【作者】 袁侨英；

【导师】 黄晶；

【作者基本信息】 重庆医科大学 ， 内科学， 2007， 博士

【摘要】 背景和目的:近年,国内外学者尝试用基因治疗恢复心肌梗死(MI)病人的血流灌注、改善预后。基因的转染表达是治疗关键,基因直接以裸露DNA的形式转移比病毒载体安全,但缺点是转染和表达效率低,难以达到治疗效果。研究发现,超声的生物物理效应可明显促进基因转染表达,但用传统的体外超声辐照心脏,因声窗等因素影响,最终到达心肌的声能量、辐照效果难以准确控制。另外,在基因治疗缺血性心脏病的临床运用中,需要建立安全简便的基因靶向导入方法。针对基因治疗效应性、投递途径等存在的问题,本研究将基因治疗与介入超声结合,在介入导管顶端安装超声换能器,并配置投送基因的特制微型注射针,研制成超声导管:深入心腔内部经导管中微型针将基因注射到心肌局部,之后在心腔内发射超声近距离辐照心肌促进基因转染表达;实验中,我们也观察超声造影剂微泡(MB)携基因直接注射到心肌后的成像效果,探讨微泡携基因直接注射到心肌后协同心腔内超声辐照对基因表达的影响。方法:资助渠道:国家自然科学基金:心腔内超声调控基因表达的实验及分子机制研究(10604068)1、正常活体犬介入实验:犬17只,分为5组(n=3-4)分别为:①绿色荧光蛋白裸质粒(EGFP)+ MB +心腔内超声辐照组(US),②EGFP + US,③EGFP + MB,④单纯EGFP注射组,⑤空白对照组(注射等量生理盐水)。在X线影像引导下,超声导管经左颈总动脉的9F血管鞘进入犬左心室腔,经微型针分5个点向心肌注入MB和/或EGFP的混合液,每个注射点注射量为100μL(含EGFP100ug), EGFP/MB/US及EGFP/US组在注射完毕后,即刻以超声导管在心腔内发射超声辐照每个注射点部位的心肌1分钟(4.3MHz/1.0 W/cm2),其他2组仅注射基因不行超声辐照。观察:①超声导管经皮心肌内注射、在心腔内辐照心肌的可行性及准确性;②MB结合基因注射到心肌后的成像效果、分布范围。术后48h:③检测心肌注射部位EGFP mRNA、蛋白表达;④探讨心腔内超声单独辐照以及协同心肌内注射MB对EGFP基因表达的影响;⑤观察心腔内超声辐照对心肌组织细胞的生物效应,评估超声导管的安全性;⑥探讨心腔内超声辐照增加EGFP在心肌表达的生物机制。2、犬MI模型的介入实验采用冠状动脉结扎法建立犬MI的动物模型,分为5组(n=3-5):①肝细胞生长因子裸质粒(HGF)+ MB +US组,②HGF + US组,③单纯HGF治疗组,另设假手术组(Sham组)与MI无干预组。用上述注射方法,在HGF基因治疗组分5个点向心肌内注入MB和/或HGF混合液(每个点100μL,含HGF100ug),HGF/MB/US、HGF/US两组注射后行心腔内超声辐照,单纯HGF治疗组仅注射HGF不行超声辐照,术后28天检测:①HGF的mRNA及蛋白在犬缺血心肌的表达;②心肌梗死面积、左右心室重量指数、心肌羟脯氨酸含量(HC)和心肌胶原容积分数(CVF)变化;③缺血心肌中血管内皮细胞生长因子(VEGF)、CD31、血小板八因子的表达,计算微血管计数。结果:①研制的超声导管经常规介入法顺利进入犬左心室腔,向心肌内注射基因、在心腔内发射超声辐照心肌的实验过程中犬生命体征平稳,无严重并发症;②微泡携基因注射到心肌可在局部清晰显影,诊断超声可清楚观察微泡在心肌内的成像效果、分布层次和范围;③与单纯EGFP注射组比较,EGFP/MB/US组EGFP的mRNA表达增加约8倍,荧光强度增加约8.6倍(P<0.01),EGFP/US组EGFP mRNA表达增加约6倍,荧光强度增加约5.1倍(P<0.01);单纯EGFP注射组与EGFP/MB组的EGFP mRNA表达、荧光强度无统计学差异( P >0.05);对照组无EGFP表达。④EGFP仅在注射部位心肌表达,其他脏器无EGFP沉积及表达,各实验组犬肝、肾、肺脏组织无异常病理改变;⑤心腔内超声辐照后心肌轻微充血,少量红细胞溢出;透射电镜提示心肌细胞及线粒体膜的通透性增加,心肌细胞浆内可见小空泡形成;心肌内直接注射微泡协同心腔内超声辐照后上述生物效应更明显;⑥心腔内超声辐照心肌后增加内源性VEGF及HSP70表达;VEGF及HSP70表达均以EGFP/MB/US组最明显,其次为EGFP/US组(P<0.01),EGFP/MB组仅有少量VEGF及HSP70表达,3组间差异有统计学意义(P<0.01);单纯EGFP注射组及对照组无VEGF、HSP70表达。⑦EGFP/MB/US组心肌中EGFPmRNA表达与VEGF及HSP70mRNA表达呈明显正相关,相关系数分别为0.76、0.75(P<0.05);EGFP/US组心肌中EGFPmRNA表达也与VEGF及HSP70mRNA表达呈明显正相关,相关系数分别为0.75、0.77 (P<0.05);EGFP/MB组EGFPmRNA表达与VEGF、HSP70mRNA表达无相关性(P>0.05)。在犬MI实验中:①心腔内超声辐照后增加HGFmRNA及蛋白在犬缺血心肌的表达;与单纯HGF治疗组比较,HGF/MB/US组HGF的mRNA表达增加约6倍,蛋白增加约4.7倍(P<0.01);HGF/US组HGF mRNA及蛋白表达则分别增加约4倍、3.3倍(P<0.01)。②HGF基因治疗能上调心肌中CD31、VEGF的mRNA表达,增加犬MI后毛细血管新生,以HGF/MB/US组最明显(P<0.01),其次为HGF/US组(P<0.01),单纯HGF裸质粒注射组CD31、VEGF的mRNA表达及血管新生较MI组增加,但低于其他2个HGF治疗组(P<0.01)。③HGF基因治疗也降低左右心室重量指数、HC和I型、III型胶原的CVF,改善犬MI后心脏重塑;所有治疗效应均以HGF/MB/US组最明显。结论:超声导管经皮介入向心肌内注射基因,在心腔内辐照心肌相对安全可行,可产生增加心肌生物膜及血管通透性等多种生物效应,并且明显增加外源性EGFP裸质粒在正常心肌、HGF裸质粒在缺血心肌的表达;MB携基因直接注射到心肌后,能够作为示踪剂显示基因在心肌内的分布,并与心腔内超声辐照协同进一步增加基因表达。心腔内超声辐照心肌增加基因表达可能与超声的空化、机械、热效应等综合作用有关;HGF基因干预对犬MI有部分治疗效应。更多还原

【Abstract】 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.更多还原