytochrome b (Cytb) gene. Methods Cytb genes from Marmots himalayana were sequenced and phylogenetic analysis were performed combined with existing Marmots Cytb gene sequences. Results The Cytb gene of Marmots himalayana was composed of 1140bp nucleotides, in which the average content of A, T, C and G was 33.2%, 25.4%, 28.8%, 12.6% respectively. Significant nucleotides and amino acids bias in Marmots Cytb genes were found: the content of G was significantly lower than that of A, T and C, the usage frequencies of leucine (Leu) and isoleucine (Ile) (11.87% and 15.69% respectively) were significantly higher than that of cysteine (Cys) (1.08%) in all marmots. However, the usage frequencies of isoleucine (Ile, 11.87%) and cysteine (Cys, 1.32%) in Cytb gene of Marmots himalayana from Qinghai-Tibet Plateau were significantly higher than that of other Marmotas. Phylogenetic analysis showed that Chinese M.himalayana, M.sibirica, M.caudata and M.bobak has a close genetic relationship with North America Marmots (Woodchuck), suggesting they were originated from the same species group.Conclusion A close genetic relationship was found between Marmots Himalayana and woodchuck, which provide new evidence for establishing an alternative model for evaluating nucleotide analogue drugs using Marmots Himalayana.



Preparation and Labeling of Anti-IgG in Tupaia belangeri, Cricetulus migratorius and Merionesun guiculatus

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

2012, 32(5):  441-445.  DOI: 10.3969/j.issn.1674-5817.2012.05.015

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Objective To prepare anti-IgG of Tupaia belangeri, Cricetulus migratorius and Merionesun guiculatus, and label them by using FITC and HRP. Method The IgG of these three animals were purified using Protein G and the anti-IgG were obtained from immunized rabbits. The anti-IgG were purified through affinity chromatography and labeled by FITC and HRP. Results The anti-IgG concentration of Cricetulus migratorius, Tupaia belangeri and Merionesun guiculatus was 2.2 mg/ml, 2.3 mg/ml and 2.3 mg/ml respectively. The IgG content of FITC labeled anti-IgG for each animal was 1.19 mg/ml, 1.36 mg/ml and 1.55 mg/ml respectively. The IgG content of HRP labeled anti-IgG for each animal was 0.84 mg/ml, 1.09 mg/ml and 1.31mg/ml respectively. Conclusion The study successfully obtained the FITC and HRP labeled anti-IgG, which can be used for the establishment of microorganism detection in these three animals.



Preliminary Probe of Detection of Simian Varicella Virus Antibodies in Cynomolgus Monkey

SANG Chuan-lan, DUO Hai-gang, ZHANG Yong-Bin, CHEN Jia, Rao Jun-hua

2012, 32(5):  446-448.  DOI: 10.3969/j.issn.1674-5817.2012.05.016

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Objective To find out epizootic of simian varicella virus occurring in cynomolgus monkey. Methods Using varicella-zoster virus IgG ELISA kit to detect cynomolgus monkey sera Ł¬from three facilities,which is B virus antibodies negative. Result The positive rate of simian varicella virus antibodies were 14.55%,30.95%,27.66% in three guangxi breeding facilities, respectively. During the serological tests, all the positive cynomolgus monkeys didn’t develop a rash. Conclusion After infection with Simian varicella virus,the virus becomes latent in ganglia, and can reactivate to produce zoster. This potential risk shoud be paid more attention.