Abstract:Objective To investigate the effect of single dose intraosseous injection of simvastatin on tumor vascular structure and function in murine breast cancer. Methods BALB/c mice and 4T1 murine breast cancer cells were used to establish a subcutaneous xenograft model. The mouse model of orthotopic breast cancer received intraosseous injection of a single dose of simvastatin (50 μg) or vehicle only. Frozen tumor tissue sections were prepared for co-immunostained with CD31 and α-SMA. Evans blue dye was injected into the tail vein to observe the vascular permeability. The expression level of HIF-1α was detected by immunohistochemistry. Results Immunofluorescence dual staining showed that intraosseous injection of simvastatin increased the number of perivascular pericytes in the tumor vessel(P < 0.05), Evans blue dye content showed that in vivo vessel permeability in the tumor tissue was significantly decreased(P < 0.05),and the immunohistochemistry results showed that local hypoxic area was significantly improved. Conclusions Single dose intraosseous injection of simvastatin can promote the normalization of tumor vasculature by improving the coverage of pericytes.

Abstract:Objective To compare the effects of two different samples and different test methods on blood biochemical indexes and blood glucose values in rats. Methods Glucose levels in the serum and plasma samples were detected with a blood glucometer, and the biochemical parameters in the serum and plasma were determined by routine blood biochemistry.The data were statistically compared and analyzed to determine if there are some significant differences.Results Among the 19 biochemical indexes of serum and plasma specimens, nine parameters, i.e., ALB,TP,ALP,CHOL,URIC,GLU,Mg,LD,Ca showed significant differences(P< 0.05),while the other 10 indexes showed no significant difference (P> 0.05).Different samples and different methods had significant differences in the detection of blood glucose (P< 0.05). Conclusions Different method and samples impact on the detection of blood glucose and some other serum and plasma biochemical indexes.

Abstract:Objective To identify the characteristics of the subtype of PMA-induced THP-1 macrophages by flow cytometry analysis. Methods THP-1 monocytic cells differentiate into macrophages promoted by PMA, then induced into M1 and M2 by adding different cytokines, such as LPS,IL-6 and IFN-γ for THP-1-M1, IL-4,IL-13 and IL-6 for THP-1-M2. Morphology of cells were observed under a microscope and the expression of CD14, CD68, CD16, CD80, CD86, CD163, CD206, CD209, CD83, CD1a, CD11c, HLA-DR were detected by flow cytometry. Results The macrophages stimulated by PMA became adherent; THP-1-M1 and THP-1-M2 lost their spherical morphology, appeared more irregular with many obvious projections. The expression of CD83, CD1a, CD11c, HLA-DR which had the function of antigen presenting on the surface of THP-1-Mφ were very low, and most of them were negative, but those of THP-1-M1 and THP-1-M2 were very high. Conclusions The macrophages differentiated from THP-1 stimulated by PMA are weak in antigen presenting function.

Abstract:Objective To identify the differential proteomic expressions between the liver tissues of male and female mice, and investigate the mechanisms underlying gender differences in liver diseases. Methods Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) were used to identify the differentially expressed proteins in the liver tissues of male and female C57BL/6J mice. The differentially expressed proteins were validated by Western blot and further analyzed by bioinformatics, including Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG). Results Among the auto-detected 1767 protein spots by 2D-DIGE, 325 protein spots were differentially expressed (|ratio| ≥ 1.5, P< 0.05) between the liver tissues of male and female mice, in which 78 spots were randomly selected for MALDI-TOF-MS identification and finally 48 distinct proteins were obtained. Compared with females, 14 and 34 proteins were up- or down-regulated in males, respectively. Among them, 6 differentially expressed proteins were validated by Western blot which confirmed the reliability of 2D-DIGE results.GO analysis showed that the differentially expressed proteins in the liver tissues of male and female mice are associated to various cellular component, molecular function and biological process. 6 pathways were significantly different between the liver tissues of males and females depending on KEGG analysis. Conclusions The proteomic data and related analysis of the liver tissues of C57BL/6J mice offer crucial clues for elucidating the underlying mechanisms of different gender effects on liver diseases.

Abstract:Objective To explore the influence of Mac-1 deficiency on tumor growth of melanoma. Methods The population of Mac-1 gene knock-out (Mac-1^-/-) mice was expanded. B16-F10 cells were subcutaneously injected into the C57BL/6J mice (control group) and Mac-1^-/-mice (experiment group), respectively. Subsequently,the survival rate, tumor volume and body weight were recorded. The proliferation and infiltration of macrophages were detected by immunohistochemistry. Results The survival rate of Mac-1^-/- mice was significantly improved compared with the C57BL/6J mice (P<0.001). The tumor volume and body weight were remarkably decreased in the Mac-1^-/- mice compared with the control group (P<0.001). Meanwhile, the tumor cell proliferation index was decreased in the Mac-1^-/- mice compared with the control group (P<0.01).Furthermore, the infiltration of macrophages in the tumor tissues was also decreased in Mac-1^-/- tumor mice compared with control group. Conclusions Mac-1 gene deletion can significantly suppress melanoma growth.

Abstract:Objective To investigate the effects of vitamin D on synthesis and secretion of lubricin in chondrocytes at the cellular level. Methods Rat articular chondrocytes were stimulated by TNF-α. Normal and inflammatory chondrocytes were treated by different doses of vitamin D respectively. ELISA and Western Blot were used to detect the secretion of lubricin in the supernatant and the synthesis level in the cells. Results TNF-α significantly reduced the activity of both normal chondrocytes and chondrocytes in inflammatory state. TNF-α also significantly reduced the expression of lubricin in the cells and supernatant.1α,25(OH)2D3 increased the activity of both normal chondrocytes and chondrocytes in inflammatory state. 1α,25(OH)2D3 significantly elevated the secretion and expression of supernatant and intracellular lubricin only in chondrocytes stimulated by TNF-α in a dose-dependent manner, but not in normal chondrocytes. Conclusions Vitamin D can promote the secretion and expression of lubricin in inflammatory state chondrocytes, which may act as one of the mechanisms of vitamin D protecting the cartilage surface in osteoarthritis.

Abstract:Objective To establish osteoarthritis model of the knee joint in mice on the basis of knocking out SIRT1 gene and to observe the differences in the morphology of the cartilage tissue using single staining and compound staining. Methods The knee joint specimens were divided into two groups:SIRT1^-/- control group (group A, n=6) and SIRT1^-/- osteoarthritis model group (group B, n=6). The knee anterior cruciate ligament was traversed, and the ipsilateral medial meniscus was cut to establish an osteoarthritis model of knee joint. HE staining, safranin O-fast green staining, safranin O-alcian blue staining, safranin O staining, fast green staining, alcian blue staining were used to observe the morphological changes in the articular cartilage of the knee. Results Safranin O-fast green staining and safranin O-alcian blue staining showed better results in observation of the morphology of chondrocytes, the structure of cartilage layers, the presence of type Ⅱ collagen, tide line and the changes of subchondral bone. While the safranin O staining and alcian blue staining had certain advantages in the observation of the defects of cartilage tissue. Conclusions Compared with the single staining, the compound staining used in this study have obvious advantages in obtaining useful information of the cartilage structure in the observation of morphology of cartilage tissues in SIRT1 gene knock-out mice.

Abstract:Objective To observe the intervention effect of salvianolic acid A (SAA) on retinopathy of Zucker diabetic fatty (ZDF) rats and explore the possible action mechanism of SAA to prevent and treat diabetic retinopathy (DR). Methods Thirty-two 7-8-week old ZDF rats were taken and fed with Purina rat chow for 4 weeks. The ZDF rats were equally divided by the blood glucose into model group, 0.5 mg/kg SAA group, 1.0 mg/kg SAA group and metformin (Met) group. 8 Zucker lean (ZL) rats were taken as control group. After 12-week administration, incidence of cataracts and retinal pathology was observed, and levels of GLU, TC and HbA_1c in blood, transferrin (TRF) and retinol binding protein (RBP) in urine and levels of interleukin-1 (IL-1), interleukin-6 (IL-6), oxidized low density lipoprotein (ox-LDL), malondialdehyde (MDA) and lipoprotein related phospholipase A2 (Lp-PLA2) activity in serum were measured. Results In the model group, GLU, TC, HbA_1c, diabetic cataract incidence rate, retinal basement thickening and microangiopathy appeared in most of the rats. The levels of TRF and RBP in urine and levels of IL-1, IL-6, ox-LDL, MDA in serum were significantly increased, and Lp-PLA2 activity was also significantly increased. After SAA administration, the morbidity rate of cataract was reduced, and retinal pathological changes were improved in different degrees. The levels of TRF, RBP, IL-1, IL-6, ox-LDL, MDA and Lp-PLA2 activity was decreased. Conclusions SAA can slow down the process of diabetic retinopathy in ZDF rats and reduce the incidence of cataract. The mechanisms of action may be related to inhibition of chronic inflammation, prevention of lipid peroxidation and reduction of Lp-PLA2 activity.

Abstract:Objective To investigate the synergism and attenuation effects of Pini Pollen on paclitaxel and cisplatin (TP) chemotherapy in model mice. Methods Forty-eight model nude mice with H456 cell line xenograft were divided into 8 groups:model group, TP chemotherapy group, Pini Pollen (150, 300 and 600 mg/kg)group and TP chemotherapy plus Pini Pollen (150, 300 and 600 mg/kg)group. The tumor inhibition rates, body weight, food intake, hematology and blood biochemistry indexes were selected to evaluate the synergism and attenuation effects of intragastric administration of Pini Pollen (150,300,600 mg/kg) on TP chemotherapy. Results Compared with the TP chemotherapy group, the tumor inhibition rates, body weight, food intake, white blood cell count were increased and liver and kidney function damage were alleviated significantly in the TP chemotherapy plus Pini Pollen groups. Conclusions Pini Pollen has a significant synergism and attenuation effects on TP chemotherapy.

Abstract:Objective To explore the body temperature thresholds of shivering in febrile rats during therapeutic hypothermia, and establish rat models of shivering in different degrees. Methods A randomized controlled trial was adopted. Rats weighted 200±20 g and basal body temperature of 36.8-38.3℃ were induced with fever using 20% dry yeast solution through dorsal subcutaneous injection. 40 rats were randomly divided into 4 groups:10 mL ice pack group, 20 mL ice pack group, 40 mL ice pack group and control group, 10 rats in each group. Physical cooling was implemented for 30 minutes in the neck and armpits. No cooling measures was given to the control group. The shivering of rats were observed, and the threshold of anal temperature was monitored. Results In the rats with hyperthermia, shivering was not observed in any part of the rats in the control group and in the therapeutic hypothermia group of 10 mL ice pack. The rats in the therapeutic hypothermia group of 20 mL ice pack developed mild shivering, which manifested as piloerection, head and neck trembling, with or without upper limbs trembling. The threshold of the average rectal temperature of mild shivering was 37.25℃, The incidence of mild shivering was 100%. The rats in the therapeutic hypothermia group of 40 mL ice pack developed severe shivering, which manifested as piloercetion, head, neck, limbs and trunk were trembling, and tail muscle tension increased. The threshold of the average rectal temperature of severe shivering was 37.07℃, severe shivering occurred in 90% of the rats. Conclusions No shivering, mild shivering, and severe shivering models can be established by intervention with ice pack in rats with high fever during therapeutic hypothermia.

Abstract:Objective To observe the changes of rat microglial inflammation and migration after exposure to sodium metavanadate(NaVO₃·2H₂O), and to analyze the possible mechanisms of vanadium neurotoxicity. Methods Primary cultured rat microglial cells were incubated with NaVO₃·2H₂O. Morphological changes and the Iba1 expression of microglia were tested by immunofluorescence assay. iNOS, Cox-2, ERK and p-ERK protein expressions were determined by western blotting. The levels of TNF-α and IL-1β in the culture medium were tested by enzyme-linked immunosorbent assay. The migration of microglia was tested by immunofluorescence staining using wound-healing assay. Results Microglia changed from resting state with ramous shape to round shape in activated state after NaVO₃·2H₂O exposure, and the expression of Iba1 increased obviously. The protein expressions of iNOS and COX-2 increased significantly compared with the control. The levels of TNF-α and IL-1β were also increased significantly. NaVO₃·2H₂O promotes the migration of microglia through ERK pathway. Conclusions Exposure to NaVO₃·2H₂O promotes primary cultured rat microglial inflammation and migration. These results suggest that the inflammatory reaction of microglia may be one of the possible mechanisms of neurotoxicity caused by vanadium exposure.

Abstract:Objective To explore whether CD4⁺T cells in mice can secrete exosomes and the possibility of exosomes to participate in the inflammation and immune response process. Methods Spleen samples were taken from mice, and CD4⁺T cells were isolated from the spleen tissue using magnetic bead separation method. Exosomes of CD4⁺ T cells were extracted from the supernatant and observed under transmission electron microscope. PCR assay was used to detect the expression of miR-23a and miR-214. Results Exosomes were observed under elecron microscope,and the expression of miR-23a and miR-214 were detected by PCR assay.Conclusions CD4⁺T cells in mice can secrete exosomes,which offer possibility to participate in the inflammation and immune response.

Abstract:Objective To analyze the possibilities of screening SPF rabbits and guinea pigs from conventional animals, viral antibodies of the conventional rabbits and guinea pigs bred by licensed companies in Guangdong province during 2014-2016 were determined according to the standard of SPF animals in GB14922.2. Methods Nine batches of 167 rabbit sera and 155 guinea pig sera were sampled from 6 companies. Serum antibodies to virus were determined by ELISA according to GB14922.2. Results Positivity of antibody to rabbit hemorrhagic disease virus (RHDV) was 82.2% (129/157) for the vaccinated rabbits, and negative result were obtained for unvaccinated rabbits. Positive rate of rabbit rotavirus (RRV) was 42.5% (71/167). No positive antibody responses to Sendai virus were detected out in all rabbits. The positive rates of guinea pig reovirus type Ⅲ (REO-3) and pneumonia virus of mice (PVM) were 52.9%(82/155)and 20% (31/155) respectively. Antibody responses to Sendai virus (SV) and lymphocytic choriomeningitis virus (LCMV) were negative in all guinea pigs. Conclusions Although the conventional rabbits and guinea pigs could meet the national standard, higher infection rates of virus excluded that SPF animals emerged in conventional animals, indicating that selection of SPF animals from conventional colonies is impracticable.

Abstract:Objective To conduct a microbiological and parasitological investigation of experimental minipigs in Guangdong province. Methods Four major experimental minipig production units in Guangdong province were included in this investigation. Samples were taken from a total of 154 pigs of 4 brreds, i.e., Bama minipigs, Juema minipigs, Tibet minipigs and Wuzhishan minipigs. Pig fur, scales, serum, rectal swabs and feces samples were collected for detection of 20 pathogens. The data were analyzed and compared among the production units and breeds. Results Mixed infections were detected in all the four institutions. The infection rates of 7 pathogens were rather high:Streptococcus suis type 2 (50.7%), Actinobacillus pleuropneumoniae (40.3%), Mycoplasma hyopneumoniae (100%), Japanese encephalitis virus (41.3%), porcine circovirus type 2 (74.8%), porcine transmissible gastroenteritis virus (73.8%),gastroenteritis virus (44.7%).Porcine parvovirus (26.0%), pseudorabies virus(15.6%) and intestinal worms (3.2%) were also detected in some animals. The immune qualified rates of classical swine fever virus (62.8%) and foot-and-mouth disease virus (35.8%) were rather low. The immune qualified rate of pseudorabies virus was as high as 98.4%. Besides, Salmonella, Brucella, swine dysentery snake like spirochetes, dermatophytes, influenza virus. Toxoplasma gondii, ectoparasites, and coccidia were not detected. Conclusions The results of this investigation indicate that epidemiological quality control of pathogens in experimental minipigs and efforts to establish high grade minipig population in Guangdong province remain to be strengthened. Our study also provides a basis for revision of local and even national standards for experimental minipigs.

Abstract:Obejectives To explore the effect of miR-424 on migration and invasion of non-small cell lung cancer A549 cells. Methods Expression of miR-424 in NCI-H460, NCI-H1975, NCI-H446, A549, NCI-H1299, NCI-H157 lung cancer cells and embryonic lung fibroblasts MRC-5 cells was examined by RT-PCR.miR-424 inhibitor and miR-424 NC were transfected into A549 cells with liposome Lipofectamine^TM2000. Cell viability was measured by MTT assay. Cell migration and invasion was measured by wound scratch assay and transwell migration assay,respectively.The expression of matrix metalloproteinase 2 (MMP2), MMP9, transforming growth factor-β (TGF-β1), p-Smad3 was measured by western blot. Results The expression of miR-424 in NCI-H460, NCI-H1975, NCI-H446, A549, NCI-H1299, NCI-H157 cells (1.78±0.13, 1.69±0.10, 1.89±0.18, 2.88±0.27, 2.52±0.20, 2.49±0.23) was significantly higher than that in MRC-5 cell 0.58±0.05(P< 0.01). Compared with the miR-424 NC group, the cell viability was reduced (P<0.01), cell migration and invasion ability was decreased (P< 0.01), and the expression of MMP2, MMP9, TGF-β1, p-Smad3 protein was down-regulated in miR-424 inhibitor group (P< 0.01). Conclusions miR-424 inhibitor can inhibit the migration and invasion in lung cancer A549 cells via inhibition of TGF-β1/Smad3 signal pathway.

Abstract:Objective To explore the effect of silencing metastasis-associated in colon cancer 1 (MACC1) gene on proliferation and invasion of cervical cancer Hela cells. Methods siRNA MACC1 and siRNA NC were transfected into cervical cancer Hela cells with liposomes. The expression of MACC1 protein and mRNA was detected by western blot and RT-PCR. Cell viability and invasion ability were measured by MTT and transwell assay, respectively. The expression of cyclin D1, cyclin E matrix metalloproteinase 2 (MMP2), MMP9 and the level of extracellular regulated protein kinases (ERK) phosphorylation was detected by western blot. Results Compared with siRNA NC, the expression of MACC1 protein and mRNA was down-regulated, cell viability and invasion ability were reduced (P< 0.01), cell cycle was arrested at G1 phase (P< 0.01), the expression of cyclin D1, cyclin E, MMP2, MMP9 and p-ERK1/2 was down-regulated (P< 0.01). Conclusions siRNA MACC1 can inhibit proliferation and invasion of cervical cancer Hela cells, which migiht be related to down-regulation of p-ERK.

Abstract:Ventricular remodeling is one of the main causes of heart failure. A large number of studies have shown that inflammation plays an important role in the occurrence and development of ventricular remodeling. Recent studies found that chronic inflammation mediated by T cells is closely related to the progression of ventricular remodeling. This review summarized the recent research progress of T lymphocyte subsets in ventricular remodeling.

Abstract:Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by a selective loss of upper and lower motor neurons that lead to paralysis and even death. Mutations in a large number of genes, including FUS/TLS, EPHA4, SS18 L1, ATXN2 and C9ORF72, are identified to the casual genes of ALS, which broadens our understanding of the role of RNA modulation in ALS pathogenesis. This review summarized ALS-associated genes and the related ALS rodent models.

Abstract:Acute myocardial infarction is one of the major causes of death in the world. Research on animal models of myocardial infarction has a great significance in improving the pathophysiology, diagnosis and treatment of human myocardial infarction. At present, problems such as low survival rate and complicated preparation still exist. In recent years, some progress has been made in the preparation of animal myocardial infarction models, which can improve the survival rate, qualified rate and speed. This paper analyzes the preparation of rat models of acute myocardial infarction and related research, combined with actual factors to simplify the process of preparation and improve the skills of scientific researchers, so as to provide a source of stable and reliable acute myocardial infarction models for medical research.

Abstract:In vitro method as indispensable biological research and testing tools, has been widely used in cosmetics compliance test, biological mechanism research and functional materials screening. These methods include alternative methods which have been accepted by reguations and standardized test guide, non-testing method used for risk assessment and prediction, and a lot more diversification and individualization of in vitro methods.With the consensus formation of the whole industry and increase of application experience, innovation and optimization in vitro method will be constantly emerge. These technologies will be beneficial to improve competitiveness and to develop products to meet the needs of consumers.

Abstract:Morphine is a widely used opioid analgesic, but great individual differences in response to morphine such as sensitivity to analgesia and susceptibility to tolerance, which due to chronic morphine treatment, are great challenges for clinicians to optimize treatment strategy. Genetic factors play an important role in individual variability to morphine treatment. Individual responses to morphine are influenced by various gene-encoded-proteins implied in pharmacokinetics and pharmacodynamics. Variants of P-glycoprotein encoding gene ABCB1 regulate transportation and distribution of morphine and affect analgesic effect. Diversity in UDP-glucuronosyl transferase encoding gene UGT2B7, whose encoding product catalyzing morphine to glycosylated metabolites contribute to different response to morphine in a pharmacokinetic way. Nevertheless, variants in μ-opioid receptor encoding gene OPRM1 and catechol-O-methyltransferase encoding gene COMT regulate morphine-induced downstream signaling and influence morphine analgesia in a pharmacodynamic way. It is necessary to employ individuals with more complex genetic diversity and screen in a larger scope through a more comprehensive system to find the key genes involved in individual differences of morphine analgesia in future research. Elucidating the association between genetic variability and individual differences will help to figure out the mechanism of pharmacogenetic regulation in morphine analgesia. It will provide basis for personalized and accurate utility of morphine or even combining with gene therapy to improve the analgesic effect.

Abstract:Animal model is an animal material with human mimic performance established in biomedical scientific research. It can be used as experimental basis for studies of experimental hypothesis and clinical hypothesis. It can shorten the research time and observe the whole process of disease occurrence, development or prevention and treatment.Human biomedical research is largely limited by the biological complexity. In order to overcome this limitation, based on the immunosuppressive characteristics of a severely immunodeficient (SCID) or recombinant activated gene (Rag^null) in mice, humanized mouse models of human diseases can be established and have been widely used to study the underlying principles of human immunobiology and complex pathological mechanisms of human diseases. This approach has become one of the important ways to promote the development of medical sciences, with practicality and foresight. In this paper, the application and research progress of humanized mouse models are reviewed.

Abstract:Biosafety hazard has been paid high attention with increasing use of animal species and quantities in medical colleges. In the present paper, we analyze potential biosafety risks in medical colleges from characteristics of biosafety environment,experiment activity,zoonosis,laboratory animal management,etc., then give some advices on prevention and control of biosafety hazards.

Abstract:Objective Mutated inbred animal model is introduced to the practical course of genetic diagnosis in the hope that medical students are able to apply what they have learned to clinical cases, based on a deep understanding of principle and technology on gene mutation detection. Methods We integrated DNA extraction, polymerase chain reaction, agarose gel electrophoresis, and gel imaging analysis into a comprehensive experiment and arranged 4-year-programme undergraduates majoring in preclinical medical sciences to conduct it with the purpose of investigating the internal relations between phenotype and genotype in a hairless Uncv mouse model. Subsequently, the questionnaire aimed at evaluating learning effect on the part of students was handed out and their feedbacks were analyzed. Results More than 90% of respondents are satisfied with the general learning effect. Especially, 98.7% of students support the enhancing effect of the new teaching mode on their research skills and 96% consider the practical course helpful to their problem-solving ability. Conclusions The introduction of mutated inbred animal model to the practical system of molecular diagnostics proves beneficial to boost students' learning effect and scientific research quality. Our practice also provokes thoughts on the further utilization of animal models in teaching system of medical sciences.