Effect of Map3k1 gene down regulation on proliferation and migration abilities of B 6 mouse eyelid keratinocytes / 重庆医学

Objective To study the effect of targeted inhibition of mitogen-activated protein kinase kinase kinase 1(Map3k1) gene on the cell proliferation and migration abilities of B6 mouse eyelid keratinocytes.Methods An artificial microRNA(amiRNA) interference vector targeting silent Map3k1 gene was constructed in vitro in the test.Lipofectamin 2000 was used to transfect the B6 mouse eyelid keratinocytes(the Ctrl Map3k1 group was transfected with empty vector,while the Map3k1 amiRNA-3 group was transfected with Map3k1 amiRNA-3 interference vector).The Map3k1 mRNA and protein expression levels were respectively de-tected by real-time PCR and Western-blot for determining the interference efficiency.The B6 mouse eyelid keratinocytes prolifera-tion level was detected by MTT.The migration ability of keratinocytes was detected by the scratch experiment.Results After the keratinocytes were transfected with Map3k1 amiRNA interference vector,the levels of Map3k1 mRNA and protein were effectively inhibited,and the interference efficiency was up to 70%(P<0.05).The proliferation level of keratinocytes in the Map3k1 amiRNA-3 group was lower than that in the Ctrl Map3k1 group(P<0.05).The migratory ability of keratinocytes in the Map3k1 amiRNA-3 group was also significantly lower than that in the Ctrl Map3k1 group(P<0.05).Conclusion Targeted inhibition of Map3k1 gene expression in B6 mouse eyelid keratinocytes significantly inhibits cell proliferation and migration,thus influence the cellular bi-ological behaviors.

Potential Vaccine Targets against Rabbit Coccidiosis by Immunoproteomic Analysis

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.

Establishment of cerebral infarction model in rabbits induced by photochemical injury / 中国组织工程研究

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.