Table of Content

25 June 2018, Volume 38 Issue 3

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The Dynamic Changes of Mitochondria in Endothelial Cells of Immunological Coronaritis in Mice

PU Xiang-qiang, WANG Xiang, QIANG Guang-hui, MA Jin, DING Yue-yue, LV Hai-tao

2018, 38(3):  169-175.  DOI: 10.3969/j.issn.1674-5817.2018.03.002

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Objective To observe the dynamic changes of morphology of vascular endothelial cell mitochondria in mice with immunologic coronaritis,in order to simulate the corresponding changes in endothelial cells and mitochondrial with coronaritis of Kawasaki disease.Methods C57BL/6 mice were randomly divided into experimental group (n=30) and control group (n=30).Mice were peritonealy injected with Candida albicans water soluble fraction or phosphate buffer (PBS) for 5 d.After the last injection of 24 h,3 d,7 d,14 d and 28 d,6 mice in each group were sacrificed to detect the blood routine and the level of interleukin (IL)-6 and tumor necrosis factor (TNF)-αin the plasma.Cardiac and coronary artery specimens were examined by hematoxylin eosin (HE) staining.The structure of mitochondria in the endothelial cells of coronary artery was observed by electron microscopy.Results The levels of blood white blood cells,platelets,plasma IL-6 and TNF-αin the experimental group were increased at the time of 3 d and 7 d after injection (P<0.05).The histopathology of the early stage in coronary artery inflammation (3 d),the endothelial cells were swollen.The fracture of elastic fiber and hemangioma appeared after the mid-term (14 d).Under the perspective of electron microscopy,coronary artery endothelial cells showed swelling and vacuolar degeneration at middle stage (24 h,3 d,7 d and 14 d),and the number of mitochondrion decreased,the crest blurred,and the electron density decreased.Conclusion The mouse model constructed can simulate characteristics similar to Kawasaki disease immunity coronaritis,suggesting that mitochondria in the endothelial cells involved in the immune process of coronary arteritis.



Development of Loop-mediated Amplification for Detection of Eimeria stiedai

WEN Fu-li

2018, 38(3):  176-181.  DOI: 10.3969/j.issn.1674-5817.2018.03.003

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Objective Loop-mediated amplification (LAMP) approaches was developed for detecting Eimeria stiedai (E.stiedai).Methods Specific primers were designed and synthesized according to a part of the sequence of ITS1-5.8sRNA-ITS2 region of E.stiedai.The LAMP methods of detecting E.stiedai was established by optimizing the reaction conditions.Results In 25 µL reaction system,the optimal dosage of Bst polymerase is 1.5 µL,and the optimal dosage of DNA is 75 ng.The optimal concentration of ring primers,internal primers and external primers is 0.8 µmol/L,1.6 µmol/L and 0.15 µmol/L respectively.The target E.stiedai strip can be specifically amplified when reacted for 1h at 63 ℃.The sensitivity of LAMP was high for determing E.stiedai,and this method could specifically detect the DNA of E.stiedai. Conclusions The LAMP method for detecting E.stiedai has been established,which provides a new method for rapid detection of E.stiedai infection.



Investigation on Defect Length in a Model of Radius Critical-size Defect in Adult Rabbits

SONG Zi-Jian, ZHENG Xin, WANG Jin, ZHANG Xing-Chen, GUO Kai-Jin

2018, 38(3):  182-187.  DOI: 10.3969/j.issn.1674-5817.2018.03.004

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Objective To establish experimental models with different defect lengths of adult rabbit radius and to determine the optimal length of bone critical-size defects in adult rabbits.Methods Eighteen male New Zealand White rabbits aged 8 months were included in this study.They were randomly assigned into 3 groups,according to the defect lengths of radius:group A (10 mm),group B (12 mm) and group C (15 mm).There were 6 rabbits with a total of 12 sides in each group.These rabbits underwent bilateral radial surgery.Computed tomography (CT) scans were taken immediately after the surgery and at 4-week intervals,respectively.The healing of the bone defect was evaluated with the CT-Hedberg scores.The rabbits were sacrificed at 12 weeks postoperatively and the forearms were harvested for gross and histological observations of new bone formation.Results All rabbits survived after the operation.Twelve (100%) of the radial bones in group A and eleven (91.7%) in group B revealed continuous bridged callus with a smooth surface.In contrast,the defect area was filled with fibrous tissue in group C with the closure of both ends.CT images showed that five out of twelve sides in group C appeared ulnar fracture at fourth weeks postoperatively.We excluded the fracture sides out of the statistical analysis.At twelve weeks postoperatively,recanalized marrow cavity of the bridged radius in group A and group B were observed on CT images.However,a small amount of callus along with closed marrow cavity was found at both ends in group C.Based on the CT-Hedberg scoring system,we found that the scores of group C (1.14±0.38,1.29±0.49,1.57±0.53) were lower than group A (2.42±0.51,3.17±0.58,3.75±0.45) and group B (2.25±0.45,2.67±0.78,3.50±0.67),respectively.There were significant statistical differences among the three groups at each time point postoperatively (P<0.05).However,there were lacks of significant differences at various postoperative time points between group A and group B (P>0.05).Histological analysis showed that the bone defects in group A and group B were perfectly repaired with the recanalization of marrow cavity.It was observed that in group C the ends of the defects were closed and the defect area was filled with fibrous tissue.Conclusions Eight-month-old male New Zealand White rabbits could be selected when radius defect in adult rabbit was established and 15 mm was considered the most appropriate critical-size defect length.However,it should be taken into account that fractures would be one of the complications at such a age of rabbits with heavier weight.



Comparative Medical Significance of Mechanism on Radiation Induced Heart Disease in Rats

MA Jin-ke, GAO Shi-le, HU Zong-tao, WANG Chong, GAO Shan, DONG Liu-yi, LIU Yang, TANG Zheng-zhong

2018, 38(3):  188-193.  DOI: 10.3969/j.issn.1674-5817.2018.03.005

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Objective To investigate the model establishment about radiation induced heart (RIHD) in rat and observed the changes of echocardiography,electrocardiogram and serum biomarkers at different time points.Methods Ninety clean grade male SD rats were divided into normal control group and irradiation group randomly,the irradiation group was divided into four groups different dosage which 5Gy,10 Gy,20 Gy and 30 Gy,18 rats in each group,irradiate the heart with different dosage through varian linear accelerator,the rats were killed after irradiation on 2,4 and 12 weekend respectively.Weight of rat before euthanized,left ventricular ejection fraction (LVEF),left venteicular systolic pressuer (LVSP),left ventricular end diastolic pressure (LVEDP),electrocardiogram(ECG),serum creatine kinase isoenzyme-MB (CKMB),cardiactroponin Ⅰ (cTnI) and weight of whole heart are all detected with different tools.Results Compared with normal control group,with the irradiation dose increased,LVEF and LVSP decreased gradually in irradiation group.LVEDP rise gradually (P<0.05 or P<0.01).When the increase of irradiation dose,the heart rate increased and the ST segment elevated significantly (P<0.05 or P<0.01).Serum CKMB and CTnI increased in different degrees (P<0.05 or P<0.01),reach statistical significance.there were no significant differences in all the indenes between 30 Gy group and 20 Gy group (P>0.05),no statistical significance.Conclusions Radiation induced heart injury model was established on rat after four weeks irradiation at 22 Gy dosage.



Efficacy and Safety Evaluation of Combination Treathment with Endostar and Cisplatin in Malignant Peritoneal Effusion

YAN Jun, LU Guo-feng, WANG Chun, MIN Xin

2018, 38(3):  194-201.  DOI: 10.3969/j.issn.1674-5817.2018.03.006

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Objective To evaluate efficacy of the combination of Endostar and cisplatin in malignant peritoneal effusion and its underlying mechanism(s).Methods The H22 tumor ascites mice model was successfully established and randomly divided into control group,Endostar group (10 mg/kg/day),cisplatin group (2.5 mg/kg/5 day),combination group 1(Endostar was given every day) and combination group2 (Endostar was given every 2 days).Each group consist of 18 animals.At the end of the treatment,6 animals in each group were sacrificed.The ascites volume,erythrocytes number in ascites,number and apoptosis rate of tumor cells in ascites were recorded.The survival time was observed and the endpoint was determined as all control animals died.Results (1) Compared with the saline group,the body weight,the growth rate of the abdominal circumference and the ascites volume were significantly decreased in the Endostar group,cisplatin group and the Endostar-cisplatin combination group (P<0.01).The growth rate in the body weight and abdominal circumference and ascites volume were significantly lower than those in the Endostar group and the cisplatin group (P<0.01).(2) The number of erythrocytes in Endostar-cisplatin combination group 2 was significantly lower than that in saline group,Endostar group and cisplatin group (P<0.01).The number of tumor cells in the ascites of the saline group was significantly higher than that in the Endostar group,the cisplatin group and the Endostar-cisplatin combination group (P<0.01).The apoptosis rate of tumor cells in Endostar-cisplatin combined group was significantly higher than that in saline group and Endostar group (P<0.01),There was no significant difference between control and cisplatin group.(3) Log-rank analysis showed that combination group had a significant effect on the survival time of mice (P<0.01).Conclusion The combination of Endostar and cisplatin effectively inhibits the production of ascites,reduces the red blood cell permeability,and induces the apoptosis of tumor cells in ascites,which may extend the survival time of animal.The two drugs has synergistic effect.The effect of daily administration of Endostar is better than administration each other day.



Construction of Lyst Gene Defective C57BL/6 Mice by CRISPR/Cas9 Technology

AI Dong-xu, ZHONG De-gang, SUN Fei, Li Yu, LI Chong, QIN Tong-tong, GAO Meng-qiao, DONG Shi-shi, SUN Zhao-zeng, LI Lian-rui

2018, 38(3):  202-206.  DOI: 10.3969/j.issn.1674-5817.2018.03.007

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Objective To obtain the animal model of NK cell deficient mice and study the function of the lysosomal trafficking regulator (Lyst) gene.Method The CRISPR/Cas9 technique was used to knockout Lyst gene on C57BL/6 mice.On fiftieth exons of mouse Lyst gene coding region,CRISPR/ Cas9 target primer (sgRNA) was designed pUC57-Lyst-sgRNA vector was constructed.The recombinant plasmids and Cas9 plasmids were transcribed into mRNA in vitro separately by using T7 RNA polymerase.mRNA was microinjected to mouse fertilized egg proportionally.We use embryo transplant to obtain Lyst knockout mice.PCR amplification and sequencing were used to screen the individual animal of Lyst gene change.Result Seven F0 generation knockout mice were obtained after embryo transplant with the coat color change obviously.Conclusion Lyst gene-deficient mice can be constructed by using CRISPR/Cas9 technology.The phenotype in morphology is consistent with those reported in the literature.



Observing on Laboratory Animal Welfare Related Indexes in Chronic Stress Rats

LIANG Lei, DONG Min, YOU Jin-wei, HU Wen-juan, CHEN Li, YUN Shi-feng

2018, 38(3):  207-211.  DOI: 10.3969/j.issn.1674-5817.2018.03.008

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Objective To observe the effects of chronic stress on animal welfare related indexes in rats.Methods Eight weeks old SPF SD rats were subjected with chronic stress by electric shock observing.Body weight gain and blood pressure of the rats were recorded throughout the experiment.The expressions of mRNA and protein of nuclear factor-κB (NF-κB) p65 in liver cells were measured by real-time quantitative PCR and immunohistochemical method respectively when the experiment ended.Interleukin-2 (IL-2),epinephrine (EPI),norepinephrine (NE),adrenocorticotropic hormone (ACTH) and corticosterone (CORT) in blood were measured by enzyme-linked immunosorbent assay (ELISA).Results Related indexes of group test were compared with group control,the body weight gain were lower and blood pressure were higher (P<0.05).The expressions of mRNA and protein of NF-κB p65 were higher (P<0.05).The indexes of NE,EPI,ACTH and CORT were higher and IL-2 got lower (P<0.05).Conclusion The animal welfare would be infected by chronic stress,and should be paid attention to during the animal experiment studies.

The Effect of Sleep Deprivation on Growth and Development in Mice

ZHANG Ya-quan, YANG Wen-jing, LIN Li-fang, RUAN Wen-jie

2018, 38(3):  212-216.  DOI: 10.3969/j.issn.1674-5817.2018.03.009

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Objective Detection on weight,blood parameters,hormone levels,and immune levels after sleep deprivation was conducted,to examine the impact of sleep deprivation on adolescents’various systems and growth.Methods A mouse model of chronic sleep deprivation was constructed using a platform-based sleep deprivation device for 20 days.During the period,normal daily routine mice were used as controls.The weight,hematological parameters,serum corticosterone,growth hormone,interleukin-2,and spleen corticosterone receptor levels.of mice in the sleep deprivation group were detected.And bone mass was measured using MicroCT,too.Results The body weight,hematological parameters of mice corticosterone,growth hormone,interleukin-2,and spleen corticosterone receptor levels in the sleep deprivation group were lower than controls’s.MicroCT test found that bone mass was significantly worse in the sleep deprived group than the control group.Conclusion Studies have shown that lack of sleep affects the normal growth and development of the main organs and body systems,which can potentially damage the overall immune function and nervous system function of mice.This result suggests that sleep plays an important role in the growth and development of adolescents.



Using Several Alternative Test Methods Strategically Combination to Assess Eye Irritation/Corrosion of 1,2-hexanediol

CHEN Xiu-juan, HUANG Yu-feng, LIU Xiang-mei, PANG Zeng-xiong, JIANG-Yi, LI Pei-ning, LI Guang-xian, CHEN Zi-ling, SUN Xia, WANG Qiang

2018, 38(3):  217-221.  DOI: 10.3969/j.issn.1674-5817.2018.03.010

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Objective Using several alternative test methods strategically combination to assess eye irritation/corrosion of different concentrations of 1,2-hexanediol.Methods With the bottom-up approach,two different sensitive degree of tests,the hen’s egg test - chorioallantoic membrane (HET-CAM) and the bovine corneal opacity and permeability (BCOP) in vitro were adapted to test eye irritation of 1,2-hexanediol in different concentrations,and finally the safe range of eye irritation was confirmed by Draize eye test.Results HET-CAM test:1,2-hexanediol was non-irritant when the concentration was below volume fraction 0.625% and light irritant at volume fraction 1.25% concentration,moderate irritation at volume fraction 2.50% concentration.In the concentration of volume fraction 5.00% and 10.00%,1,2-hexanediol induced serious eye damage.BCOP test:1,2-hexanediol at volume fraction 2.50% and 5.00% concentration induced light irritation or irritation,and severe corrosive in the concentration of volume fraction 10.00%.Confirmed by the Draize test,1,2-hexanediol was non-irritant at volume fraction 5.00% concentration,light irritation at volume fraction 10.00% concentration.Conclusion In this condition,1,2-hexanediol was non-irritant with the concentration lower than volume fraction 5.00%.



Generation and Genotyping of Monocyte-Peroxisome Proliferator-activated Receptor γ Conditional Knockout Mice

ZHENG Xin-xun, ZHANG Zhi-jing, HUANG Bao-yi, LIAO Zi-jun, LIU Jian-jun, LUO Tao

2018, 38(3):  222-226.  DOI: 10.3969/j.issn.1674-5817.2018.03.011

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Objective To generate monocyte- peroxisome proliferator-activated receptor γ(PPARγ) conditional knockout mice and to identify the genotype.Methods The imported PPARγ-loxp mice and CX3CR1-Cre mice were hybridized to generate F1 generation,and the genotypes were identified by RT-PCR.Using the same method to generate F2 generation and detect their genotypes.Then the effect of PPARγgene knock-out were identified by using immunofluorescence and Western blotting.Result The monocyte-PPARγknockout mice (PPARγ^loxp/loxp Cx3cr1^cre/- or PPARγ^loxp/loxp Cx3cr1^cre/cre) were as test group(PPARγko),and the control group(PPARγwt) of mice (PPARγ^loxp/loxp Cx3cr1^-/-) were also identified.Conclusion Successfully established the monocyte-PPARγknock-out mice,and provide a experimental tool to further study the funtions of PPARγon monocyte .



Analyzing the Variation of Air Changes and Pressure Parameters in Individually Ventilated Cage

ZHANG Jian-ping, FU Jiang-nan

2018, 38(3):  231-235.  DOI: 10.3969/j.issn.1674-5817.2018.03.013

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Objective To assess the variation of air changes and pressure parameters in the same equipment of individually ventilated cage (IVC) during the daily operation.Methods Taking the onsite single-side IVC as the test object which was placed in the barrier environment,and the parameters were set like this:ventilation rate 50 times/h,40 times/h;the pressure:+ 20Pa,+ 10Pa.The sample points were set at the air inlet,which was used to test the air speed,meanwhile testing the pressure between the cage inside and outside.Result During the IVC equipment daily operation,the variation of air changes among the cages were huge,the number ranged from 30 times/h to 60 times/h,but the parameter of pressure between inside and outside has little difference among the cage,and within the set value ±5Pa range fluctuations.Conclusion The structure and ventilation system of IVC equipment had big impact on the air changes,the temperature,humidity,airflow velocity and ammonia concentration in IVC cage were closely related to the ventilation rate.In order to reduce the environmental differences on the experimental results,controlling the ventilation rate and pressure difference between internal and external of cages are necessary.