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Objective To study the effect of microRNA-204 (miR-204) on the biological characteristics of breast cancer cells.Methods Real-time PCR was used to detect the expression of miR-204 in human breast cancer cell MDA-MB-231 after transfection of miR-204 mimics and inhibitor for 48 h.Flow cytometry was used to analyse the effect of miR-204 on the proliferation and apoptosis of MDA-MB-231 cells.The effect of miR-204 on the migration of MDA-MB-231 cells was detected by Transwell migration assay.Results Real-time PCR analysis showed that miR-204 mimics and inhibitors had significant effect compared with normal control group(P<0.01).Flow cytometry analysis showed that compared with normal control group,the number of G1 phase cells of miR-204 mimics group was significantly decreased(P<0.01),while the number of G2/M cells of miR-204 mimics group was significantly increased(P<0.01).In contrast,the number of G1 phase cells of miR-204 inhibitor group was significantly increased(P<0.01),while the number of G2/M cells of miR-204 inhibitor group was significantly decreased(P<0.01).miR-204 mimics group significantly promoted apoptosis,while the inhibitor group significantly inhibited apoptosis(P<0.01).Transwell migration analysis showed that the number of cells of miR-204 mimics group were significantly reduced,while the number of cells was significantly increased in the inhibitor group(P<0.01).Conclusion We find miR-204,which can promote cell apoptosis and inhibit cell proliferation and migration,is a negative factor in the breast cancer cell line MDA-MB-231.
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The aim of this study was to identify antigens for a vaccine or drug target to control rabbit coccidiosis. A combination of 2-dimensional electrophoresis, immunoblotting, and mass spectrometric analysis were used to identify novel antigens from the sporozoites of Eimeria stiedae. Protein spots were recognized by the sera of New Zealand rabbits infected artificially with E. stiedae. The proteins were characterized by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/TOF-MS) analysis in combination with bioinformatics. Approximately 868 protein spots were detected by silver-staining, and a total of 41 immunoreactive protein spots were recognized by anti-E. stiedae sera. Finally, 23 protein spots were successfully identified. The proteins such as heat shock protein 70 and aspartyl protease may have potential as immunodiagnostic or vaccine antigens. The immunoreactive proteins were found to possess a wide range of biological functions. This study is the first to report the proteins recognized by sera of infected rabbits with E. stiedae, which might be helpful in identifying potential targets for vaccine development to control rabbit coccidiosis.
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Rabbits , Coccidiosis , Computational Biology , Eimeria , Electrophoresis , HSP70 Heat-Shock Proteins , Immunoblotting , Mass Spectrometry , SporozoitesABSTRACT
Background Mutant C57BL/6 mouse with corneal opacity (B6-Co) appears eye open at birth (EOB) phenotype,which is a good animal model in the study of developmental mechanism of eyelid.Investigating the relationship between serum response factor (SRF) and EOB phenotype can provide theoretical support for the research on the mechanism of innate defects in eyelid development in humans.Objective This study was to assess the dynamic expressions of SRF in eyelid of embryonic B6-Co mouse.Methods Total RNA was extracted from B6 and B6-Co mice eyelid tissue at embryonic day 16.5 (E16.5 d),E17.5 d and E18.5 d.The relative expression levels of SRF mRNA and protein in the eyelid tissue of B6 and B6-Co embryonic mice were assayed by real-time quantitative PCR and Western blot,respectively.In situ expressions of SRF protein in eyelid of B6-Co mice and B6 mice were detected using immunofluorescence technique.The use and care of the animals complied with the Regulation for the Administration of Affair Concerning Experimental Animals of Nantong University.Results The relative expression levels ofSRF mRNA in the eyelids were 0.41±0.06 and 0.24±0.17 in E16.5 d and E17.5 d of B6-Co mice,showing a significant decline in comparison with 1.03 ±0.17 and 1.01 ±0.09 in the B6 mice (P =0.025,0.017).The expression levels of SRF protein in the eyelids of E16.5 d and E17.5 d B6-Co mice were 0.08±0.01 and 0.08± 0.01,which were significantly lower than 0.12 ±0.03 and 0.13 ± 0.02 of B6 mice (P =0.036,0.024).However,there were no significant differences in the expression levels of SRF mRNA and protein in E18.5 d between the B6-Co mice and B6 mice (P =0.387,0.774).Immunofluorescence assay displayed that SRF was expressed in the keratinocytes of eyelids in both mice,but the fluorescence intensity was weaker in the B6-Co mice.Conclusions SRF probably interrupts the developing process of eyelid in early embryo of B6-Co mice.
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Objective To study the clone sequencing and expression of fibroblast growth factor 10(Fgf10) gene in corneal opaci-ty (B6-Co) mouse .Methods Normal mice mate with B6-Co mice ,the skin tissue separation from B6 and B6-Co mice at embryo 16 . 5 d ,total RNA extraction and reverse transcription ,the target gene was fragment amplification by RT-PCR ,connection with T vec-tor ,transformed to competent cells ,selection positive clone ,sequencing analysis .The gene expression in B6-Co mice was detected by real-time PCR .Results The base A inserted between 1 914 and 1 915 in Fgf10 gene by sequencing .The expression of Fgf10 was significant down regulation in B6-Co mice by real-time PCR(P< 0 .05) .Conclusion Fgf10 is relevant with phenotype of B6-Co mouse ,and the regulation mechanism was expected further study .
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Background An ethylnitrosourea(ENU)-induced mutant strain C57BL/6 mouse model has been established by our research group.This model is proved to have the spontaneous phenotype of corneal opacity and the typical pathological process similar to human keratitis.Therefore,this model is expected to be a good animal model in the research of the mechanism,hereditary property,and development of drugs for corneal infectious diseases.Objective The present study is to investigate the biological features of opportunistic pathogens using a mouse Staphylococcus-infected corneal model(C57BL/6 mouse) induced by N-ethyl-N-nitrosourea(ENU),and offers an evidence of stability in this animal model.Methods Ten-week-old male C57BL/6 mice were treated with ENU at 150mg/kg by intraperitoneal injection,and then mated with female mice after 60 days.Corneal opacity mutant mice in the F1 generation were selected to backcross with C57BL/6 mice.The bacteria were isolated from the eyeballs of the mutants and cultivated,purified and identified.Drug sensitivity assay was carried out to screen for effective antibiotics for clinic medical care.Results The staphylococcus-infected corneal mouse model(B6-Co) was established successfully,and the Staphylococcus sciuri strain was separated and purified,and then the sensitive antibiotics were distinguished from resistant ones.The sensitive drugs for Staphylococcus sciuri included azithromycin,clindamycin,chloramphenicol,gentamicin,rifampicin,tetracycline,amikacin,sulfamethoxazole compound sinomin,minocycline,levofloxacin,cephalothin,cefotaxime,and furazolidone;whereas this Staphylococcal strain was resistant to cefoxitin,penicillin,ampicillin,novobiocin.Nitrofurantoin showed an intermediate sensitivity.Conclusion The C57BL/6 mouse model is a spontaneous-derived animal model that is infected by coagulase-negative staphylococci,among which the most abundant strain is Staphylococcus sciuri.
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BACKGROUND: Cerebral infarctional animal model provide basis for studying human cerebral infarction(CI). There are two traditional CI models, one is reproduced by craniotomy or electro-coagulation by which supplying artery are blocked, another is achieved by embolus or water gelatin micro-thrombosis. But both are difficult to perform and results were instable, which limit the application. Photochemical injury is a novel way to reproduce CI model on experimental animals.OBJECTIVE: To explore a new method of experimental research of local cerebral infarction model which is induced by photochemical injury in rabbits.DESIGN: Single sample studySETTING: Experimental Animal Center of Nantong University.MATERIALS: This study was conducted at the Experimental Animal Center of Nantong University from May to December 2003 (secondary laboratory). Totally 63 Japanese flap-eared rabbits, with birth age of 10-12 month, 33 females and 30 males, with body mass of 1.7-3.3 kg, were randomly selected.METHODS: After anaesthetized, rabbits were cut at the skin for 2 cm long at the crossing of skull center and posterior canthus, skull was exposed and periosteum was separated, then a round skull window with diameter of 0.5 cm was drilled at 0.5 cm left or (right) to sagittal suture and 0.5 cm posterior to coronal suture, after that, 35 g/L rose Bengal was slowly injected from ear-edge vein in dosage of 1 mL/kg by once. About 3minutes later, cold light source (wave length of 540 nm, power of 140 lx)was used to cast light directly onto the skull window for consecutively 8minutes, then incision was sutured. At postoperative 24 hours, neurological defects were scored in five grades [0 score represent no neural impairments; 1 score: the left posterior limbs displayed decreased muscular tension and attenuated contraction reflex; 2 scores: the left posterior limbs were paralyzed, displaying obvious abduction; 3 scores: rabbit displayed obvious adductive drag with body leant to the opposite side; 4 scores: unable to walk and unconsciousness], rabbits were put to death at postoperative 48 hours, infarctional area and volume were determined and pathological changes was also observed.MAIN OUTCOME MEASURES: Limb movement, infarctional area and volume and pathological changes.RESULTS: CI mode was successfully established on 59 rabbits, the sucmean infarctional area was (0.465±0.012) cm2, and the mean volume changes: Infarctional focus displayed typical pathological changes such as impairment, effusion and inflammation. Gentle impairment could be observed in 22 rabbits (37%), medium in 32 rabbits (54%) and severer in 5rabbits (9%).infarction model has multiple advantages, such as easy performance, quick and good repeatability, it can be used to reproduce experimental models for for a long time with low mortality, benefiting for researches on chronic tional size and depth are under control, meeting the need of researches on observed in photochemical injury, which provide basis for study on the efBut there was still some limitations: Since thrombosis was induced at the terminal artery, unfit for the study of lateral circulation and reperfusion;however it was found more similar to human microvascular diseases, thereby incapable of explaining the pathogenesis of other ischemic strokes.