Abstract:Objective To investigate the influencing factors involved in the establishment of a C57BL/6 J model of metastatic melanoma in the lung, including the way of tumor inoculation, the number of inoculated cells and the time of tumor formation. Methods Mouse melanoma B16F10 cells were cultured in vitro. 1) Eighteen healthy male C57BL/6 J mice were randomly divided into three groups. Mice in each group received 100 μL cell suspension (including 3 × 10⁶ melanoma cells) via intravenous, intraperitoneal and subcutaneous injection, respectively. After two weeks, the mice were killed and dissected, and the tumor growth and metastasis were observed. 2) Eighteen male mice were randomly divided into three groups. Mice in each group were injected with 3 × 10⁶ cells, 1 × 10⁶ cells, and 3 × 10⁵ cells through the tail vein, respectively. After two weeks, mice were killed and dissected, and the tumor growth and metastasis were observed. 3) Eighteen male mice were randomly divided into three groups. Mice in each group were injected with 1 ×10⁶ cells though the tail vein. Mice were killed and dissected after one week, two weeks and three weeks, respectively. The growth and metastasis of tumor were observed. Results 1) The success rate of lung metastasis was 100% in the mice with intravenous injection, but not in mice receiving intraperitoneal injection and subcutaneous injection. 2) The size of metastatic melanoma nodules were moderate in mice inoculated by 1 ×10⁶ cells. The number of melanoma metastatic foci was too high in the mice inoculated with 3 ×10⁶ cells, but too low in the mice inoculated with 3 ×10⁵ cells. 3) Significant metastatic melanoma foci were observed in the mice killed and dissected after two weeks with no death. The number of melanoma foci in the lung was too high in the mice killed after three weeks, while was too low in the mice killed at one week after tumor cell inoculation. Conclusions Intravenous injection of 1 ×10⁶ mouse melanoma cells into C57BL/6 J mice and killed after two weeks is an optimal method for establishment of a mouse model of metastatic melanoma in the lung, and is worth of recommendation.

Abstract:Objective To study the expression of lipoic acid synthase (LIAS) in the liver and kidney of Lepr ^db/db mice with deficient leptin receptor. Methods Eight 10?week old male Lepr ^db/+ mice and Lepr ^db/db mice were included in this study. The body weight of rats in the two groups was measured. Fasting blood glucose (FPG) was measured with blood glucose test strips for all mice after fasting for 8 hours. Blood samples were obtained from the abdominal aorta and the animals were sacrificed. The liver and kidney were weighed. The right lobe of liver and the left kidney samples were fixed in 4% paraformaldehyde for pathological examination. Serum samples were separated and the sereum contents of CHO, TG, HDL and LDL were detected. The mitochondria of liver and kidney tissues were extracted with a mitochondrial isolation kit, and the protein was extracted. The expression of LIAS protein was detected by western blot. Results Histopathological observation showed that the liver and kidney tissues of Lepr ^db/+ mice have intact and clear structure. But the liver tissue of Lepr ^db/db mice showed fatty degeneration, the kidney tissue showed glomerular hypertrophy, basement membrane thickening, mesangial area widened, including mesangial cells and mesangial matrix increased. The GLU, CHO, TG, LDL and AST of Lepr ^db/db mice were significantly increased compared with those of Lepr ^db/+ mice ( P < 0.05). Compared with Lepr ^db/+ mice, the LIAS protein expression was significantly increased in the liver and kidney mitochondria of Lepr ^db/db mice ( P <0.05). Conclusions There is impaired glucose and lipid metabolism in the Lepr ^db/db mice which has defect leptin receptor, and the expression of LIAS protein in liver and kidney of the Lepr ^db/db mice is higher than that of Lepr ^db/+ mice.

Abstract:Objective To observe the structure of the visual organ and functional development of marine medaka and to provide a theoretical basis for further studying its behavior. Methods The structure of visual organ of marine medaka was examined, with emphasis on the variations of its eyeball and retina. Results The medaka visual organ developed very rapidly. At the embryonic period, the pigment layer of the eyeball appeared, and there already had a significant eye area. At the first day, the structural development of the eyeball and retina were basically completed. At the 6th day, there was a significant difference of pigment layer and photoreceptor layer between light and dark vision. At the 23th day, the retinal structure was greatly changed, which can explain the reason about the change of medaka from the floating stage to the benthic stage. During from 9 to 14 month-old, the thickness of all layers in the retina decreased, indicating that the vision was degraded. Meanwhile, the kernel layer differentiation was obvious, as vertebral cells showed photoreceptor layer structure closely inlaid with rod cells, that medaka has good light sensitivity and strong visual acuity. The rapid development of medaka visual organ can be recognized as it needs to adapt the surroundings and to keep the life events such as feeding. Conclusions The development process of the visual organ of marine medaka is obvious and can provide a theoretical basis for further study of its behavior, and it may be a good experimental method for studying other related species.

Abstract:Objective To establish the expression profiles of differentially expressed miRNA and mRNA in oral squamous cell carcinoma (OSCC) and to investigate the roles of miRNA and mRNA associated with the occurrence and development of OSCC. Methods The expression profiles of miRNA and mRNA were constructed using a new generation of high-throughput sequencing techniques. The miRNA and mRNA associated with the occurrence and development of OSCC were predicted by Gene Ontology enrichment analysis and KEGG pathway analysis. Results We successfully constructed the differentially expressed miRNA and mRNA profiles of Chinese hamster buccal pouch squamous cell carcinoma. 11 known and 3 novel significantly differentially expressed miRNAs and 194 differentially expressed mRNAs were found. A miRNA can regulate multiple mRNAs, and multiple miRNAs can control one mRNA. Conclusions Differential expression of miRNA play a an important role in the carcinogenesis and development of OSCC through regulating mRNA and forming a complex regulatory network. It provides theoretical data for the occurrence, pathogenesis, clinical treatment and prognosis of OSCC.

Abstract:Objective To compare the differences in the intestinal microflora of WHBE rabbit and JW rabbit models of diarrhea-predominant irritable bowel syndrome (IBS). Methods 16 WHBE rabbits and 16 JW rabbits were randomly divided into normal control (NC) group and IBS model group, respectively (n = 8). The diarrhea-predominant IBS model was established by wet-heat stress combined with intragastric gavage of senna decoction. The abdominal circumference index, water content of feces and colonic transit function were observed. After sacrifice, colon tissue samples were taken for histopathological examination and colon contents for intestinal flora diversity analysis. Results Compared with the NC group, the IBS model rabbits showed an increased abdominal circumference index and fecal water content, and a shortened colon transit time, but no obvious pathological changes were observed in the colon tissues. Meanwhile, the Shannon index and Chao1 index of IBS model rabbits were significantly decreased ( P <0.05). According to the result of OTU classification analysis, Firmicutes and Bacteroidetes are the dominant bacteria in the intestinal microflora of rabbits. Compared with the NC group, the Firmicutes, Verrucomicrobia, Chloroflexi, Akkermansia, and Streptococcus in the WHBE rabbit IBS model group were significantly reduced ( P < 0.05, P < 0.01), while Bacteroidetes and rc4-4 significantly increased ( P < 0.05, P < 0.01). However, in the JW rabbit IBS model group, Eubacterium and Subdoligranulum were significantly increased ( P < 0.05), while Lactobacillus, Coprobacter, Veillonella and Streptococcus were markedly decreased ( P <0.05). Compared with the JW rabbit NC group, the abundance of Firmicutes, Odoribacter, Veillonella, Streptococcus, Oscillospira and Pseudoflavonifractor were significantly decreased ( P < 0.05, P < 0.01), but Bacteroidetes, Verrucomicrobia, Eubacterium, Akkermansia and Coprobacter were significantly increased ( P <0.05, P <0.01) in the WHBE rabbit NC group. Compared with the JW rabbit IBS model group, the abundance of rc4-4, Bacteroidetes, Coprobacter and Clostridium were significantly higher ( P < 0.05, P < 0.01), while the Firmicutes, Dorea, Coprococcus and Subdoligranulum were significantly lower ( P < 0.05) in the WHBE rabbit IBS model group. Conclusions There is an intestinal microflora imbalance in rabbits with IBS, resulting in a decrease of microflora diversity. The changes of intestinal microflora in the WHBE rabbits and JW rabbits with IBS have their own characteristics, and have apparent differences.

Abstract:Objective To evaluate and discuss the changes of biomarkers of abnormal Savda syndrome rat model in Uyghur traditional medicine by external feature observation and urine metabolomics assessment. Methods The abnormal Savda syndrome rat model was established according to the theory of Uyghur traditional medicine. Its external characteristics such as hair, tongue, sleep, feces, emotion and weight growth rate were observed and scored, and their urine was detected and analyzed by proton nuclear magnetic resonance (¹H-NMR) spectroscopy. Results Compared with the healthy control group, there were significant changes in the external features in the abnormal Savda syndrome rat model group, including dry and hard stools, reduced urine output and darker color, dry fur, dark purple tongue with ecchymosis, and decreased weight growth rate. Moreover, 23 urinary metabolites were significantly reduced, including propionate, lactic acid, pyruvic acid, acetic acid, alanine, acetamide, glycoprotein, acetone, methyl guanidine, sarcosine, ornithine, glycine, creatine, creatinine, aminoanhydride, β-galactose, urocanate, tyrosine, phenylalanine, hippuric acid, aminohippuric acid, formic acid and lysine. However, urea, citric acid, allantoin and α-ketoglutaric acid were significantly increased. Conclusions During the development process of Savda syndrome, there are not only abnormal changes in external appearance in the model rats, but also evident changes of many internal metabolic pathways. The obvious abnormalities of the urine metabolites may be related to the biological mechanisms of abnormal Savda syndrome.

Abstract:Objective The aim of this experiment was to explore the effect and mechanism of intestinal microbiota on shaping the growth performance by fecal microbiota transplantation from pigs to pseudo-germ-free mice. Methods Thirty-six barrows with a similar initial body weight of 30 kg were raised for 42 days (ad libitum) within individual metabolic cages. Feed intake and body weight of each pig were recorded every week to calculate the feed conversion rate and average daily gain. At the end of the experiment, feed conversion ratio and average daily gain were integrated to divide the pigs into 3 groups, namely, high growth performance (HP), moderate growth performance (MP) and low growth performance (LP) groups. Feces were collected to calculate the total intestinal nutrient digestibility and prepare for fecal microbiota transplantation to pseudo-germ-free mice, which were induced with several antibiotics for four weeks. Fecal microbiome structure was assayed by profiling V3-V4 region of the 16S rRNA gene. Results Fecal microbiota transplantation from pigs to pseudo-germ-free mice resulted in reappearance of the original phenotype. Compared with the LP pigs, the microbial species richness and microbial diversity in feces were higher in the HP pigs. The HP pigs had improved digestibility of gross energy ( P = 0.01) and higher abundance of Methanobrevibacter. Enterococcus and Akkermansia were also more abundant in the recipient pseudo-germ-free mice from the HP pigs which may be correlated with a high energy utilization. Conclusions Fecal microbiota transplantation from pigs to mice results in reappearance of the original phenotype and microbial species richness, microbial diversity, and their growth ability. Different nutritional metabolism is shown among pigs with different feed efficiency and the HP pigs have improved energy utilization ( P =0.01). At the same time, the bacteria correlated with high energy utilization are more abundant in feces of HP pigs than in LP pigs.

Abstract:Objective To compare the differences of bacterial distribution of intestinal flora in Microtus fortis living under laboratory feeding and wild survival conditions. Methods The 16S rDNA?V4?V5 region of bacteria in the ileocecal contents from Microtus fortis raised in lab and captured in wild were measured by high?throughput sequencing. The number of operational taxonomic units (OTUs) were sorted and calculated, and the species abundance and distribution and difference were analyzed. Results The rarefaction curves indicated that adequate sampling was achieved. At the phylum level, the distribution of intestinal flora between two groups was similar. The experimental group had a unique phylum, Lentisphaerae. The wild type group had 3 unique phylums, Fusobacteria, Thaumarchaeota and an unclassified phylum. At the genus level, the kind of intestinal flora in the wild type group was more abundant than the experimental group. Ruminococcus is the largest differential genus. Conclusions The microbial community structure and differences of Microtus fortis living under different conditions are obtained. It may further enrich the basic biology data of Microtus fortis.

Abstract: Objective To explore the relationship between gut microbiota and anti-dsDNA antibody in systemic lupus erythematosus (lupus) TC mice. Methods ELISA was performed to detect serum anti-dsDNA antibody in the lupus TC mice. Then, the feces of both dsDNA-positive and -negative mice were collected, and 16S rRNA of the stool sample was sequenced by Illumina HiSeq 2500 high-throughput sequencing, to analyze the relationship between gut microbiota and anti-dsDNA antibody level in the two groups. Results The result of ELISA and mouse fecal high-throughput sequencing showed that the species diversity of gut microbiota in the dsDNA-positive TC mice was significantly lower than that in the dsDNA-negative TC mice. The gut microbiota of TC mice in the two groups showed significant differences at different classification levels: Chloroflexi at phylum level, Betaproteobacteria, Deltaproteobacteria and Ktedonobacteria at class level, Burkholderiales, Desulfovibrionales and Ktedonobacterales at order level, Alcaligenaceae and Desulfovibrionaceae at family level, and Parasutterella and Desulfovibrio at genus level ( P <0.05 for all). Conclusions The flora composition of gut microbiota in lupus TC mice is highly correlated with anti-dsDNA antibody. The results of our study provide a basis for further elucidation of the relationship between gut microbiota and the pathogenetic mechanism of systemic lupus erythematosus.

Abstract:Objective To compare the biological characteristics of eyeballs between Zmu-1:DHP and DHP guinea pigs, and to explore the retinal mechanism of myopia in Zmu-1:DHP guinea pigs. Methods To measure the refraction, corneal curvature and axial length of the two guinea pig strains at age of 4 -12 weeks. Those spontaneous myopic Zmu-1: DHP guinea pigs were chosen to take the retina for pathological examination. The pathological changes in the retina were determined and compared with the DHP guinea pigs. The expression of RALDH, ALDHTH, TH, TK, iNOS, nNOS, bFGF and TGFβ mRNA in the retina were detected by real time-PCR. Results The myopic rate of 3-week old Zmu-1: DHP guinea pigs was 90.21%, while of the DHP guinea pig was only 18.00%. From 4 to 12 weeks, compared with the DHP guinea pigs, myopia and axial length of the Zmu-1:DHP guinea pigs were significantly increased ( P <0.01), and the corneal curvature of Zmu-1:DHP guinea pigs was significantly less than the DHP guinea pigs ( P <0.01). The retina outer nuclear layer of Zmu-1:DHP guinea pigs was reduced in thickness, the cell volume was smaller, and the cell number was less compared with the DHP guinea pigs. The choroid of Zmu-1:DHP guinea pigs showed atrophy and became thinner. There were few pigment granules in the pigment epithelium of Zmu-1:DHP guinea pigs, while there were plenty of pigment granules in the DHP guinea pigs. Compared with the DHP guinea pigs, the expression of TH mRNA was significantly down-regulated in the retina of Zmu-1:DHP guinea pigs (P <0.01), and the expression of TK, iNOS, nNOS, bFGF and TGFβ was significantly down-regulated ( P < 0.01, P < 0.05, P < 0.05, P < 0.05, P < 0.05). Conclusions Zmu-1:DHP strain guinea pig has a high rate of spontaneous axial myopia. The retinal mechanism of myopia has a relationship with the regulation of several myopia factors.

Abstract:Objective To establish an efficient method of genotyping for Lepr ^{db/ +} mouse offsprings by TaqMan probe quantitative fluorescence PCR. Methods Genome DNA was extracted from tails of 228 Lepr ^{db/ +} mouse offsprings. PCR primers and TaqMan probes were designed according to the mutation sites of Lepr gene (rs1801133). Real time PCR assay was applied and SNP loci were typed with SDS software. The genotyping of 2-month old Lepr ^{db/ db} mice was validated by the phenotype and Hardy-Weinberg equilibrium test was performed. Results 228 samples were detected by the established TaqMan fluorescence quantitative PCR assay. 64 mice were of GG genotype, with a genotype frequency of 0.1929. 123 mice were of GT genotype, with a genotype frequency of 0.5395. 41 mice were of TT genotype, with a genotype frequency of 0.2807. Compared with the phenotype typing, the sensitivity of the TaqMan fluorescence quantitative PCR was 97.56% and the specificity was 99.47%. Conclusions TaqMan probe quantitative fluorescence PCR assay is a simple and efficient method, and can be used to detect the genotype of Lepr ^{db/ +} mouse offsprings.

Abstract:Objective To optimize the detection test of Pig-a gene mutation in peripheral blood of rats by enriching and detecting mutant erythrocytes, using immunomagnetic separation technique in combination with flow cytometry. Methods SD rats were administered with 20, 40 and 80 mg/ (kg·bw) doses of N-ethyl-N-nitrosourea (ENU) continually for 3 days. The peripheral blood samples of rats were collected on the 7th, 14th and 28th days, respectively, after treatment. Immunomagnetic separation columns were used to enrich RET ^{CD59 -} and RBC ^CD59- cells, and then flow cytometry was used to count the number of pre-column and post-column peripheral erythrocytes. Results Compared with the control group, the frequencies of RET ^CD59- and RBC ^CD59- were significantly increased in each ENU group ( P <0.05). With immunomagnetic separation technique, the test of Pig-a gene mutation of a sample could be completed within 3 minutes, and the number of detected RET ^{CD59 -} or RBC ^{CD59 -} cells was up to 2 ×10 ⁴ or 9 ×10 ⁴ , respectively. Conclusions In this study, immunomagnetic separation in combination with flow cytometry is used to establish and optimize the Pig-a gene mutation test in rat peripheral blood, showing a high-throughput detection and improved accuracy and efficiency of the experiment.

Abstract:Objective SIV30 protein of simian immunodeficiency virus ( SIV) was prepared by genetic engineering technique as an antigen diagnostic reagent, to establish an immune comb method for the specific detection of anti SIV IgG in monkey serum. Methods Recombinant expression plasmid of SIV SIV30 gene was constructed by prokaryotic expression vector pGEX-4T-1, and expressed in the competent BL21 cells. The recombinant protein was purified as a diagnostic antigen, and a standardized procedure for the detection of immune comb was established and applied for clinical detection. Results The optimum coating amount of antigen was 0.02 mg/ mL. The prepared IC was able to specifically detect the positive serum of SIV. There was no cross reaction between the sera of other viruses. It showed a high specificity of the detection method. Sensitivity analysis showed that the SIV30 protein was able to detect 1∶400 times diluted SIV positive sera. The result of stability and repeatability test (the same sample was repeated 3 times) showed that the coefficient of variation (CV) was less than 10%. The serum samples of 10 suspicious monkeys were detected by this method , showing a consistent rate of comb method and ELISA test result of 100%, Kappa = 1.000. Conclusions SIV30 protein is expressed in prokaryotic cells. The immune comb is prepared, and is successfullyl applied in clinical examination. It shows that the method has a high sensitivity, strong specificity, good reproducibility and practicability.

Abstract:Objective To investigate the changes of coagulatory function in septic rats induced by cecal ligation and puncture (CLP). Methods Cecal ligation and puncture (CLP) were performed to induce sepsis in SD rats. Coagulation indexes were detected at 8, 16 and 48 h after operation, and histopathological changes of the lung, kidney, liver and spleen were examined using HE staining. Results The 12-day survival rate of the CLP-induced septic rats was 30%, with an acute onset and high mortality. In the acute phase of disease development of the CLP rats, the activated partial thromboplastin time (APTT) was prolonged ( P <0.05) at 8 h, the prothrombin time (PT) was prolonged at 16 h ( P <0.05), the factor XII activity in the endogenous coagulation pathway and the factor VII activity in the extrinsic coagulation pathway showed a transient inhibition, the thrombin time (TT) was prolonged at 48 h ( P < 0.01), and the content of fibrinogen (FIB) was increased gradually from 16 h ( P < 0.001). Among the other important coagulation and anticoagulation indexes, the number of platelets (PLT) was decreased gradually from 8 h ( P <0.01), while the number of vWF:Ag increased gradually from 8 h ( P <0.001). The D-dimer amount gradually increased from 16 h ( P <0.05), and the amount of PS:Ag significantly decreased until 48 h ( P < 0.001). However, there was no significant change in the antithrombin-III (AT-Ⅲ) content. The histopathological examination showed that there are different degrees of damages in the lung, kidney, liver and spleen tissues, but no obvious venous thrombosis and bleeding were found. Conclusions In the acute phase, there is coagulatory dysfunction in the septic rats, however, no histopathological changes such as venous thrombosis and bleeding were observed in the lung, kidney, liver and spleen tissues due to coagulatory dysfunction.

Abstract:Objective To study the effect of noise pollution on serum hormone and heat shock protein-70 (Hsp-70) levels in rats. Methods Forty male Wistar rats were randomly divided into control group (normal), experimental group (further divided into 35, 65 and 85 dB three groups), each group 10 animals, stimulated for 30 min once a day, continually stimulated for consecutive 20 days. On the 21st day of experiment, the serum noradrenaline (NA), testosterone (T), dopamine (DA) and Hsp-70 levels were determined by enzyme-linked immunosorbent assay (ELISA). Results < /b> Compared with the control group, the body weight of experimental group (35, 65 and 85 dB groups) was reduced by 23.45%, 30.13%, and 35.64%, respectively The serum T and DA levels were decreased by 9.12%, 20.06%, 37.99% and 15.49%, 8.31%, 24.88%, respectively; while the serum NA and Hsp-70 levels were increased by 35.08%, 171.52%, 197.86%, and 39.34%, 195.09%, and 285.25%, respectively. All the result showed a significant difference ( P <0.01). Conclusions Noise pollution can significantly affect the serum levels of hormone and heat shock protein-70 expression in rats.