Table of Content

25 October 2015, Volume 35 Issue 5

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Anti-metastatic Actions of Realgar Nanoparticles on Hepatic and Pulmonary Metastasis of Murine Malignant Melanoma with in vivo Bioluminescence Imaging System

XI Xiao-xia, XI Dong-bin, FAN Lin-lan, WEI Hu-lai

2015, 35(5):  345-350.  DOI: 10.3969/j.issn.1674-5817.2015.05.001

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Objective To study the anti-metastasis action and mechanisms of realgar nanoparticles (nano-realgar) on malignant melanoma B16-luc cells in mice by application of in vivo bioluminescence imaging assay. Methods The firefly luciferase (luc) gene was transferred into murine B16 melanoma cells with a lentiviral vector(B16-luc cells). B16-luc cells were injected into tail vein of BALB/c mice to establish the pulmonary and hepatic melanoma metastasis model. The model mice were treated with 4 mg/kg and 8 mg/kg nano-realgar once a day by ig for 24 days, and the optical in vivo imaging system was used continuously and dynamically to observe the tumor metastatic situation in mice. At the end of the treating period the mice were sacrificed, and the lungs and liver were removed, and the fluorescence images of the organs were acquired directly with the optical in vivo imaging system, and the metastasis nodules on the lung and liver surface were also observed for perusal. Finally the lung, liver and tumor stained with HE staining, and the morphological changes were observed under microscope. Results Observation with optical in vivo imaging system showed that nano-realgar administration could markedly suppressed the pulmonary and hepatic metastasis of B16-luc cells (P<0.05). After nano-realgar treatment, the numbers of pulmonary and hepatic metastasis nodules significantly reduced even disappeared, and pathological examinations revealed that, in the nano-realgar treated mice, the metastasis nodules in lungs seems small and little, the liquefactive necrosis appeared in the central area of most nodules, and no metastasis nodules was found in liver. Conclusion Realgar nanoparticles are able to inhibit significantly the abilities of pulmonary and hepatic metastasis of B16-luc malignant melanoma cells in mice.



Comparison on Two Models of Epiphyseal Plate Injury in Rat

JING Jin-zhu, LI Qing-hai, LV Xue-min, LIANG Zhen, WANG Meng

2015, 35(5):  351-355.  DOI: 10.3969/j.issn.1674-5817.2015.05.002

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Objective To compare two methods for constructing animal models of epiphyseal plate injury, and therefore to learn more about the disease of epiphyseal plate injury. Methods Fourty-five Wistar rats aged between 4-5 weeks were divided into three groups. Group A was control group, without operation; Group B was direct-injury group, in which the upper part of right tibial epihpyseal plate were cut; Group C was manual-rotation group, in which the knees of rats were grabbed valgusly. The rats were sacrificed after 4, 10 and 21days of operation. Micro-CT scanning and histological assessments were done. The differences between the length of left and right tibia, the width of tuberositas of left and right tibiae, and the minimum distance between tibia and fibula were examined by CTAn(Comprehensive TeX Archive Network) software. Results Micro-CT scanning and histological assessments showed that, after 4 days of operation, the width of epiphyseal plate were larger in Group B and Group C, immature chondrocytes were found in Group B, and the bone mineral density around operation site were lower than control in Group B. After 10 days of operation, the epiphyseal plate became narrower in Group B, and the bone mineral density around operation site appeared to be lower than normal in Group C. After 21 days of operation, the bone bridge was found in Group B, and only small new-born sclerotin appeared in Group C. According to the length of tibia, the width of tibial tuberosity, and the maximal interval between tibia and fibula, all of these values in Group B and Group C were significantly higher than Group A (control group) (P<0.05); but the values after operation of 21 days of Group B were significantly higher than those of Group B (P<0.05), other values were not significantly different between the two groups (P>0.05). Conclusion It is proved that the method of direct-injury is a much better one for constructing rat models of epiphyseal plate injury. This method has good stability and variation which more proper for studying disease of epiphyseal plate injury.



Study on Vaginal Irritation of dl-Mandelic Acid Thermosensitive in situ Gel in Miniature Pig

XIA Min-jie, HUANG Chao, TIAN Fang, WANG Yu-zhu, ZHI Rui-na, DING Xun-cheng, Yan Qian-Qin, LI Wei-hua

2015, 35(5):  356-361.  DOI: 10.3969/j.issn.1674-5817.2015.05.003

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Objective To study the vaginal irritation of dl-Mandelic acid thermosensitive in situ Gel(dl-Mandelic acid-TISG) in miniature pigs. Methods Nine miniature pigs injected with estradiol benzoate (400 μg/kg) were randomly divided into three groups: control group, dl-Mandelic acid -TISG group and nonoxynol-9 gel (N-9 gel) group. Each miniature pig was administrated with 5 mL gel through vagina once daily for 7 days. Cervicovaginal lavage (CVL) fluids were collected before administration and at the 24 h and 72 h after the last administration. The number of inflammatory cells and neutrophilic granulocytes in the CVL were detected by the flow cytometry. The miniature pigs were sacrificed at 72 h after the last administration. Histopathological changes of the vaginal mucosa were observed under light microscope. Results The number of inflammatory cells and neutrophilic granulocytes in the dl-Mandelic acid-TISG group were significantly lower than that in the N-9 group (P<0.05). Histological scoring of vaginal mucosa showed that there were no obvious mucosal epithelium damage, vascular congestion and inflammatory cell infiltration (5.10±0.47) in the dl-Mandelic acid-TISG group.There was no significant difference (3.30±1.60, P>0.05) compared with the control group (P>0.05) but significant difference compared with the N-9 group (20.50±3.50, P<0.05). Conclusion The vaginal irritation of dl-Mandelic acid-TISG in miniature pigs is mild.



Estabilishment of Cardiotoxicity Model in Zebrafish Induced by Doxorubicin

ZHANG Cheng-da, ZHANG Li-li, ZHANG Li-jiang, CHEN Yun-xiang

2015, 35(5):  362-366.  DOI: 10.3969/j.issn.1674-5817.2015.05.004

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Objective To establish zebrafish model of cardiotoxicity induced by doxorubicin (DOX). Methods The wild type zebrafish was divided into 6 groups: 4 different concentrations of DOX solution, control group and 0.9% NaCl groups. To assay the expression of cardiac troponin T(cTnT) and myoglobin (Mb) by Western blot at 72 hpf (72 hours post fertilization). Morphologic changes in the cardiovascular system were observed under stereomicroscope. The expression of miRNA146a in zebrafish was tested by RT-PCR. Results DOX had no cardiotoxicity in zebrafish within 0~MNLC/10; when the concentration of DOX ≥MNLC/3, it can be induced cardiovascular toxicty in the zebrafish, manifested as blood flow slows down, the lack of blood flow, venous sinus congestion, venous sinus edema and it is dose-dependent. Tissue expression of miRNA146a on zebadfishes of 3 concentrations were all increased compared with control group (P<0.01), and it is dose-dependent. There was no significant differences in the expression of cTnT and Mb between DOX groups and control group (P>0.05). Conclusion The cardiotoxicity induced by DOX was significant and ithad a similar toxic effect to mammal. So the zebrafish model of cardiotoxicity induced by DOX was successfully established.



Establishment of S180 Tumor Multidrug Resistance Mouse Model by Increasing PFC and Observation on Stability

GU Yun-hao, CAO Chen-jie, HU Bi-yuan, WANG Jun, HAN Dong-dong, XU Ai-hua

2015, 35(5):  367-373.  DOI: 10.3969/j.issn.1674-5817.2015.05.005

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Objective To establish aquired S180 multidrug resistance (MDR) mouse model and study on its stability. Methods To mimic the clinical PFC (cis-Dichlorodiamineplatinum+5-Fluorouracil+cyclophosphamide) scheme, gradually increase the dose in three phases to induce S180 ascites tumor mice and establish the aquired S180 MDR mouse model. The induced cells resistance factor to the chemotherapeutics drugs in different stages, the accumulation of adriamycin (ADR) and the functional activity of P-glycoprotein (P-gp) were detected by MTT assay and flow cytometry. And then through detecting the above indicators to monitor the resistance drug stability of induced cells in different stages. The mRNA expression of MDR-1 and multidrug resistance-associated protein 1 (MRP-1) of induced cells in different stages were detected by real time quantitative PCR (RT-qPCR). Results Compared with the parent cells, with induction time extending and dose increasing, the resistance factors in each stage of induced S180 cells to chemotherapeutics drugs were gradually increased, the accumulation of ADR was gradually reduced, and the functional activity of P-gp was strengthened. The mRNA expression of MDR-1 and MRP-1 of induced cells in different stages had a positive correlation to the induction time and dose. The stable resistance time of induced cells in the first, second and third phase are respectively for about 1 week, 2 weeks and 3 weeks. Conclusion To mimic the clinical PFC scheme, using the dose gradually increasing by phased can establish a high resistant strength, long stable time aquired S180MDR experimental model.



Improvement on Kidney Transplantation Model in Mice

HUANG Jian-bing, DING Fang-bao, LIU Hao, MEI Ju

2015, 35(5):  374-377.  DOI: 10.3969/j.issn.1674-5817.2015.05.006

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Objective To improve the technique of kidney transplantation model in mice and to increase the success rate of operation. Methods Totally 25 pairs of C57BL/6 mice were used for kidney transplantations, and 8 C57BL/6 mice were used for control after nephrectomy. To simplify the donor kidney acquisition method, begin to separate the donor kidney from the dorsal side, reduce the separation range of the renal arteriovenous. Obtain the artery at a vascular patch. Improve the arteriovenous anastomosis technique. The donor renal artery and vein is sutured to the recipient abdominal aorta and inferior vena cava respectively by end to side anastomosis. The urethral ends of the donor were put into the recipient bladder to reconstruct the ureter, and fix the ureteral wall to the outer membrane of the bladder. The natural kidneys of the recipients were excised by two phases. Results The success rate of operation was 92%, the total operation time was 47.6±8.7 min, warm ischemia time was 3~5 s, cold ischemia time was 31.1±6.3 min. Conclusion The improvement technique of kidney transplantation model in mice can improve the success rate of operation. It is easy to learn and is worth to be widely applied.



Histological Observation on Digestive Tract of Sub-plateau Acclimatized SD Rats and KM Mice

XU Wen-mang, WANG Yuan-yuan, LI Gui-yun, LI Tao, CAI Lin, WANG Li, CUI Jing, LI Xia

2015, 35(5):  378-382.  DOI: 10.3969/j.issn.1674-5817.2015.05.007

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Objective To explore histological characters of the digestive tract of Sub-plateau acclimatized SD rats and 20 KM mice and to provide comparative data for pathological inspection, toxicological studies and drug safety evaluation. Methods Twenty SD rats and 20 KM mice were divided into flatland group and sub-plateau group, each group include 10 ones. Animal quality and breeding condition complied with the state standard without any infectious disease after quarantine. Tissues of digestive tract were collected after euthanasia and autopsy . Tissues were fixed with 10% neutral formalin, the pathologic slide specimens were prepared by HE staining after paraffin imbedding, and then which were observed and studied under the light microscope. The same and different histological structure characters of them were summarized by comparative study. The histological structures of digestive tract were observed by light microscopy. Results (1) Inflammation was a common spontaneous lesion of digestive tract in all groups, but imflammation in the sub-plateau groups were more serious than the flatland groups. (2) Widening of space between lingualis, and more nuclear chains of lingualis were seen in sub-plateau groups. (3) Goblet cell metaplasia existed in intestinal mucous membrane of sub-plateau groups. (4) The mast cells were observed much easier in muscle stomach of sub-plateau groups. Conclusion The digestive tract characters of SD rat and KM mice are basically the same, but there are some histological differences in digestive tract between the flatland and sub-plateau group, which is an adaptive response of cell and tissue, and this difference should be fully considered in experimental research, toxicological studies and drug safety evaluation.



The Forelimb Bone Mineral Density and Expression of Calcitonin and Osteocalcin in Forelimb Malformed White Hair & Black Eyes Rabbits

CHEN Fang-ming, LIN Lin, CAI Yue-qin, CHEN Cheng, CHEN Min-li

2015, 35(5):  383-389.  DOI: 10.3969/j.issn.1674-5817.2015.05.008

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Objective To observe bone mineral density and expression level of calcitonin (CT), osteocalcin (also named bone gla protein, BGP) proteins in forelimb malformation WHBE rabbits (FMWHBE) and to explore possible cause of forelimb malformation and possibility to be an animal model for bone biomechanical studies. Method FMWHBE rabbits, WHBE rabbits and Japan white rabbits(JW) at 1, 2, 3 and 4 months of age were selected with six rabbits for each age. Contents of CT, BGP in serum were detected after body weighing and a small animal imaging system was used to measuring bone mineral density of proximal end, middle end and telecentric end in forelimb humerus, radius and ulna. CT and BGP immunohistochemical staining were used for pathological observation after decalcification. Result No significant difference of body weights of each month old was observed among all groups. Bone mineral density of whole length in 1 month old and proximal end, middle end in 2 months old and telecentric end in 3 months old of humerus, middle end and telecentric end in 1 month and telecentric end in 2 and 3 months old of radius and whole length in 1 and 2 months old and telecentric end in 3 months old of ulna in FMWHBE rabbits were notably lower than that in WHBE and JW rabbits (P<0.05 or P<0.01). CT and BGP contents in serum of 1 month old FMWHBE were notably decreased (P<0.05 or P<0.01). BGP level in 2 months old was obviously higher than that of in JW rabbits(P<0.01). CT level in 3 month olds was notably higher than that in WHBE rabbits (P<0.05). Immunohistochemical staining of CT, BGP showed that positive parts of CT expression in FMWHBE rabbit was less and weaker, and expression of BGP in FMWHBE rabbits was slightly weaker than that in JW rabbits and WHBE rabbits. Conclusion Forelimb malformation formation and bone mineral density changes in FMWHBE rabbits may be related with the special stress in forelimb bones and expression character of CT and BGP induced by lying posture during growth.



Experimental Observation on Repair of Calvarial Defects by Hydroxyapatite Nanotube

ZHU Yan-cheng, ZHENG Xin, CHEN Yi-xin, WANG Xiao-bo, QIU Xu-sheng, SHI Hong-fei, WANG Jun-fei, GUI Xue-yang

2015, 35(5):  390-393.  DOI: 10.3969/j.issn.1674-5817.2015.05.009

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Objective To investigate the ostegenesis by hydroxyapatite nanotube(HANT) in a calvarial defect model. Methods Bilateral full-thickness defects (5 mm in diameter) of parietal bone were created in twelve three -month-old SD rats. The left defected side was filled with 0.5 g HANT (patent number: Zl201010123277.5) as experimental side and the right side was set as control. CT were taken at 12 weeks postoperation. The rats were euthanized at 12 weeks after surgery and the specimens were harvested for patholologic analysis. Results There was no infection or death on the surgical models. CT examination demonstrated that the residual HANT powder could be seen on the experimental side of the skull defect, but the density is lower than bone. Pathologic analysis showed that bone defect in the control side was filled with fiber cells. The powder partly degraded in the experimental side, few chondrocytes were found, and a large number of fiber cells enwrapped the powder. Conclusion The HANT has good biocompatibility, but singly implanting the nanotube can't completely repair the skull defect of rats. Therefore, its osteogenic ability need to be refined in further experiment.



Continuous Monitoring on Blood Glucose and Insulin Levels in Obesity and Diabetes Cynomolgus Monkeys

WEI Zhu-mei, YANG Bo, LI Zhen-ming, HUAN Yin-hua, SU Ke-long, YANG Ji-hong

2015, 35(5):  394-397.  DOI: 10.3969/j.issn.1674-5817.2015.05.010

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Objective To explore the dynamic changes of the glucose and insulin levels of obesity and diabetes cynomolgus monkey during 24 hours cycle. Methods Blood glucose and insulin levels from 36 adult male cynomolgus monkeys, which were uniformly selected from obesity, diabetes and normal group, were examined. Parameters including body weight, abdominal circumference, skin fold thickness, calculated body mass index (BMI), heart rate, blood pressure, breathing and body temperature were detected. Glucose, insulin, glycosylated hemoglobin and lipids levels were recorded by a interval of 1.5 hours in all individuals. Results The blood glucose value in normal, obesity and diabetes cynomolgus monkeys were 3.94±0.65 mmol/L, 4.33±0.84 mmol/L, and 12.09±4.58 mmol/L respectively. The blood glucose level of diabetes cynomolgus monkeys reached the peak during 21∶00-22∶30 with 13.68±0.55 mmol/L, and reached lowest point during 18:00-19:30 with 10.94±0.49 mmol/L. The insulin values in normal, obesity and diabetes cynomolgus monkeys were (29.50±12.36)-(62.50±21.98) mU/L, (31.21±0.98)-(123.42±3.38) mU/L, and (12.60±3.16)-(30.62±3.16) mU/L respectively. The maximum insulin value of diabetes monkeys was 30.62±11.71 mU/L during 9∶00~10∶30, and minimum of 12.60 ±3.16 mU/L during 03∶00~04∶30. Conclusion This study provides an immediate quantitative basis for investigating of glucose and insulin levels in cynomolgus monkeys, and it sets up a fundamental reference for primate models of diabetes or related diseases.

Analysis on Pathogen Infection of Mice and Rats in Shanghai from 2010 to 2013

FENG Li-ping, TAO Ling-yun, FENG Jie, XIE Jian-yun, LIN Jin-xing, WANG Sheng-chang, ZHOU Jie, WEI Xiao-feng, GAO Cheng, XING Zheng-hong

2015, 35(5):  398-402.  DOI: 10.3969/j.issn.1674-5817.2015.05.011

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Objective To investigate the pathogen infection status of laboratory mice and rats in Shanghai. Methods The quality monitoring for viruses, bacteria and parasites in SPF or clean rats and mice was conducted according to national standards, in which the method, equipment and reagents were referring to the requirements of national standards. Results From 2010 to 2013 in Shanghai, the mainly identified pathogens infecting the mice and rats included the Staphylococcus aureus, Pseudomonas aeruginosa, Mycoplasma and Tyzzer's organism. And the mainly identified viruses infecting the mice and rats were mouse hepatitis virus and Sendai virus etc. Meanwhile, the mainly identified parasites were all Helminths, Flagellates and Ectoparasites. During this period, the detection rate of Staphylococcus aureus presented the downtrend in mice, but the detection rate of Tyzzer's organism and Mycoplasma showed uptrend. In addition, the detection rate of Tyzzer's organism, Sendai virus and all Helminths in rats increased year by year during this period. Conclusion In Shanghai region the quality of laboratory mice and rats needs to be improved. And the regulatory authorities in laboratory animal should strengthen the administration and quality monitoring of the rats and mice in the Shanghai.

A Survey on Epizoic Parasites Infection and Evaluations of Purification Techniques in Albino Marmota himalayana

LIU Hai-qing, WU Ke-mei, ZHANG Jing-xiao, LIU Yu-fang, FAN Wei, LIU Ba-rui, TAO Yuan-qing

2015, 35(5):  403-405.  DOI: 10.3969/j.issn.1674-5817.2015.05.012

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Objective To investigate the epizoic parasites infection in albino Marmota himalayan, and provide basis for setting up the quality standard of Marmota himalayana. Methods Twenty-one wild albinism and 30 wild normal Marmota himalayana from the same origin were detected by the method as transparent tape, operation scraping, inverse brush bug and naked eye observation for epizoic parasites of each animal, then detected the epizoic parasites and eggs under platoscope and microscope. Results The natural infectious rate of the epizoic parasites in the wild albinism and the normal Marmota Himalayana were respectively C.dolabris (85.7%/93.3%), O.silantiewi (90.1%/90.0%), R.Liventricos (71.4%/96.7%), L.laeviusculus (85.7%/86.7%) and I.crenulatus (100%/9.7%), but the infection rate of R.Li ventricos has significant difference (P<0.05), and there was no significant difference in the rest of the four kinds of the epizoic parasite infection rate between the two groups; The efficay is significant by using Deltamethrin and Ivermectin injection for purification. Conclusions The monitoring results and the cure method can be used as an index of animal parasite quality for albino Himalayan marmot.



Probe on Orthotopic Liver Transplantation in Rats by Double-Cuff Technique

ZHANG Jie, DOU Min, BAO Xing, DAI Jin-yu, YAN Yu-kuang, PENG Hai-feng

2015, 35(5):  406-408.  DOI: 10.3969/j.issn.1674-5817.2015.05.013

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Objective To investigate the surgical techniques for establishing the rat model of orthotopic liver transplantation. Methods On the basis of the double-cuff technique of Kamada, Some of the skills of the techniques for the veins of donors and the portal vein and the suprahepatic inferior vena cava of recipients were modified. Results Of the 50 rats that underwent orthotopic liver transplantation, 92% survived longer than 24 hours and 88% over 7 days. Conclusion The operator can successfully establish the rat model of orthotopic liver transplantation by streamline operations and improving the details on the basis of skillful surgical techniques.



Research Progress of Murine Norovirus in Characteristics, Immunology and Detection Methods

GAO Jie, HE Zheng-ming

2015, 35(5):  414-420.  DOI: 10.3969/j.issn.1674-5817.2015.05.016

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Murine norovirus (MNV) is the only norovirus that can be cultivated in vitro. The isolation of the strains and its successful proliferation in the mouse macrophage RAW264.7 cell lay the foundation for molecular mechanism research of norovirus, which also provide help for the pathogenic mechanism research and preparation of vaccine of human norovirus (HuNoVs). MNV is one of the most widely spread infection factors in mice. The replication of murine norovirus in cell culture reveals a tropism for dendritic cells and macrophages. The virus can make lethal effect on immune-deficient mice. It's of great significance to strengthen the control of the virus infection.

The Current Research Status of Gut Microbes in Mice

JING Ya-xing, BAI Lu, GAO Huan-yun, ZHENG Zhen-yu

2015, 35(5):  421-424.  DOI: 10.3969/j.issn.1674-5817.2015.05.017

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The current research status on factors influencing gut microflora in mice and gut microflora by using germ free mice were reviewed. Those may be provide references for future study on gut microbiota in human and animals.

Discussion on Aspects of Animal Lateralization

ZHAN Hong-wei, WANG Ai-guo, WANG Liang, QIU Ze-wen, GAO Wen-ting, WANG Fu-jin, WANG Jing-yu

2015, 35(5):  425-430.  DOI: 10.3969/j.issn.1674-5817.2015.05.018

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Lateralization is caused by the structural and functional difference of hemisphere, which is the general characteristics widely existing in human and other chordate. In this paper, we summarized the existence of lateralization, the different function of the left and right hemisphere, the category of lateralization, the practical application and the influence factor of the lateralization.