| Objective: Investigating the expression of Zinc finger protein 580(ZFP580)and endothelial NOS(eNOS)in the mouse model of myocardial ischemia,to provide new experimental evidence for elucidating the pathogenesis of ischemic diseases.Method:1 50 adult male mice were randomly divided into five groups:(1)normal control group(NC group),(2)isoproterenol(ISO),0.25mg/kg group,(3)ISO,1mg/kg group,(4)ISO,2.5mg/kg group,(5)ISO,5mg/kg group,10 in each group.Administered ISO by intraperitoneal injections.After injection of ISO5 minutes,BL-420 captured mice lead ECG?.When found waveform in st-elevation,T wave high,show the typical myocardial ischemia ECG waveform,prove successful,and then follow-up experiments.Respectively picked eyeballs bleed before put to death.Control mice after intraperitoneal injection of equal parts salt water method executed.2 Detection of serum CK and LDH activity: 3000r/min centrifuged serum,according to kit instructions spectrophotometer detected activity of LDH and CK.3 Myocardial tissue morphological examination: apex organization for fixation,dehydration,embedding,which was made of tissue section observation,changes of the acquisition of ischemic myocardium in form.4 Quantitative RT-PCR expression ZFP580mRNA: apex myocardial tissue tatal RNA was extacted using Trizol reagent.Preparation of total RNA quantification(400?g)system of 10?l cDNA synthesis RT first chain.Taked quantitative cDNA template(10?l)was added the target gene ZFP580 primer.With ?-actin as an internal reference,compared the relative expression of the gene ZFP580.5 Myocardial immunohistochemical staining: myocardial tissue paraffin sections were dewaxed.After using HE and immunohistochemical staining,myocardial cells was observed under an optical microscope coloring cases.Each slide be positive cell counts at least 200 cells.The positive rate with the percentage of positive cells accounted form the same field of view expressed cardiac cells.The positive rate of the average taked per slide positive rate of different horizons.Negative control: with 0.01mol/L PBS instead of primary antibody.6 Statistical analysis: data expressed as SX ?,SPSS11.5 software was used for statistical analysis.Differences between groups was analyzed by one-way ANOVA,there were significant differences by using the StudengtNewmen-Kuele q test for pairwise comparison.Result:1 Effect of ISO on mouse ST segment and T wave in ECG and effect of serum LDH and CK activity.After peritoneal injection in ISO,mice showed typical ischemic ECG changes.That Indicated myocardial ischemia model in mice was successful.Compared with the control group,model mice serum LDH and CK activity markedly elevated.2 Effect of ISO on morphology of myocardium in mice.Masson staining showed normal control group myocardial cells arranged in neat rows,myocardial been dyed yellow.After the injection ISO,myocardial fibers derangement sarcoplasmic swelling,endocardial,epicardial myocardial interstitial and there were many scatter Irregular necrosis foci,significant inflammatory cell infiltration,ischemic myocardium was stained red.3 The expression of ZFP580 and eNOS in ischemic myocardial cells.ZFP580 and eNOS in myocardial ischemia injury in the expression are lower,prompt ZFP580 and eNOS in myocardial ischemia exert the function of a certain regulation,on myocardial protection.At the same time,the results showed that the ISO ZFP580 transcription and translation could affect myocardial cells,its specific mechanism is not fully clear.4 ZFP580 localization in the nucleus and cytoplasm,yellow staining cellsrepresent positive reaction,in experimental mice myocardial ZFP580 positive cells was significantly lower in proportion.All appear brownish-yellow granules in the cytoplasm of eNOS protein staining positive cells,compared with the control group is significantly lower.Conclusion: Mouse model of myocardial ischemia in isoproterenol induced expression of ZFP580 reduced with myocardial ischemia injury. |
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