Table of Content

25 February 2016, Volume 36 Issue 1

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Establishment of Escherichia coli O127:H6 Infected Mouse Model and Expression of Regulated upon Activation Normal T-cell Expressed and Secreted

FAN Jun-wen, SUI Li-hua, XU Qin, LIU Yi, LIU Hui-fang, SUN Zhao-zeng, YAN Liang

2016, 36(1):  1-5.  DOI: 10.3969/j.issn.1674-5817.2016.01.001

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Objective To investigate the changes of jejunal pathology and expression of regulated upon activation normal T-cell expressed and secreted (RANTES) in BALB/c mice infected with Escherichia coli O127:H6. Methods Five groups of BALB/c mouse were administered intragastrically with different doses of Escherichia coli O127:H6 and one group administered with saline as control. The jejunum of the mouse were embedded in paraffin, and pathological changes were observed by HE staining. The jejunal RANTES expression level in the all the mice were determined by using Real-Time PCR. Results The enteritis mice model was successfully established, and significant changes of jejunal inflammatory pathology were found in model groups compared with the control group. Real-Time PCR results showed that the expression level of jejunal RANTES in the model groups were significantly higher than that of control group. Conclusion Escherichia coli O127:H6 can cause the changes of jejunum pathology in mice by intragastrical administration, and the RANTES expression level can be used as parameter to evaluate the in enteritis mice.



Androgenic Effects of Synthetic Progesterone (Medroxyprogesterone acetate) on Gambusia affinis

JIN Zi-yue, FANG Zhan-qiang

2016, 36(1):  6-7.  DOI: 10.3969/j.issn.1674-5817.2016.01.002

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Objective To investigate the androgen effects of synthetic progesterone (Medroxyproges-terone acetate, MPA) exposure on the female mosquitofish (Gambusia affinis). Methods The mature female mosquito fish were randomly divided into four groups, with one control group and three experimental groups exposed with 40 ng/L, 200 ng/L and 1 000 ng/L MPA respectively. Parallel experimental groups were also established. After 28-day-long exposure, five indexes including the body length (BL), body weight (BW), health index (CF), the section number (FJ) of the 3rd anal fin, the length (FL) change of the 4rd, 5rd, 6rd anal fin, and the morphological changes in the 14, 15 and 16 vertebral ribs in mosquito fish were observed and measured. The mRNA expression levels of vitellogenin gene (VTG〈), cytochrome P450 gene (CYP19〈) in livers, and androgen receptor gene (AR〈) in anal were also determined after 7, 14, 21 and 28 days exposure. Results The BL, BW and CF of experimental groups exposed in concentrations of 200 ng/L and 1000 ng/L MPA for 28 d were not changed significantly (respectively, P>0.05) when compared with those of the control group. The 3rd anal fin of FJ and the 6rd anal fin of FL were not changed significantly (P>0.05), however, the 4rd, 5rd of FL in experimental groups had been increased in varying degrees (P<0.01). When exposed in 200 ng/L and 1000 ng/L MPA, the L, D values of the 14th vertebral ribs, the P, P:D value of the 15th vertebral ribs, and the P, L:D, P:D value of the 16th vertebral ribs in female G. affinis were appeared very significantly different (P<0.05 or P<0.01), respectively. Centrum haemal spine elongation and with the spine nearly vertical were displayed significant changes in morphological masculinization. Compared with the control group, low concentration groups (40 ng/L MPA) VTG〈, CYP19〈 genes expression were increased significantly (P<0.01), while AR〈 expression was inhibited (P<0.05); medium concentration groups (200 ng/L MPA) VTG〈, CYP19〈, AR〈 genes expression were increased significantly (P<0.01); high concentration groups (1 000 ng/L MPA) CYP19〈, AR〈?genes expression were increased significantly (P<0.01), however the expression of VTG〈 was inhibition (P<0.05). Conclusion MPA exposure induced skeletal development morphological masculinizing effects in female mosquitofish, and the results of target gene expression level changes showed that the effects of androgen induced by MPA in mosquitofish, indicating that the MPA was a kind of androgen effect of progesterone.



Histological Observation on Major Digestive Glands in Captive Breeding Tree Shrews

KUANG De-xuan, WANG Wen-guang, SUN Xiao-mei, HUANG Xiao-yun, LU Cai-xia, HAN Yuan-yuan, TONG Pin-feng, DAI Jie-jie

2016, 36(1):  13-18.  DOI: 10.3969/j.issn.1674-5817.2016.01.003

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Objective To understand the histological characteristics of the major digestive glands in captive breeding tree shrews (Tupaia belangeri), and to establish the normal histological atlas. Methods Histology observation on parotid, submandibular gland, sublingual gland, pancreas and liver of captive breeding tree shrews were conducted by conventional tissue processing, HE staining and microscopy. Results (1) The parotid gland was pure serous acini, with obvious intercalated ducts, the wall was composed of a single layer of low cuboidal epithelial cells, the lunmen was filled with secretions. (2) The submandibular gland was mixed gland, mainly consisting of serous acinai with longer secretory tube. (3) The sublingual gland was mixed gland, mainly consisting of mucinous acini, and a small amount of intercalated duct. (4) The surface of the liver was a thin layer capsule of dense connective tissue envelop. The liver parenchyma was several hepatic lobule with unconspicuous boundary. The central vein was located in the center of hepatic lobule. Liver cells were polygonal, and the large and round nucleus was located in the center of the cell, the majority was mononuclear, the minority was binuclear. The cytoplasm was red, containing vacuoles and small lacuna. The interlobular artery, vein and bile duct were clearly visible in the portal area. (5) The surface of pancreas was a layer of loose connective tissue envelope. The pancreatic parenchyma consisted of exocrine portion and endocrine portion. The exocrine portion contained acini and ducts. The acini was composed of serous cells, and the ducts consisted of intercalated, intralobular, interlobular and pancreatic ducts, which was some cell clusters and cell cords irregularly scattered among exocrine acini with variable size. The cytoplasm of the endocrine cells was stained lighter than that of the exocrine cells. Conclusion Digestive gland of captive breeding is similar to that of the primate animal in the morphology. Current research would provide histological evidence for the functional study of digestive gland and pathological changes in tupaia ,as well as setting up the animal model of human diseases.



Determination of Serum Calcium, Phosphorus and Alkaline Phosphatase Levels and Forelimb Bone Mineral Density in Forelimb Malformed White Hair and Black Eyes Rabbits

CHEN Fang-ming, XU Jian-qin, ZHU Ke-yan, YANG Qin-qin, CHEN Min-li

2016, 36(1):  19-23.  DOI: 10.3969/j.issn.1674-5817.2016.01.004

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Objective To observe the physiological and pathological characteristics of forelimb and serum calcium (Ca) and phosphorus (P) levels in forelimb malformed white hair and black eyes (FMWHBE) rabbits, to discuss the possible causes of forelimb malformation in white hair& black eyes (WHBE) rabbits, and to evaluate possibility of FMWHBE rabbit as a potential model in bone biomechanics research such as spontaneous osteoporosis animal model. Method Totally 1, 2, 3 and 4 months old FMWHBE rabbits, WHBE rabbits and Japan white (JPW) rabbits were used respectively (6 rabbits in each group, totally 72 rabbits).The levels of serum Ca, P and alkaline phosphatase (ALP) were tested in all rabbits. The bone mineral density (BMD) of fore humerus, radius and ulna were measured by using X-ray imaging system. Pathologic analysis was performed by H&E staining. Results Compared with JWP rabbits, the serum Ca content of 1, 2 and 4 months old FMWHBE rabbits was significantly lower (P<0.05 or P<0.01). The serum P content of 4 months old FMWHBE rabbits was significantly higher than JPW rabbits at the same age(P<0.05 or P<0.01). The serum ALP levels of FMWHBE rabbits was significantly lower than JPW rabbits and WHBE rabbit (P<0.05 or P<0.01). Compared with the same period WHBE rabbit, the BMD of fore humerus of 1-4 month old FMWHBE rabbit , radial of 2, 3, 4-month-old rabbit and ulna of 2-month-old FMWHBE rabbit were significantly lower (P<0.05 or P<0.01). Compared with the same period JPW rabbits , the BMD of fore humeral and radius of 1, 3 months old rabbits, the ulna of 1, 2-month old rabbits were significantly lower (P<0.05 or P<0.01). Compared with WHBE and JPW rabbits, the cortex of fore humerus, radius and ulna in FMWHBE rabbits was thinner. There were less osteoblasts on local bone tissue surface and none of them were arranged in a layer trimly. Bone canalicules and new bone tissue were less. Conclusion The malformation of FMWHBE rabbits may be related with the low levels of Ca and P in blood leading the loss of osteoblasts and new bone tissue.



Application of Single Nucleotide Polymorphism Genotyping Panel in Genetic Monitoring of Laboratory Mice

ZHAO Li-ya, ZHANG Rong, ZHAO Ying, XING Zheng-hong, CHEN Guo-qiang

2016, 36(1):  24-31.  DOI: 10.3969/j.issn.1674-5817.2016.01.005

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Objective To develop multiple PCR-LDR genotyping protocols for monitoring genetic contamination in inbreed mice in a fast and reliable way. Method In total 51 single nucleotide polymorphisms (SNPs) from public SNP database were chosen and divided into five groups. These 51 SNPs are wildly distributed and there are least one SNP on each autosomal chromosome and the sex chromosome. A multiple PCR-LDR genotyping protocol was established as a genetic quality control approach to monitor the genotypes of 7 inbred mice. Results The homozygosity is 100% for the 7 tested mice. The genetic background of the inbred mice from the same source is identical. For the same inbred mice from different companies, several SNPs are different. The SNPs are different at a9、a10、b5、b9、c1、c12、e1、e2 for CBA/Ca/Bkl and CBA/JSlac, while the SNPs are different at c7, c10 for C57BL/6/BKl and C57BL/6JSlac. However, there are five loci which are the same in the 7 inbred mice of different companies. So there are 46 effective loci which are different in the 7 inbred mice of different companies. Conclusion The multiple PCR-LDR genotyping protocol is used to monitor the genetic differences of inbred mice from two companies. The results of this study provide a rapid and high-throughput genotyping approach, which is sufficient for genetic contamination monitoring and strain identification.



Comparison on Organ Parameter, Blood Parameter, Heart Rate and Ventricular Pressure between Male and Femele New Zealand White Rabbit

ZHANG Xiao-yu, ZHANG Xiu-yan, ZHAN Chun-lie

2016, 36(1):  32-36.  DOI: 10.3969/j.issn.1674-5817.2016.01.006

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Objective To determine and compare the parameters of organ and blood, heart rate and ventricular pressure in New Zealand white rabbit. Methods A total of 60 New Zealand white rabbits about 70-80 days old were fed for a week. Weight of rabbit and main organ, blood physiological and biochemical index and blood gas, ventricular pressure and heart rate were determined on respiratory support condition. Result The thymus gland and lung organ coefficient differences were significant between male and female rabbits (P<0.05 or P<0.01). Percentage of eosinophils, neutrophils, mononuclear cell and lymphocyte, neutrophils, mononuclear cell, mean corpuscular volume, average amount of hemoglobin, serum albumin /globulin, blood glucose, complement C3, glutamyl- transpeptidase, value of pH, temperature correction pH, oxygen saturation differences were significant (P<0.05 or P<0.01). The left and right ventricular diastolic blood pressure differences were also significant(P<0.05). Conclusion Some of the organ coefficient, blood physiological and biochemical index, blood gas analysis and ventricular pressure exist significant difference between male and female New Zealand white rabbit, it should be paid attention in related research.

Experimental Study on Hematological Parameters of New Zealand White Rabbits at Different Ages

YUN Shi-feng, DONG Min, ZHAO Zhi-gang, CHEN Li, LIU Biao, TIAN Xiao-yun

2016, 36(1):  37-40.  DOI: 10.3969/j.issn.1674-5817.2016.01.007

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Objective To investigate the hematological parameters of outbred colony New Zealand white rabbits at different ages. Methods Blood samples from 160 New Zealand white rabbits (Female, 80 and male, 80) in different age groups were analyzed with automatic blood cell analyzer. Result There was no statically significant difference between the blood samples of different age experimental closed colony New Zealand white rabbits in different sex sets (P>0.05). Significant differences (P<0.05) of hematological indexes except monocyte (P>0.05) were observed among New Zealand white rabbits in different age groups. Conclusion The outbred colony New Zealand white rabbits had stable biological characteristics and less difference among individuals. In this study, the biological hematologic parameters database for New Zealand white rabbits at different ages was established, which will be used as basic reference for New Zealand white rabbits experiment.

Effect of Environmental Enrichment on Growth and Hematology in SD Rats

LIU Shuai, LI Jun-yan, CHEN Wei, ZHANG Qi, LV Sai

2016, 36(1):  41-47.  DOI: 10.3969/j.issn.1674-5817.2016.01.008

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Objective To study the effect of environmental enrichment on the growth and hematology of Sprague-Dawley (SD) rats. Method Forty-eight weaned 3-week old SD rats (male:female = 1:1) were allocated randomly to standard laboratory cages and enriched cages which were consisted of small sticks for grinding and wood-wool as nesting materials. The growth rate and food consumption were monitored. The rats were sacrificed at 32 weeks of age, and the indexes of organ weight, hematology and biochemistry were measured and analyzed. Results The food consumption of male rats in enriched cages was significantly increased between 10 and 19 weeks, but the weight gain was not significantly different compared to the standard group. The total serum protein, albumin and cholesterol were also increased significantly. No significant difference was found in the growth rate, food consumption or serum biochemistry in the female rats. The weights of epididymis of male rats and the weights of heart, liver of female rats were increased significantly. However, there was no significant difference for other organs. In addition, environmental enrichment showed significant impact on the erythrocyte, hemoglobin and platelet in the female rats, but not the male rats. Conclusions The environmental enrichment can alter the growth and hematology in SD rats to some degree, so the researchers need to pay attention on designing environmental enrichment strategies .



A Perspective on Self-imposed Regulatory Burden in a Laboratory Animal Care and Use Program

LU Jia-qi, LI Ning, GE Li-jun, LIU Ji-hong, ZHU Feng-xian, WANG Jian-fei

2016, 36(1):  61-65.  DOI: 10.3969/j.issn.1674-5817.2016.01.013

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Any institution which cares and uses laboratory animals must comply with national and local regulations and standards by enhancing management and compliance. The institutions always attempt to minimize the risks of non-compliance when they establish the “laboratory animal care and use program”, this would accelerate the development and standardization of the global and local industry of laboratory animal science, on the other hand, the program often leads to redundant and superficial, the costs and budget of the program will increase dramatically, and finally it results in “self-imposed regulatory burden”. In this article, we will discuss about the major resources of “self-imposed regulatory burden”, how to evaluate “self-imposed regulatory burden” in the institution, and how to avoid “self-imposed regulatory burden”. At the end, we expect that the domestic institutions could make these as a guide, to understand, evaluate and balance their own managing goals and capabilities, to avoid the “self-imposed regulatory burden” at all.



Influence of Cobalt Chloride Induced Hypoxia on Proliferation and Apoptosis of Hepatic Stellate Cells in Naked Mole Rat

XIAO Bang, LI Li, YU Chen-lin, ZHAO Shan-min, LIN Li-fang, YANG Wen-jing, CONG Wei, CUI Shu-fang

2016, 36(1):  66-71.  DOI: 10.3969/j.issn.1674-5817.2016.01.014

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Objective To compare and study the influence of cobalt chloride (CoCl₂) on proliferation and apoptosis of hepatic stellate cells in the naked mole rat and C57BL/6J mouse, and preliminarily investigate the possible mechanism of hypoxic tolerance in naked mole rat. Methods Evaluating the effects of different concentrations of CoCl₂ on proliferation activity and apoptosis level of hepatic stellate cells in the naked mole rat and mouse by Cell Counting Kit-8 assay (CCK8) and flow cytometry. Detect the expression of hypoxia inducible factor 1〈 (HIF-1〈) and apoptosis-related proteins (BCL₂, BAX)of hepatic stellate cells in the naked mole rat after CoCl₂ treatment by Western blot assay. Results CoCl₂ significantly inhibited the proliferation and increased the apoptotic rate of hepatic stellate cells in C57BL/6j mice at a low concentration of 50 μmol/L. However, a same dose of CoCl₂ can significantly improve the proliferation of hepatic stellate cells in the naked mole rat but had no significant effect on its apoptotic rate. While high concentrations of CoCl₂ (> 200 μmol/L) showed a inhibition of proliferation and a rise of apoptotic rate of hepatic stellate cells in naked mole rat and mice, but the CoCl₂-induced changes in hepatic stellate cells of the naked mole rat were much smaller than those of mice. Simultaneously, the expression of HIF-1〈 and the ratio of apoptosis-related proteins (BCL₂/BAX) in hepatic stellate cells of the naked mole rat upregulated or accumulated significantly after treating with CoCl₂ (P<0.05). Conclusion Compared with C57BL/6J mice, the naked mole rat hepatic stellate cells had a greater ability of resistance to hypoxic damage caused by CoCl₂. It was speculated that HIF-1〈 may be involved in the regulation of resistance to damage caused by hypoxic environment in naked mole rat.



Establishment of Method for Separation, Cultivation of Bone Marrow Macrophage in Naked Mole Rats and Preliminary Research on Function

CHENG Ji-shuai, LI Li, XIAO Bang, ZHAO Shan-min, LIN Li-fang, CONG Wei, TANG Qiu, SUN Wei, YU Chen-lin, YANG Wen-jing, XU Chen, CUI Shu-fang

2016, 36(1):  72-75.  DOI: 10.3969/j.issn.1674-5817.2016.01.015

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Objective To establish the method for the primary culture of bone marrow macrophage in naked mole rats and study the phagocytosis compared with mice. Method The bone marrow cells from the hing leg of naked mole rats were detached. The cells were induced by 60 ng/mL Macrophage colony-stimulating factor (M-CSF) and cultured for 6~7 days in vitro. During the culture, the cell was observed in microscope. After induced, the flow cytometry was conducted to detect the purity of macrophage and the phagocytosis of macrophage with mouse as control. Result Through this method, the adherent cells with typical morphological characteristics were obtained and the positive rate of the cell surface antigen F4/80 and CD11b were higher than 80%. The phagocytosis rate of naked mole rats bone marrow macrophage was 99.87%±0.13%, but the ICR mice was 88.79%±0.90%. Conclusion This method is simple and practical for separation and culture of highly purified and functional naked mole rats bone marrow macrophage in vitro. It indicates that phagocytosis of naked mole rats bone marrow macrophage is higher than that of mice.



Establishment of PCR Method Using Fluorescence Labeled Universal Primers for Screening Microsatellite Loci in Naked Mole Rat

LIN Li-fang, LI Li, XIAO Bang, CHENG Ji-shuai, YANG Wen-jing, CONG Wei, ZHAO Shan-min, TANG Qiu, CUI Shu-fang

2016, 36(1):  76-80.  DOI: 10.3969/j.issn.1674-5817.2016.01.016

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Objective Fluorescent labeled universal primers PCR in two-steps was established to screen microsatellite loci of naked mole rat. Methods A fusion of the forward primer and universal primers in 5' end was created. The first PCR was conducted with reverse primers and forward primer extended with universal primers. The second PCR was initiated after the adding of fluorescent labed universal primers. With conventional fluorescent labeled primers PCR as a control, PCR product was detected by agarose gel electrophoresis and capillary electrophoresis. Results Fluorescent labeled universal primer PCR had strong specificity and bands in agarose gel electrophoresis was sharp. Furthermore, the product had high consistent polymorphism with those in traditional PCR, with size being about 15 bp. They were in line with expectations. After analysis of capillary electrophoresis, it also showel equal number of allele with these two different method. As well, this fluorescent labeled universal primer PCR had high amplification efficiency and being specific, operational. Conclusion The fluorescent labed universal primers PCR in two-steps was applicable to screen a high-flux of microsatellite loci in naked mole rat.