| Background and Objective: It is important that resume blood stream perfusion of ischemic myocardium in treating ischemic heart disease such as myocardium infarction, and researchers have been making efforts during past few years to develop gene therapy for revascu1arization, relieving symptoms, and ameliorating prognosis.The key factor of gene therapy is gene transfection and expression. Nudity DNA is a more safe transfusion method than viral vector, but with a poor transfection efficiency and therapeutic efficacy. Recent studies have proved that ultrasound could increase gene expression obviously, but there were some deficiencies of ultrasound wave irradiate heart in vitro because of the sharp attenuate of ultrasound energy. In addition, it is necessary to find a simple and safe method for gene introduction in clinical application.Therefore, we hypothesized to make interventional catheter technology and ultrasound therapy integrated to compose a novel ultrasound catheter system. This system has an injection needle and a mini-ultrasound transducer on the top of catheter, which can be inserted into ventricular chamber for percutaneous intramyocardial delivery of gene, and intracardiac ultrasound irradiation for facilitating uptake of therapeutic gene and avoiding acoustic window interference. Besides, ultrasound contrast agents (microbubbles) have been used for gene carriers and enhance ultrasound efficiency except for diagnostic imaging, so we will also observe the efficiency of directly intramyocardial injection microbubble.Methods:1 Interventional Experiment of Normal Dogs Seventeen dogs were randomly divided into five groups(n=3-4):①EGFP plasmid administration in combination with microbubble and intracardiac ultrasound irradiation (EGFP/MB/US);②EGFP/US;③EGFP/MB;④EGFP gene alone;⑤blank group.The ultrasound catheter tip was passed through the aortic valve and into the target area of left ventricle by a 9F arterial sheath under fluoroscopic and echocardiography guidance. In the first and second groups, the ultrasound catheter was used to inject 0.1 ml of microbubble attaching to EGFP gene or EGFP gene alone, then insonation for 60s at 4.3MHz/1.0 W/cm2. The other two groups received the same media injection without ultrasound irradiation. The blank group was injected with normal saline without EGFP gene and irradiation.Thus, we observed:①the feasibility and safety of catheter-based transendocardial injection and intracardiac ultrasound insonation in normal myocardium;②the local deposition and diffusion of a microbubble loading gene from the injection site into myocardial tissue under the real time echocardiography guidance. Forty-eight hours later,③the expression amount of EGFP mRNA and protein in myocardium were measured by RT-PCR technique and laser confocal microscope;④whether gene expression is at higher level underwent intracardiac insonation or combined with intramyocardium injection microbubble were evaluate;⑤the biological effect and safety of intracardiac ultrasound irradiating myocardium were explored;⑥and the biomechanism of intracardiac ultrasound exposure enhancing gene expression was analyzed.2 Interventional Experiment of MI DogsMI was made in dogs by legation of the left anterior descending coronary artery. Dogs treated with HGF were randomized divided into three groups (n=3-4), which were①HGF/MB/US,②HGF/US,③HGF alone. Besides,④Sham group,⑤MI group without therapy.In the first and second groups, the ultrasound catheter was used to inject 0.1 ml of microbubble attaching to HGF gene or HGF gene alone and insonation for 60s at 4.3MHz/1.0 W/cm2 after injection. The third group were received the same HGF injection without ultrasound irradiation, and the others were control group without HGF gene therapy and ultrasound irradiation.Twenty-eight days later,①the expression amount of HGF mRNA and protein in ischemic myocardium were detected;②the infact size, total and regional ventricular weights, hydroproline content (HC) and collagen volume fraction (CVF) of type I and III of all the five groups were recorded.③the expression of VEGF, CD31 was detected and the microvascular density was evaluated.Results:①the ultrasound catheter system could be navigated to the ventricle chamber of dogs by intervention technology, deliver therapeutic solution to the border of interested area, and no serious adverse events occurred during the injection and intracardiac insonation procedure;②the image indication, distribution of microbubble loading gene in myocardium could be observed by transthoracic echocardiography;③compared with the alone EGFP injection group, the EGFP mRNA expression of EGFP/MB/US group was about 8 times higher, the fluorescence intensity was 8.6 times higher(P<0.01);and of the EGFP/US group, those were about 6 times and 5.1 times higher respectively(P<0.01);there were no statistic differences between the EGFP/MB group and alone EGFP group, (P>0.05).④expression of EGFP occurred only in the injected myocardium, and nowhere else found EGFP deposition. All the dogs'liver, kidney and lung did not be found abnormalities pathology alteration. No detectable levels of EGFP were found in blank group;⑤HE dyeing showed that lightly congestion of myocardium occurred after intracardiac ultrasound irradiation. With transmission electron microscopic investigation, we found red blood cells oozed from the capillaries in dogs of intracaidac ultrasound irradiation group, also did the permeability of biomembrane. All these biological effects were more conspicuous in the group of MB combined with US;⑥VEGF and HSP70 expression were found in some dogs; the EGFP/MB/US group had the highest VEGF and HSP70 expression, next is the EGFP/US group, the less in EGFP/MB group(P<0.01), and no VEGF and HSP70 expression in alone EGFP group or blank group;⑦the expression of EGFP mRNA and VEGF , HSP70 mRNA were positive correlation in the EGFP/MB/US group, and the correlation coefficient were 0.76 and 0.75 respectivly(P<0.05); which was the same to the EGFP/US group, but the correlation coefficient were 0.75 and 0.77 respectivly (P<0.05). There was no correlation in that of MB/EGFP group.In the MI dogs'interventional experiment:①the expression of HGF gene and protein in ischemic myocardium were increased after intracardiac ultrasound irradiation. Compared with the alone HGF group, the expression of HGF mRNA in HGF/MB/US group was about 6 times higher, and the protein was about 4.7 times higher; and of the HGF/US group, those were about 4 times higher and 3.3 times higher respectively(P<0.01).②HGF gene therapy could simultaneously up-regulate the expression of CD31 and VEGF mRNA, and increase angiogenesis of ischemic myocardium;③the HGF gene therapy could enhance the resuming of capillary vessel of MI dogs, ameliorate LV remodeling, decrease the HC and the CVF of type I and III. The therapeutic effectiveness of MB/HGF/US group was the best, and the next was the HGF/US group. The HGF group wasn't so good as the former two HGF gene therapy groups (P<0.01).Conclusion: The ultrasound catheter system is safe and reliable.Using this catheter system, transgenes can be effectively transfected into myocardial sites and gene expression enhanced by intracardiac ultrasonic irradiation. Except for enhancing the permeability of vascular and biomembrane, intracardiac ultrasonic irradiation can also obviously increase the expression of exogenous EGFP plasmid in normal myocardium and HGF plasmid in ischemic myocardium, especially at the site of microbubble combination gene injection. The distribution and diffusion of the microbubble loading gene in the myocardium can be visualized by echocardiography. The biological effect of ultrasound such as cavitation, heat energy and mechanical vibration may be the comprehend reason for intracardiac ultrasound irradiation increasing gene expression. The HGF gene transfer can provide partly cardioprotective effects by enhancing new blood vessel formation in ischamia myocardium and amelioratind LV remodeling in MI dogs.
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