Enhancement Of Ultrasound Targeted Microbubbles Destruction Mediated Angiogenesis With Nuclear Localization Signal Petide In Canine Myocardial Infarction

To improve the canines myocardial infarction curative effect by using ultrasound targeted microbubble destruction(UTMD)combining with nuclear localization signal(NLS)peptide to increase hAng-1 gene transfection efficiency,and evaluate its application and feasibility.MethodFifty canines were randomly divided to 5 groups after the models of myocardial infarction were prepared.Group A:blank control group(n=10),group B:UTMD+pDNA(n=10),group C:UTMD+pDNA+cNLS(n=10),group D:UTMD+pDNA+mNLS(n=10),group E:UTMD+pDNA+cNLS+WGA(n=10).The therapeutic agents were injected by intravenous one week after myocardial infarction before ultrasonic irradiated at the precardium with group B?C?D?E.? Three days after gene transfection,the hAng-1 gene expression was detected in the frozen sections of the myocardium,the expression of hAng-1 was detected by RT-PCR and Western Blot from the gene and protein level.? Twenty-eight days after gene transfection,a-SMA immunofluorescence and were used to detect capillary density of peri-infarct area and microvessel density(MVD)was detected.The microvessel density was counted under the microscope.?Masson's trichromatic staining and gross specimen were used to evaluate the degree and the area of myocardial fibrosis.?Echocardiography was used before and one week after myocardial infarction and 28 days after gene transfection.Two-dimensional echocardiography was used to detect the cardiac left ventricular ejection fraction,the left ventricular wall motion and the myocardial contrast echocardiography was used to detect myocardial perfusion of all canines in the four groups.Evaluated and compared the gene transfection efficiency and the curative effect of all the four groups.? Collected blood samples through elbow vein before myocardial infarction(pre-MI),4h after gene transfection(MI-4h),12h after gene transfection(MI-12h)?24h after gene transfection(MI-24h)?48h after gene transfection(MI-48h)and 7d after gene transfection(MI-7d).Detected the content of cTnI.Results(1)Myocardial infarction model was successfully established using gelatin sponge embolization.(2)Myocardial frozen section and RT-PCR and Western Blot results showed that the green fluorescent protein and the expression of hAng-1 gene and protein were significantly higher in the C group than in other groups(p<0.05).(3)Immunofluorescence showed the capillary density of group C were the highest,the differences of group C was statistically significant compared with other groups(p<0.01).(4)Masson's trichromatic staining and cardiac gross specimen showed that the degree and area of myocardial fibrosis were reduced in an order of group A,B,C and increased in an order of group C,D,E.(5)Before myocardial infarction,four groups of canine ventricular wall motion and cardiac function were normal,myocardial filling defect was not showed by myocardial contrast echocardiography.One week after myocardial infarction,the left ventricular wall motion amplitude and the left ventricular ejection fraction of the four groups were significantly decreased.Myocardial contrast echocardiography showed anterior and interval walls myocardial filling defect.Twenty-eight days after gene transfection the left ventricular ejection fraction of group C were slightly increased and slightly decreased in B and D groups while significantly decreased in A and E groups.Myocardial contrast echocardiography showed much contrast filling in the infarction and surrounding area in group C and a little contrast filling in B and D groups while filling defect in A and E groups,there was significant difference comparing group C with other groups separately(p<0.01).(6)The level of cTnI in each group showed the same trend.It increased at 4 hrs after MI induction and reached the peak level 12-24 hrs after MI and reached the normal range on day 7 after surgery,which showed no statistical differences among the five groups(p>0.05).ConclusionThe present study shows that this method of UTMD combined with NLS can be used to deliver plasmid DNA to the myocardium selectively and effectively,and it can promote neovascularization and improve myocardial fibrosis.This noninvasive technique is a promising method for cardiac gene therapy.