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Comparison of human and animal oral microbiota by Illumina sequencing of 16S rRNA tags

Dongshu GU ; Bangzhu CHEN ; Xia JIANG ; Haiyue LIU ; Bayaer NASHUN ; Hongwei ZHOU ; Weiwang GU

Chinese Journal of Comparative Medicine.2016;26(8):96-102. doi:10.3969/j.issn.1671-7856.2016.08.016

Objective To provide original reference data for oral ecosystem research, Tibet minipigs, beagle dogs, rhesus monkey, New Zealand white rabbits and Wistar rats were selected to study their respective characteristics of oral microbial mmunities and compared with normal data of humans.Methods Total DNA was extracted from the specimens of oral microbial communities of Tibet minipigs, beagle dogs, rhesus monkey, New Zealand white rabbits and Wistar rats, and used to amplify 16S rRNA V4 fragments with labeled universal primers.The diversity and structure of microbial communities from those animals were compared with that of humans using BIPES and QIIME analysis after Illumina sequencing of 16S rRNA V4 fragments.Results The richness of the oral microbial communities of humans and the five species of laboratory animals was significantly different (P <0.05).Different species of animals have their own unique oral flora, among which the oral flora of the monkey is the most similar to that of humans.Conclusions Among the five species of laboratory animals, the oral microbial communities of rhesus monkeys and humans have highest similarity. Specifically, the Fusobacterium and Porphyromonas levels of rhesus monkeys is most similar to those of humans.Our findings indicate that rhesus monkeys may be suitable animal model for studies of human oral microbial communities.Tibet minipigs may be suitable animal model for Proteobacteria studies, while beagle dogs may be appropriate for modeling of diseases related to Spirochaetes.

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Genetic monitoring and analysis of highly immunodeficient mice from different sources by microsatellite DNA markers

Tao FAN ; Hong WANG ; Jie WEI ; Shuya ZHOU ; Bingfei YUE ; Baowen LI

Chinese Journal of Comparative Medicine.2016;26(8):91-95. doi:10.3969/j.issn.1671-7856.2016.08.015

Objective To test and analyze the genetic background of highly immunodeficient mice from different sources.Methods Four highly immunodeficient mouse strains from different sources of NOD background were collected. 30 microsatellite DNA sites were detected, and the genotype can be displayed by gel electrophoresis and STR scanning. Results 17 microsatellite sites exhibit polymorphism in 20 mice of the four groups.There were 30 homozygous loci in the mice of groups A and B, and heterozygous in the other two groups.The genetic distance is minimum between groups A and B, showing a higher genetic similarity.Conclusions The genetic backgrounds are different in highly immunodeficient mice from different sources.

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Study on the lmna gene knockdown in zebrafish embryo with morpholino oligonucleotides

Feng LIU ; Huimin HUANG ; Zhihua WANG ; Xijun WU ; Zhixu HE ; Liping SHU

Chinese Journal of Comparative Medicine.2016;26(8):85-90. doi:10.3969/j.issn.1671-7856.2016.08.014

Objective Lamins are the major components of nuclear lamina underneath the inner nuclear membrane (INM).Lamins express in most cells and are involved in the whole process of growth, also play a major role in cell stability and embryonic development.Mutant in human LMNA gene may lead to a series of disorders, which are similar to progeria or other aging-associate syndrome.In this study, we report a new lmna knockdown animal model generated in our laboratory in order to provide a useful tool for studying laminopathies.Methods Two plasmids tagged to zebrafish lmna gene were designed based on morpholino oligonucleotides technology.Co-microinjected the plasmids into zebrafish embryos to knockdown lmna gene.Imagining and western blot detection were used to identify the mutants.Results Two different proteins, Lamin A/C, were expressed in the zebrafish embryos.Two plasmids lmna-MO and lmna-EGFP-pCS 2 + were generated and co-microinjected into embryos.The results of imagining and western blot showed that the expression of lmna gene was downregulated in the zebrafish embryos.Conclusions Lamin A/C are expressed in zebrafish.lmna gene can be knocked down by the injection of lmna-MO and lmna-EGFP-pCS 2 +.This new animal model may be a powerful tool for study on laminopathies.

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Influence of protein phosphatase 5 on the lipid metabolism in mice

Jun WANG ; Bin QIU ; Ming LIU ; Chao WANG ; Weidong YONG ; Zhongwen XIE

Chinese Journal of Comparative Medicine.2016;26(8):79-84. doi:10.3969/j.issn.1671-7856.2016.08.013

Objective To investigate the effect of protein phosphatase 5 (PP5) on lipid metabolism in the PP5 knockout (KO) mice.Methods Male PP5 KO and wild type (WT) mice at the age of 6 weeks were used in this study. In order to study the effect of high fat diet ( HFD) feeding, the body weight was measured.The liver histology was examined by HE and oil red O staining.To further verify PP5 functions in the adipogenesis, in vitro experiment was carried out using mouse embryonic fibroblasts (MEF).Western blotting and real-time PCR were performed to quantified the expression of lipid metabolism-related genes in the liver tissues.Results Compared with the WT mice, the body weight gain was slower in the KO mice.The size of the lipid droplets was smaller and the quantity was less in the KO mouse liver tissue.In vitro study revealed that the KO mouse MEF cells showed less differentiated adipocytes with smaller lipid droplets than the WT MEF cells.This observation was further confirmed by detecting the expression of adipogenesis-related genes in the HFD liver.The markers of adipocyte differentiation, such as CD36, AP2, PPARγ2, and Glut4, were significantly decreased, while energy expenditure-related markers, such as phosphorylation of GR and expression of UCP1, were significantly increased.Conclusions Protein phosphatase 5 may play a regulatory role in the mouse lipid metabolism through regulating the de-phosphorylation of p-GR and enhancing the expression of UCP1.

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Construction of a Hr mutant knockout mouse model and phenotypic analysis

Kuicheng ZHU ; Cunshuan XU ; Chunyao WANG ; Yingying CHEN ; Jingtao ZHANG

Chinese Journal of Comparative Medicine.2016;26(8):75-78. doi:10.3969/j.issn.1671-7856.2016.08.012

Objective To establish a Hr mutant knockout mouse model to study the function of Hr gene.Methods Transcription activator-like effector nucleases ( TALENs) technique was used to disrupt the mouse Hr locus,creating heritable mutations that eliminate Hr function to explore the effects of Hr on hair development and provide a good model to study the function of Hr gene.The phenotype of Hr -/- mice was observed after birth and skin histology of the transgenic mice was studied by light microscopy.Results It was shown that a F0 mouse with the 2 -bp deletion in Hr gene ranging from 86 to 87 base pairs was obtained.The male mice with clear deletion of the Hr fragment and with obvious frame shifting were mated with wild-type female mice, and F1 mice were achieved.The heterozygous males mated with females to generate the F2 homozygous mice.The first hair coat of Hr-/- mice developed normally.Beginning from 14 days after birth, however, there was a rapid hair loss.The mices were completely hairless except for a few vibrissae at 30 days. Histologically, two characteristic structures appeared, the utriculus and dermal cyst.Conclusions The results suggest that Hr -/- mice are successfully created using TALENs, and Hr is important for regulating hair development, which could explain at least in part the hair loss and be applied to study the mechanism of hair growth and development disorder.

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A53T α-synuclein decreases the expression of type 2 vesicular monoamine transporter in neuroblastoma SH-SY5Y cells

Jianmei MAN ; Juntang GUO ; Daijuan ZHANG ; Anqi CHEN

Chinese Journal of Comparative Medicine.2016;26(8):66-69. doi:10.3969/j.issn.1671-7856.2016.08.010

Objective To investigate the effect of A53T α-synuclein on the expression of type 2 vesicular monoamine transporter (VMAT2) in neuronblastoma SH-SY5Y cells stably expressing A53T α-synuclein.Methods A53T α-synuclein eukaryotic plasmid was constructed by transfection of the SH-SY5Y cells using LipofectamineTM 2000, and a stable transfected monoclonal cell line was selected by G418.Western blotting and DCFH-DA staining were used to detect the effect of A53T α-synuclein overexpression on the expression of VMAT2 protein and level of reactive oxygen species (ROS).Results Western blotting showed that compared with the control group, the expression of VMAT2 protein was significantly decreased, and DCFH-DA staining showed that DCF signal was significantly increased (507.3 ±7.1) than that in the cell line stably expressing A53T α-synuclein (410.7 ±10.5) (P <0.05).Conclusions A53T α-synuclein can increase the intracellular ROS level by inhibiting the expression of VMAT2, thereby playing an important role in the pathogenesis of Parkinson′s disease.

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A long-term study of the toxicity of an Uyghur medicine, Kursi Kaknaq, on hematological parameters in rats

Islam RABIGUL ; Abdugheny ABDUJILIL ; Yingli LENG ; Jing WANG

Chinese Journal of Comparative Medicine.2016;26(8):59-65. doi:10.3969/j.issn.1671-7856.2016.08.009

Objective To investigate the long-term toxicity of an Uyhgur medicine, Kursi Kaknaq, on hematological parameters in the rats.Methods A total of 120 healthy Sprague-Dawley rats were randomly divided into the control, low dose (0.32 g/kg? d ), moderate dose (1.6 g/kg? d) and high dose (3.2g/kg? d) Kursi Kaknaq groups.The drug was given orally, 6 days per week for 180 days.The control group was given 0.5% sodium carboxymethyl cellulose suspension. Results No death was recorded in the rats and no obvious toxic events were observed during the experiment. Hematological parameters including RBC, HGB, MCH, MCHC, LYMP%, WBC, and PLT; biochemical parameters including ALT,AST, ALP, GLU, BUN, ALB ,TBIL, Crea, TCHO, TG, and CK; electrolyte time parameters such as K+, Na+, Cl-and prothrombin time(PT) showed statistically significant differences (P <0.05 and P <0.01), but did not show time and dose effect regularity, and no pathological significance.Conclusions No obvious toxic effects on hematological parameters are observed in the SD rats treated with Kursi Kaknaq at doses of 0.32 g, 1.6 g, or 3.2 g (crude drug) /kg? d orally administered for 180 days, indicating that this drug is safe for long-term clinical use.

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Changes and significance of metallothionein expression during hepatocarcinogenesis in C57BL/6J mice

Xu YI ; Li LONG ; Mingliang CHENG

Chinese Journal of Comparative Medicine.2016;26(8):53-58. doi:10.3969/j.issn.1671-7856.2016.08.008

Objective To investigate the dynamic changes of metallothionein(MTs)gene expression and explore the important significance of MTs during hepatocarcinogenesis.Methods One hundred and twenty-five SPF 5 -8-week old male C57BL/6J mice were randomly divided into control group and model group.Diethylnitrosamine (DEN) was given to the mice at a dose of 100 mg/kg, ip, and 50 mg/kg, ip, in the first and next week, respectively.The mice were given ethanol (53%, 5 mL/kg/day, 5 days/week) from the third week of experiment till 35 weeks.At 1, 3, 9, 13, 24 and 35 weeks of the experiment, liver samples were taken for histopathological examination of liver damages and incidence of HCC. The liver index and malondialdehyde (MDA) of liver homogenate were determined.All liver tissue samples were examined by histopathology using hematoxylin and eosin (HE), Masson and reticular fiber staining.Real-time RT-PCR was used to analyze the mRNA expression level of liver metallothionein-1 /2 (MT-1 /2) in different periods.Results Progressive liver damages in model group mice were identified in different periods.Hepatocytes abnormal tission and abnormal liver plate structure, architecture often characteristic of HCC could be seen in approximately 50% of mice at 35 weeks.In addition to these, a higher liver index also were seen at 35 weeks.Increased MDA levels in the mouse liver tissues were observed in each stage.Real-time RT-PCR analysis showed that significantly increased transcription of MT-1 and MT-2 at 1, 3 and 9 weeks, then gradually declined and even below the normal level.Conclusions MTs gene expression levels in mouse liver tissues are changed from significantly increased in the early stage of injury to decreased expression combined with distinct fibrosis. Our findings further demonstrate that the down-regulation of MTs level is closely correlated with hepatocarcinogenesis.

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Advantages and application of telemetry compared with the traditional methods in safety pharmacology

Jingjing SUN ; Quan ZHOU ; Zhenzhen QI ; Qiuping GUO

Chinese Journal of Comparative Medicine.2016;26(8):47-52. doi:10.3969/j.issn.1671-7856.2016.08.007

Objective To prove the advantages of telemetry by comparing with the traditional methods in safety pharmacology.Methods To monitor continuously the heart rate, respiratory rate, blood pressure and ECG of Beagle dogs by traditional and telemetry methods respectively, analyze and compare the changes between anesthetized and conscious dogs before and after feeding.Results Maintenance of anesthesia changed significantly the heart rate, respiratory rate, blood pressure and QT interval in the ECG of animals.The changes of physiological indicators in 24 h is not obvious in conscious animals, and showed a certain biorhythm.Compared with the conscious animals, the anesthetized dogs’ heart rate was significantly higher, blood pressure increased significantly, QRS and QTcf interval prolonged significantly, respiratory frequency decreased, heart rate increased significantly after feeding, and QTcf interval extended very significantly.Conclusions Traditional methods in safety pharmacology affect animal physiological indicators such as heart rate, blood pressure, respiratory rate and QT interval, which affect the objectivity of drug evaluation.Using conscious animals by telemetry can reduce these errors, however, the interference from outside should be eliminated.

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Comparison of the efficiency of tail vein injection and intramyocardial injection of adenovirus vector in mice

Linshan YANG ; Zhenzhen ZHAN ; Bo WANG ; Junqi YANG ; Huimin FAN ; Zhongmin LIU

Chinese Journal of Comparative Medicine.2016;26(8):42-46. doi:10.3969/j.issn.1671-7856.2016.08.006

Objective To investigate the efficiency of target gene transfection of the heart and liver after tail vein or intramyocardial injection of adenovirus vector (GFP-Ad).Methods GFP-AD was constructed at first.A total of 20 male 8-week old C57BL/6 mice were randomly and equally divided into tail vein injection of GFP-AD group and intramyocardial injection of GFP-AD group.The mRNA levels of GFP in the heart and liver tissues were detected by Q-PCR at different time points.Fluorescence microscopy was performed to visualize the expression of GFP fluorescence.Results Compared with the tail vein injection group, the GFP mRNA level in mouse heart tissue was apparently higher in the intramyocardial injection group.In both groups, the GFP mRNA levels in liver tissue were significantly increased compared with that in the heart tissue.In the tail vein injection group, the GFP mRNA level in liver tissue reached a peak on day 7;but in the intramyocardial injection group, the mRNA level of GFP in liver tissue reached apeak on day 3.We also observed the same trend of GFP fluorescence expression in the tail vein injection group compared with that in the intramyocardial injection group.Conclusions Intramyocardial injection of adenovirus vector is suitable to achieve a higher transfection efficiency in mouse heart tissue compared with the tail vein injection method.Although both injection methods are suitable for transfection of mouse liver, the tail vein injection method is preferential for it is simple and less invasive.

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