The aim of gene therapy for coronary heart disease (CHD) is to enhance new blood vessel formation,so that the small vessels of myocardium can regeneration and collateral circulation can replace the function of emphraxised vessele,this is so-called "self-bridge" of ischamia myocardium. Works from animal model and clinical investigation demonstrated that vessel growth factors such as VEGF can make new vessels re-exist and upregulate myocardium's blood flow perfusion. But how to apply these growth factors is very difficult;veinal injection is convenient but impossible,because it may affect other organs and this will lead to bad side-effect. But if growth factors applied through coronary artery or myocardium injection directly,despite it can obtain good location expression,will be difficult to manipulate and often bring wound. Therefore,to establish a safe,efficient and non-traumatic method transfering growth factor to the reasonable spot is the best way. And gene therapy by ultrasound mediated is the hopeful method to realize the purpose. Ultrasound can make drugs release from microbubble that combined drugs in vitro and prepared in advance,what' s more ultrasound will not cause any injury. In basic research,microbubble system and ultrasound have already applied to transform therapeutic gene to the damaged spot caused by CHD. In this study,we prepared microbubbles containing TSP-l(angiogenesis inhibiting gene) type I repeat and its antisense RNA,and transform them to the ischemia spot of rat's myocardium by ultrasound,investigated the formation of new vessels,in order to analyze this new method's effect on the gene cure of CHD.Methods and Results:1. Construct recombinant vectors with interested gene-pcDNA3.1+/TSP-1-I and pcDNA3.1-/anti-TSP-1-I. The recombinant vectors identified by double restriction analyzing,DNA sequencing and Western Blotting,and then tansfected the cell line-ECV304. The growth activity of ECV304 was detected by MTT method,and the proliferation was detected by Flow Cvtometer(FCM);Cell morphological change,by transmission electron microscope (TEM). Results:after transfected by pcDNA3.1+/TSP-1-I,the growth activity of ECV304 (MTT OD) is higher than the blank (without transfection) and negative (transfect zero load vectors) controls (P=0.03 and P=0.023);while collected superum from ECV304 with pcDNA3.1+/TSP-1-I and added into another ECV304 cells,48 hours later MTT OD of the latter cells is lower than the blank and negative controls (P=0.004 P=0.016),and the MTT OD continue to decrease with concentration increasing. By the detection of FCM,the number of cells in S+G2 phase of pcDNA3.1+/TSP-1-I tranfected ECV304 is lower than the blank and negative controls (P=0.03 P=0.011). By investigation of TEM,there are chromatin agglutination,nucleus fragments and apoptotic bodies in ECV304 tranfected by pcDNA3.1+/TSP-1-I. When ECV304 transfected by pcDN3.1-/anti-TSP-1-I,MTT OD was higher than the blank and negative controls,and even the ECV304 tranfected by pcDNA3.1+/TSP-1-I(P=0.002,P=0.002 and P<0.001);48 hours later,the results is the same (P<0.001,P<0.OOland P<0.001). The cells in S+G2 phase of ECV304 transfected by pcDNA3.1-/anti-TSP-1-I increased with the clear differences against the blank,the negative and the group of pcDNA3.1+/TSP-1-I transfection (P=0.001,P=0.002 and P<0.001). By TEM,the nucleole increased in the group of pcDNA3.1-/anti-TSP-1-I transfection.2. Liposomes,liposome microbubbles were constructand. Liposome microbubble containing LacZ gene was transfected into ECV304 cell by ultrasound. Transfection rate was detected by B-Gal staining kit. Expression level was detected by B-Gal assay kit. Results:the diameter of liposomemicrobubble (95.84%) is no more than Sum,and concentration,5.5x109 /ml. The microbubbles has good effect of Myocardial imaging,with the time of imaging,7 minuites. After LacZ-carrying liposome microbubble transfected into ECV304 cell by ultrasound applied,p-Gal staining showed that tranfection rate of ultrasound + liposome microbubble + LacZ is h...
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