心包腔内注入腺病毒-血管内皮生长因子165基因治疗心肌缺血的实验研究

The effects of gene transfer into myocardium by recombinant adenovirus-VEGF165 via pericardial cavity in swine acute myocardial infarction model on angiogenesis and cardiac function

目的:探讨经心包腔途径转染血管新生基因对缺血心肌的血管生成及舒缩功能的影响。方法:第一部分:随机将12头中国小型猪分为实验组和对照组,每组6头。两组猪均采用球囊堵塞前降支第一对角支远端以建立心肌梗死模型,心肌梗死模型建立后即刻,采用经皮剑突下穿刺方法,将中心静脉导管插入心包腔内转染Ad-LacZ。以胶原酶1200 u及透明质酸酶3000u预处理心包后,在心包腔内注射含Ad-LacZ基因2.0×109pfu。对照组心包腔内注射生理盐水。分别于注射后3天、7天及28天处死动物,对缺血心肌进行染色及病理观察。第二部分:随机将20头中国小型猪分为实验组和对照组,每组10头,每组又分3天(n=2)、7天(n=6)及28天(n=6)三个亚组。注射后3天、7天及28天分别用免疫组化、超声心动图对缺血心肌血管新生情况进行检测,并以酶联免疫吸附试验(ELISA)检测血浆、心包及心肌组织中Ad-VEGF165的表达。第三部分:20头小型猪随机分为心包转染组(心包组)和冠脉转染组(冠脉组)。心包组和冠脉组均注射Ad-VEGF1651.0 ml(2×109pfu),于注射前及其后3、7、28天分别测定组织内VEGF水平、微血管密度(MVD)、心功能。结果:①实验组注射Ad-LacZ基因后第3天、第7天及28天后X-gal染色有阳性细胞,以第7天最明显,对照组无阳性细胞。②Ad-VEGF165基因经心包腔转染缺血心肌组织后,在心包及组织中成高表达,于7天达到高峰,28天降至基线水平,血浆中无目的基因的表达;28天时,实验组缺血心肌微血管密度(MVD)、心功能均明显高于对照组[MVD,517.0±75.7/mm2vs 226.5±54.1/mm2,P=0.009;LVEF72.11±5.2%vs 55.14±4.37%,P=0.005]。③心包组和冠脉组的心脏均表达有VEGF165基因,组织内VEGF水平在7天时达高峰,28天时降至基线水平,前组高于后组(702±85pg/ml vs 592±59 pg/ml,P=0.026)。而两组的MVD、心功能随转染时间延长均明显增加,但心包组优于冠脉组(28d,MVD,517.0±75.7/mm2vs 326.4±24.1/mm2,P=0.001;FS,32.9±2.2%vs 30.6±2.1%,P=0.049;LVEF,72.11±5.2%vs 65.87±2.16%,P=0.034)。结论:①应用球囊堵塞法可成功建立猪急性心肌梗死模型,胶原酶及透明质酸酶预处理心包后,腺病毒载体可转染缺血心肌,并持续表达4周。②用胶原酶及透明质酸酶预处理心包腔后,经其转染Ad-VEGF165可以诱导急性心肌梗死模型局部VEGF蛋白表达,促进缺血心肌组织血管新生并能改善心功能。③导管介导的心包腔与冠脉转染Ad-VEGF165基因治疗心肌缺血是有效的、切实可行的,而前者可能是更有前途的新方法。

Abstract： Objective： To explore the influence of adenovirus-mediated vascular endothelial growth factor 165 （Ad-VEGF165） gene transfer on angiogenesis and cardiac function in ischemic myocardium via the pericardial cavity. Methods： The first part： twelve health swine were randomly divided into two groups, experimental group（n =6） and control group（n = 6）. AMI model was constructed by balloon occlusion of the distal part of D1 branch of LAD （Left Anterior Descending） at the same time the intra-pericardial cavity injections were performed through the small incision of the abdominal wall below the xyphoid appendix using a home-made device. After succeeding, gene transfer was performed by center venous catheter. Both the experimental group and the control group, the pericardium was pre-treated by injecting a mixture of collagenase and hyaluronidase. Then 2.0 × 10^9pfu Ad-LacZ was injected into pericardial cavity. The histological changes and be- ta-galactosidase activity（X-gal） of the ischemic myocardium were observed on the 3rd, 7th and 28th day after injection. The second part： twenty health porcine were randomly divided into two groups, experimental group（ n = 10） and control group（ n = 10）. There are 2 in each group on the 3rd, 7th day after injection respectively and 6 on the 28th day. Then 2.0× 10^9pfu Ad-VEGF165 was injected into pericardial cavity in experimental group. While physiological saline was injected into control group. Immunohistochemistry assay and echocardiography were carried out to evaluate the angiogenesis in the ischemic region and cardio function on the 3rd, 7th and 28th day after injection respectively and enzyme-linked immunoassay（ELISA） was done to investigate the local expression of Ad-VEGF165 in plasma, pericardial cavity and myocardium. The third part： twenty health porcine were randomly divided into two groups： pericardium transfer group and coronary artery transfer group. Then 2.0 ×10^9pfu Ad-VEGF165 was injected into pericardial cavity. While 2.0 ×10^9pfu Ad-VEGF165 was injected into myocardium via coronary artery in the coronary group at the same time AMI mode was constructed by the same way. Tissue VEGF concentration,microv- enous density and cardiac function of the two groups were respectively observed on the 3rd, 7th and 28th day before and after injection. Results ：① The LAD was occluded completely and infarction and ischemic were detected histological assessment. In experimental group, X-gal staining positive cells were detected on the 3rd day after injection, increased markedly on the 7th day and then declined on the 28th day. In control group, no positive cells were observed. ②The peak expression of Ad-VEGF165 gene in pericardium and myocardium occurred on the 7th day after the ad- ministration of Ad-VEGF165 and decreased gradually to baseline level on the 28th day. And microvenous density and cardiac function of the experimental group significantly increased in the course of transfer and had the advantage of the control group（on the 28th day,／MVD 517.0 ± 75.7 vs, 226.5 ±54.1P=0.009, LVEF% 72.11 ±5.2vs55.14±4.37, P=0.005）. ③ VEGF165 gene was expressed in the heart of the pericardium group and coronary artery group. Tissue VEGF concentration increased markedly on the 7th day after injection and then declined on the 28th day. Tissue VEGF levels in the pericardium group were higher than the coronary artery group [ （702 ± 85 ）pg/ml vs（592 ±59）pg/ml, P = 0. 026 ]. And microvenous density and cardiac function of the two groups all significantl increased in the course of transfer. However, the pericardium group had the advantage of the coronary artery group in parameters above（on the 28th day, MVD [ （517.0 ± 75.7）/mm^2 vs （326.4±24.1）/mm^2, P=0.001; FS （32.9±2.2）% vs （30.6±2.1）%, P= 0.049; LVEF （72.11 ±5.2）% vs （65.87 ＋2.16）%, P=0.034]. Conclusion： ① Ad can be transfered into ischemic myocardium and succeed in expressing target gene in the model for 4 weeks via pericardium pre-treated by injecting a mixture of collagenase and hyaluronidase. ②Gene transfer of Ad-VEGF165 via pericardium can induce protein expression and enhance angiogenesis in ischemic region and improve cardio function in AMI after the pericardium pre-treated by injecting a mixture of collagenase and hyaluronidase. ③Catheter mediated Ad-VEGF165 gene transfer via coronary artery or pericardium is efficalions and feasible for ischemic myocardium, and the latter is more promising.