| ObjectiveMediated hTK-1 and hTIMP-1 gene were transfected with recombinant adenovirus vector to rat myocardial infarction model by coronary artery ligation,and we observed the single gene vector(TK-1 or TIMP-1)or double gene co-expression vector(TK-1 and TIMP-1)on inflammatory response after acute myocardial infarction and its possible mechanism.Methods1?In this study,the model of myocardial infarction was established by the method of coronary artery ligation.250-300 g male Sprague Dawley(SD)rats were randomly divided into sham operation group and coronary artery ligation group.We observed the electrocardiogram of rats during operation.Then rats were fed for 7 days postoperative,detected heart function by heart ultrasound.Rats were sacrificed,and we got their myocardial tissue,used HE staining to observe the myocardial infarction area of inflammation,used TTC staining to observe the area of myocardial infarction.2 ? Applying the classical coronary artery ligation method to build models of myocardial infarction.After coronary artery ligation in SD rats,250-300 g male SD rats were randomly divided into 6 groups: sham operation group,PBS group,control virus group,TK-1 group,TIMP-1 group,double genes group.Except the sham operation group,the other groups were lagaitoned coronary artery to cause myocardial infarction with mousse line,myocardial infarction area in rats peripheral injection of saline(100?l),control virus vector,TK-1 vector,TIMP-1 vector,association gene vector(1x1010pfu/ per rat,virus stored in 100?l dilutions).28 days after operation,we used echocardiography to test heart function in rats.Rats were sacrificed and got the left ventricle,and detection of co-expression of double gene by Immunofluorescence confocal,and HE staining was used to detect the Inflammatory infiltration of myocardial infarction,and TTC staining was used to analyzed the size of myocardial infarct,and myocardial apoptosis was examed by TUNEL,and detection of expression level of MIF,NF-?B,MMP-9 and MMP-2 by immunohistochemical,and western blot was used to analyzed TK-1,TIMP-1,MIF,NF-?B,MMP-2 and MMP-9 protein.Results1.During surgery,we used the standard two lead electrocardiogram(ECG)to observe ECG of rats.After ligation of the left anterior descending branch of coronary artery,we saw theST segment up,and R wave and T wave were fused into tent wave.The successful establishment of rat myocardial infarction model by coronary artery ligation method(Part 1).Postoperative 7 days,left ventricule with HE staining shows the inflammatory infiltration in the sham operation group was significantly less than in myocardial infarction group.The cardiac function in sham operation group was better than in myocardial infarction group(P<0.01).The survival rate of model of myocardial infarction was about 80%.2.Immunofluorescence staining was performed on the myocardium of double genes rats.Red fluorescence and green fluorescence were observed under confocal microscopy,indicating that TK-1 and TIMP-1 genes were successfully transfected.By western blot to detect the expression of TIMP-1 protein and TK-1 protein in myocardial tissues,results showed that only TK-1 group and double gene group had TK-1 protein expression,and only TIMP-1 group and double gene group had TIMP-1 protein expression,and the difference of protein expression between single gene group and double gene group was not statistically significant(P>0.05).The other groups were no TK-1 and TIMP-1 protein expression.3.28 days after the operation,the heart function and infarct size of rats: compared with the sham group,LVIDs and LVIDs increased,and FS and EF decreased in PBS group(P<0.05),and inflammatory cell infiltration was obvious,and the infarct size was larger(P<0.05).There was no significant difference between PBS group and control virus group(P>0.05).Compared with PBS group,LVIDd decreased but the difference was not statistically significant(P>0.05),and LVIDs decreased,and FS and EF increased,and the size of myocardial infarction and myocardial apoptosis index decreased in TK-1 group or TIMP-1 group or double genes group(P<0.05).Compared with TK-1 group or TIMP-1 group,LVIDd decreased,and FS and EF increased,and infarct size and myocardial apoptosis index decreased significantly in double genes group(P<0.05).4.28 days after the operation,the protein expression ofinflammatory factors(MMP-2 and MMP-9)and NF-?B,MIF in myocardial tissue: compared with sham group,the expression of inflammatory factors and NF-?B,MIF were significantly increased in PBS group(P<0.05).There was no significant difference between PBS group and control virus group(P>0.05).Compared with PBS group,expression of inflammatory factors and NF-?B,MIF decreased significantly in TK-1 group or TIMP-1 group or double genes group(P<0.05).Compared with TK-1 group or TIMP-1 group,the expression of inflammatory factors and NF-?B,MIF decreased in double genes group(P<0.05).Conclusion1.The successful construction of rat myocardial infarction model,and local injection with single gene recombinant adenovirus mediated over expression vector(TK-1 or TIMP-1)or co-expression of double genes vector(TK-1 and TIMP-1),both can be successfully transfected into myocardial tissue and cells,and achieve high expression.2.Compared with single gene(TK-1 or TIMP-1),double genes(TK-1 and TIMP-1)are more significant in reducing infarct size,inhibiting myocardial apoptosis and improving cardiac function after myocardial infarction in rats.3.Double genes(TK-1 and TIMP-1)improved ventricular remodeling effect in myocardial infarction rats,may be due to TK-1 and TIMP-1 protein could inhibit inflammatory pathway of MIF / NF-?B / MMP-2,MMP-9,which may present synergistic or superimposed effects. |
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