经心包腔转染腺病毒血管内皮生长因子165基因对猪急性心肌梗死的血管形成及心功能的影响
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摘要
第一部分 复制缺陷的重组腺病毒经心包腔转染猪急性心肌梗死模型的实验研究
目的 建立猪急性心肌梗死模型，评价重组腺病毒经心包腔的转染效率及持续时间。
方法 应用磷酸钙沉淀方法制备携带大肠杆菌LacZ基因复制缺陷的重组腺病毒(Ad-LacZ)，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。实验组：以含胶原酶1200u及透明质酸酶3000u的生理盐水2ml预处理心包后，在心包腔内注射含Ad-LacZ基因2.0×10~9p.f.u的生理盐水液1ml；对照组：同样方法预处理心包后，在心包腔内注射生理盐水1ml。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。
结果 冠状动脉造影证实前降支远端完全闭塞，病理显示心肌有缺血和梗死；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。
结论 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。
第二部分 经心包腔转染腺病毒血管内皮生长因子165基因对猪冠脉血管形成影响的研究
目的 探讨以腺病毒为载体的血管内皮生长因子165(Ad-VEGF_(165))基因经心包腔转染的表达规律，对缺血心肌血管生成的作用。
方法 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3d(n=2)、7d(n=2)及28d(n=2)三个亚组。采用球囊堵塞前降支第一对角支远端建立心肌梗死模型，成模后即刻，实验组：采用经皮剑突下穿刺中心静脉导管心包腔内转染，以含胶原酶1200u及透明质酸酶3000u的生理盐水2ml预处理心包后，在心包腔内注射含Ad-VEGF_(165)基因2.0×10~9pfu的生理盐水液1ml；对照组：同样方法预处理心包后，在心包腔内注射生理盐水1ml。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-VEGF_(165)的表达。
结果 Ad-VEGF_(165)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／mm~2 vs 226.5±54.1／mm~2，P=0.009；LVEF 72.11±5.2％ vs 55.14±4.37％，P=0.005]。
结论 用胶原酶及透明质酸酶预处理心包腔后，经其转染Ad-VEGF_(165)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。
第三部分 重组腺病毒血管内皮生长因子165基因心包腔与冠状动脉转染心肌的比较研究
目的 比较经心包腔与冠状动脉转染重组腺病毒血管内皮生长因子165基因(Ad-VEGF_(165))的有效性。
方法 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)，两组猪均采用球囊堵塞前降支第一对角支远端以构建心肌梗死模型，心包组在心肌梗死模型建立后即刻，采用经皮剑突下穿刺将中心静脉导管插入心包腔内，以胶原酶和透明质酸酶预先处理心包，然后注射Ad-VEGF_(165) 1.0ml(2×10~9pfu)；冠脉组在心肌梗死模型建立后即刻，经冠脉注射Ad-VEGF_(165) 1.0ml(2×10~9pfu)，于注射前后3、7、28d分别测定组织内VEGF水平、微血管密度(MVD)、心功能。
结果 心包组和冠脉组的心脏均表达有VEGF_(165)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)。而两组的微血管密度、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／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)。
结论 导管介导的心包腔与冠脉转染Ad-VEGF_(165)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。
第四部分 心包穿刺简易装置在基因转染中的应用
目的 探讨自制心包穿刺装置转染心脏的安全性、可行性。方法 应用磷酸钙沉淀方法制备携带大肠杆菌LacZ基因复制缺陷的重组腺病毒(Ad-LacZ)，将12头中国小型猪分为实验组和对照组，采用球囊堵塞前降支第一对角支远端，心肌梗死模型建立后即刻，采用自制简易心包腔穿刺装置经皮剑突下穿刺，成功后置中心静脉导管于心包腔内并行转染，28d后处死。实验组：胶原酶1200u及透明质酸酶3000u预处理心包后，在心包腔内注射Ad-LacZ基因2.0×10~9p.f.u；对照组：同样方法预处理心包后，在心包腔内注射生理盐水1ml。于注射后3d、7d及28d分别对缺血心肌进行染色及病理观察。结果 冠状动脉造影证实前降支远端完全闭塞，病理显示心肌有缺血和梗死；实验组注射Ad-LacZ基因后第3d、第7d及28d后X-gal染色有阳性细胞，以第7d最明显，对照组无阳性细胞。结论 自制的心包腔简易穿刺装置将腺病毒载体转染至缺血心肌是安全的，可行的，并且腺病毒可持续表达4周。
目的 探讨自制心包穿刺装置转染心脏的安全性、可行性。方法 应用磷酸钙沉淀方法制备携带大肠杆菌LacZ基因复制缺陷的重组腺病毒(Ad-LacZ)，将12头中国小型猪分为实验组和对照组，采用球囊堵塞前降支第一对角支远端，心肌梗死模型建立后即刻，采用自制简易心包腔穿刺装置经皮剑突下穿刺，成功后置中心静脉导管于心包腔内并行转染，28d后处死。实验组：胶原酶1200u及透明质酸酶3000u预处理心包后，在心包腔内注射Ad-LacZ基因2.0×10~9p.f.u；对照组：同样方法预处理心包后，在心包腔内注射生理盐水1ml。于注射后3d、7d及28d分别对缺血心肌进行染色及病理观察。结果 冠状动脉造影证实前降支远端完全闭塞，病理显示心肌有缺血和梗死；实验组注射Ad-LacZ基因后第3d、第7d及28d后X-gal染色有阳性细胞，以第7d最明显，对照组无阳性细胞。结论 自制的心包腔简易穿刺装置将腺病毒载体转染至缺血心肌是安全的，可行的，并且腺病毒可持续表达4周。
Objective To establish a model of acute myocardial infarction and evaluate the expression efficacy and time of adenovirus vector via the pericardial cavity. Methods Replication-deficient recombinant adenoviral vector carrying LacZ reporter genes(Ad-LacZ) was constructed by the calcium phosphate method. Twelve health porcine were randomly divided into two groups, experimental group(n=6) and control group(n=6). AMI mode was constructed by occluding by balloon the D_1 distance of
LAD then the intra-pericardial injections were performed through a small incision of the abdominal wall below the xyphoid appendix using center venous catheter. Whether the experimental group or the control group, the pericardial cavity pre-treated by injecting a mixture of collagenase and hyaluronidase. Then 2.0×10~9p.f.u Ad-LacZ was injected into pericardial cavity. The histological changes and beta-galactosidase activity of the ischemic myocardium were observed at the 3rd, 7th and 28th day after injection.
Results The LAD was occluded completely and infarction and ischemic were detected by histologic assessment. In experimental group, X-gal staining positive cells were detected at the 3rd day after injection, increased markedly at the 7th day and then declined at the 28th day. In control group, no positive cells were observed at the same time.
Conclusion Acute myocardial infarction model of pigs can successfully constructed by occluding by balloon. Ad can transfer 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.
Part II
Effect Of Gene Transfer By Adenovirus-Vascular Endothelial Growth Factor165 Via The Pericardium On Coronary Artery
Angiogensis In Porcine
Objective To explore the influence on angiogenesis and the expressing and proper dose of adenovirus-mediated vascular endothelial growth factor 165(Ad-VEGF_(165)) gene in ischemic myocardium via the percardium.
Methods Twenty health porcine were randomly divided into two groups,
experimental group(n=10) and control group(n=10). There are 2 in each group at 3rd, 7th after injection respectively and 6 at 28th. AMI mode was constructed by ballooning occlusion the D_1 distance of LAD at the same time the intra-pericardial injections were performed through a small incision of the abdominal wall below the xyphoid appendix using center venous catheter. Whether the experimental group or the control group, the pericardium pre-treated by injecting a mixture of collagenase(1200U) and hyaluronidase(3000U). Then 2.0×10~9p.f.u Ad-VEGF_(165) was injected into pericardium 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 at 3rd, 7th and 28th after injection respectively and enzyme-linked immunoassay(ELISA) was done to investigate the local expression of Ad-VEGF_(165) in plasma, pericardium and myocardium.
Results The peak expression of Ad-VEGF_(165) gene in pericardium and myocardium occurred 7th after the administration of Ad-VEGF_(165) and decreased gradually to baseline level at 28th. And microvenous density and cardiac function of the experimental group significantly increase in the course of transfer and have the dvantage of the control group(in 28 days, MVD 517.0±75.7 vs, 226.5±54.1 P=0.009, LVEF% 72.11±5.2 vs 55.14±4.37, P=0.005).
Conclusion Gene transfer of Ad-VEGF_(165) 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.
Objective To explore the efficacy of gene transfer into myocardium by recombination adenoviais -VEGF_(165) via the pericardium or the coronary artery.
Methods Twelve health porcine were randomly divided into two groups: pericardium transfer group and coronary artery transfer group. AMI mode in the pericardium group was constructed by occluding by balloon the D_1 distance of LAD then the intra-pericardial injections were performed through a small incision of the abdominal wall below the xyphoid appendix using center venous catheter. The pericardium pre-treated by injecting a mixture of collagenase and hyaluronidase and then 2.0×10~9pfu Ad-VEGF_(165) was injected into pericardium. While 2.0×10~9p.f.u Ad-VEGF_(165) was injected into myocardial cavity via coronary artery in the coronary group at the same time AMI mode was constructed by the same way. Tissue VEGF concentration microvenous density and cardiac function of the two groups were respectively observed at the 3rd, 7th and 28th day before and after injection.
Results VEGF_(165) gene was expressed in the heart of the pericardium group and coronary artery group. Tissue VEGF concentration increased markedly at the 7th day after injection and then declined at 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 significantly increase in the course of transfer. However, the pericardium group has the advantage of the coronary artery group in parameters above(in 28 days, 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 Catheter mediated Ad-VEGF_(165) gene transfer via coronary artery or pericardium is efficacy and feasibility for ischemic myocardium. And the latter is
Objective To explore the feasibility and safety of gene transfer via the pericardial cavity by home-made easy device. Methods Replication-deficient recombinant adenoviral vector carrying LacZ reporter genes(Ad-LacZ) was constructed by the calcium phosphate method. Twelve healthly porcine were randomly divided into two groups, experimental group(n=6) and control group(n=6). AMI mode was constructed by balloon occlusion of the distal part of D_1 branch of LAD at the same time the intra-pericardial cavity injections were performed through a small incision of the abdominal wall below the xyphoid appendix using home-made device. After succeeding, gene transfer were performed by center venous catheter. In both the experimental group and the control group, the pericardium was pre-treated by injecting a mixture of collagenase and hyaluronidase. Then 2.0×10~9p.f.u Ad-LacZ was injected into pericardium. The histological changes and beta-galactosidase activity of the ischemic myocardium were observed at the 3rd, 7th and 28th day after injection. Results The LAD was occluded completely and infarction and ischemic were detected by histologic assessment. In experimental group, X-gal staining positive cells were detected at the 3rd day after injection, increased markedly at the 7th day and then declined at the 28th day. In control group, no positive cells were observed. Conclusion Ad can be transfered into ischemic myocardium and succeed in expressing target gene in the model for 4 weeks via pericardial cavity pre-treated by injecting a mixture of collagenase and hyaluronidase by home-made easy device.
目的 建立猪急性心肌梗死模型，评价重组腺病毒经心包腔的转染效率及持续时间。
方法 应用磷酸钙沉淀方法制备携带大肠杆菌LacZ基因复制缺陷的重组腺病毒(Ad-LacZ)，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。实验组：以含胶原酶1200u及透明质酸酶3000u的生理盐水2ml预处理心包后，在心包腔内注射含Ad-LacZ基因2.0×10~9p.f.u的生理盐水液1ml；对照组：同样方法预处理心包后，在心包腔内注射生理盐水1ml。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。
结果 冠状动脉造影证实前降支远端完全闭塞，病理显示心肌有缺血和梗死；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。
结论 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。
第二部分 经心包腔转染腺病毒血管内皮生长因子165基因对猪冠脉血管形成影响的研究
目的 探讨以腺病毒为载体的血管内皮生长因子165(Ad-VEGF_(165))基因经心包腔转染的表达规律，对缺血心肌血管生成的作用。
方法 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3d(n=2)、7d(n=2)及28d(n=2)三个亚组。采用球囊堵塞前降支第一对角支远端建立心肌梗死模型，成模后即刻，实验组：采用经皮剑突下穿刺中心静脉导管心包腔内转染，以含胶原酶1200u及透明质酸酶3000u的生理盐水2ml预处理心包后，在心包腔内注射含Ad-VEGF_(165)基因2.0×10~9pfu的生理盐水液1ml；对照组：同样方法预处理心包后，在心包腔内注射生理盐水1ml。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-VEGF_(165)的表达。
结果 Ad-VEGF_(165)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／mm~2 vs 226.5±54.1／mm~2，P=0.009；LVEF 72.11±5.2％ vs 55.14±4.37％，P=0.005]。
结论 用胶原酶及透明质酸酶预处理心包腔后，经其转染Ad-VEGF_(165)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。
第三部分 重组腺病毒血管内皮生长因子165基因心包腔与冠状动脉转染心肌的比较研究
目的 比较经心包腔与冠状动脉转染重组腺病毒血管内皮生长因子165基因(Ad-VEGF_(165))的有效性。
方法 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)，两组猪均采用球囊堵塞前降支第一对角支远端以构建心肌梗死模型，心包组在心肌梗死模型建立后即刻，采用经皮剑突下穿刺将中心静脉导管插入心包腔内，以胶原酶和透明质酸酶预先处理心包，然后注射Ad-VEGF_(165) 1.0ml(2×10~9pfu)；冠脉组在心肌梗死模型建立后即刻，经冠脉注射Ad-VEGF_(165) 1.0ml(2×10~9pfu)，于注射前后3、7、28d分别测定组织内VEGF水平、微血管密度(MVD)、心功能。
结果 心包组和冠脉组的心脏均表达有VEGF_(165)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)。而两组的微血管密度、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／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)。
结论 导管介导的心包腔与冠脉转染Ad-VEGF_(165)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。
第四部分 心包穿刺简易装置在基因转染中的应用
目的 探讨自制心包穿刺装置转染心脏的安全性、可行性。方法 应用磷酸钙沉淀方法制备携带大肠杆菌LacZ基因复制缺陷的重组腺病毒(Ad-LacZ)，将12头中国小型猪分为实验组和对照组，采用球囊堵塞前降支第一对角支远端，心肌梗死模型建立后即刻，采用自制简易心包腔穿刺装置经皮剑突下穿刺，成功后置中心静脉导管于心包腔内并行转染，28d后处死。实验组：胶原酶1200u及透明质酸酶3000u预处理心包后，在心包腔内注射Ad-LacZ基因2.0×10~9p.f.u；对照组：同样方法预处理心包后，在心包腔内注射生理盐水1ml。于注射后3d、7d及28d分别对缺血心肌进行染色及病理观察。结果 冠状动脉造影证实前降支远端完全闭塞，病理显示心肌有缺血和梗死；实验组注射Ad-LacZ基因后第3d、第7d及28d后X-gal染色有阳性细胞，以第7d最明显，对照组无阳性细胞。结论 自制的心包腔简易穿刺装置将腺病毒载体转染至缺血心肌是安全的，可行的，并且腺病毒可持续表达4周。
目的 探讨自制心包穿刺装置转染心脏的安全性、可行性。方法 应用磷酸钙沉淀方法制备携带大肠杆菌LacZ基因复制缺陷的重组腺病毒(Ad-LacZ)，将12头中国小型猪分为实验组和对照组，采用球囊堵塞前降支第一对角支远端，心肌梗死模型建立后即刻，采用自制简易心包腔穿刺装置经皮剑突下穿刺，成功后置中心静脉导管于心包腔内并行转染，28d后处死。实验组：胶原酶1200u及透明质酸酶3000u预处理心包后，在心包腔内注射Ad-LacZ基因2.0×10~9p.f.u；对照组：同样方法预处理心包后，在心包腔内注射生理盐水1ml。于注射后3d、7d及28d分别对缺血心肌进行染色及病理观察。结果 冠状动脉造影证实前降支远端完全闭塞，病理显示心肌有缺血和梗死；实验组注射Ad-LacZ基因后第3d、第7d及28d后X-gal染色有阳性细胞，以第7d最明显，对照组无阳性细胞。结论 自制的心包腔简易穿刺装置将腺病毒载体转染至缺血心肌是安全的，可行的，并且腺病毒可持续表达4周。
Objective To establish a model of acute myocardial infarction and evaluate the expression efficacy and time of adenovirus vector via the pericardial cavity. Methods Replication-deficient recombinant adenoviral vector carrying LacZ reporter genes(Ad-LacZ) was constructed by the calcium phosphate method. Twelve health porcine were randomly divided into two groups, experimental group(n=6) and control group(n=6). AMI mode was constructed by occluding by balloon the D_1 distance of
LAD then the intra-pericardial injections were performed through a small incision of the abdominal wall below the xyphoid appendix using center venous catheter. Whether the experimental group or the control group, the pericardial cavity pre-treated by injecting a mixture of collagenase and hyaluronidase. Then 2.0×10~9p.f.u Ad-LacZ was injected into pericardial cavity. The histological changes and beta-galactosidase activity of the ischemic myocardium were observed at the 3rd, 7th and 28th day after injection.
Results The LAD was occluded completely and infarction and ischemic were detected by histologic assessment. In experimental group, X-gal staining positive cells were detected at the 3rd day after injection, increased markedly at the 7th day and then declined at the 28th day. In control group, no positive cells were observed at the same time.
Conclusion Acute myocardial infarction model of pigs can successfully constructed by occluding by balloon. Ad can transfer 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.
Part II
Effect Of Gene Transfer By Adenovirus-Vascular Endothelial Growth Factor165 Via The Pericardium On Coronary Artery
Angiogensis In Porcine
Objective To explore the influence on angiogenesis and the expressing and proper dose of adenovirus-mediated vascular endothelial growth factor 165(Ad-VEGF_(165)) gene in ischemic myocardium via the percardium.
Methods Twenty health porcine were randomly divided into two groups,
experimental group(n=10) and control group(n=10). There are 2 in each group at 3rd, 7th after injection respectively and 6 at 28th. AMI mode was constructed by ballooning occlusion the D_1 distance of LAD at the same time the intra-pericardial injections were performed through a small incision of the abdominal wall below the xyphoid appendix using center venous catheter. Whether the experimental group or the control group, the pericardium pre-treated by injecting a mixture of collagenase(1200U) and hyaluronidase(3000U). Then 2.0×10~9p.f.u Ad-VEGF_(165) was injected into pericardium 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 at 3rd, 7th and 28th after injection respectively and enzyme-linked immunoassay(ELISA) was done to investigate the local expression of Ad-VEGF_(165) in plasma, pericardium and myocardium.
Results The peak expression of Ad-VEGF_(165) gene in pericardium and myocardium occurred 7th after the administration of Ad-VEGF_(165) and decreased gradually to baseline level at 28th. And microvenous density and cardiac function of the experimental group significantly increase in the course of transfer and have the dvantage of the control group(in 28 days, MVD 517.0±75.7 vs, 226.5±54.1 P=0.009, LVEF% 72.11±5.2 vs 55.14±4.37, P=0.005).
Conclusion Gene transfer of Ad-VEGF_(165) 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.
Objective To explore the efficacy of gene transfer into myocardium by recombination adenoviais -VEGF_(165) via the pericardium or the coronary artery.
Methods Twelve health porcine were randomly divided into two groups: pericardium transfer group and coronary artery transfer group. AMI mode in the pericardium group was constructed by occluding by balloon the D_1 distance of LAD then the intra-pericardial injections were performed through a small incision of the abdominal wall below the xyphoid appendix using center venous catheter. The pericardium pre-treated by injecting a mixture of collagenase and hyaluronidase and then 2.0×10~9pfu Ad-VEGF_(165) was injected into pericardium. While 2.0×10~9p.f.u Ad-VEGF_(165) was injected into myocardial cavity via coronary artery in the coronary group at the same time AMI mode was constructed by the same way. Tissue VEGF concentration microvenous density and cardiac function of the two groups were respectively observed at the 3rd, 7th and 28th day before and after injection.
Results VEGF_(165) gene was expressed in the heart of the pericardium group and coronary artery group. Tissue VEGF concentration increased markedly at the 7th day after injection and then declined at 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 significantly increase in the course of transfer. However, the pericardium group has the advantage of the coronary artery group in parameters above(in 28 days, 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 Catheter mediated Ad-VEGF_(165) gene transfer via coronary artery or pericardium is efficacy and feasibility for ischemic myocardium. And the latter is
Objective To explore the feasibility and safety of gene transfer via the pericardial cavity by home-made easy device. Methods Replication-deficient recombinant adenoviral vector carrying LacZ reporter genes(Ad-LacZ) was constructed by the calcium phosphate method. Twelve healthly porcine were randomly divided into two groups, experimental group(n=6) and control group(n=6). AMI mode was constructed by balloon occlusion of the distal part of D_1 branch of LAD at the same time the intra-pericardial cavity injections were performed through a small incision of the abdominal wall below the xyphoid appendix using home-made device. After succeeding, gene transfer were performed by center venous catheter. In both the experimental group and the control group, the pericardium was pre-treated by injecting a mixture of collagenase and hyaluronidase. Then 2.0×10~9p.f.u Ad-LacZ was injected into pericardium. The histological changes and beta-galactosidase activity of the ischemic myocardium were observed at the 3rd, 7th and 28th day after injection. Results The LAD was occluded completely and infarction and ischemic were detected by histologic assessment. In experimental group, X-gal staining positive cells were detected at the 3rd day after injection, increased markedly at the 7th day and then declined at the 28th day. In control group, no positive cells were observed. Conclusion Ad can be transfered into ischemic myocardium and succeed in expressing target gene in the model for 4 weeks via pericardial cavity pre-treated by injecting a mixture of collagenase and hyaluronidase by home-made easy device.
引文
1．董书强；梅举；张宝仁等．复制缺陷的重组腺病毒载体直接转染猪慢性缺血心肌的研究．中国胸心血管外科临床杂志；2002，9(2)：103-106．
2. Dimmeler S, Zeiher AM, Schneider MD. Unchain my heart: the scientific foundations of cardiac repair. J. Clin. Invest, 2005; 115:572-583.
3. Hammond HK, McKirman MD. Angiogenic gene therapy for heart disease: a review of animal studies and clinical trials. Cardiovascular Research, 2001, 49: 561-567.
4. Tio RA, Grandjean JG, Suurmeijer AJ, et al. Monitoring for pericardial application of local drug or gene therapy. Int J Cardiol, 2002, 82(2): 117-21.
5. Lazarous DF, Shou M, Stiber JA et al. Adenoviral-mediated gene transfer induces sustained pericardial VEGF expression in dogs: effect on myocardial angiogenesis. Cardiovasc Res, 1999, 44:294-302.
6. Lamping KG, Rios CD, Chun JA, et al. Intrapericardial administration of adenovirus for gene transfer. Am J Physiol, 1997 Jan, 272(1 Pt 2):H310-7.
7. Aoki K, Barker C, Danthinne X, et al. Efficient in vivo gene transfer into the heart in the rat. J Mol Cell Cardiol, 1997, 29: 949-959.
8. Fromes Y, Salmom A, Wang X, et al. Gene delivery to the myocardium by intrapericardial injection. Gene Therapy, 1999, 6:683-688.
9. Li J, Li G, Huang C, et al. Comparative study of catheter-mediated gene transfer into heart. Chin Med J (Engl), 2002, 115(4):612-3.
1. Bosaris T, Lee AK, Dekellei RA, et al. Microvessel quantitation and prognosis in invasive breast carcinoma. N Engl J Med. 1991; 342:1-8.
2. Mack CA, Patel SR, Schwartz EA et al. Biologic bypass with the use of adenovirus-mediated gene transfer of the complementary deoxyribonucleic acid for vascular endothelial growth factor 121 improves myocardial perfusion and function in the ischemic porcine heart. J Cardiovasc Surg. 1998; 115:168-177.
3. Patel SR, Lee LY, Mack CA, et al. Safety of direct myocardial administration of an adenovirus vector encoding vascular endothelial growth factor 121. Hum Gene Ther, 1999; 10:1331-1348.
4. Kornowski R, Leon MB, Fuchs S, et al. Electromagnetic guidance for catheter-based transendocardial injection: a platform for intramyocardial angiogenesis therapy. J Am Coil Cardiol, 2000; 35:1031-1039.
5. Safi J, DiPaula AF, Riccioni T et al. Adenovirus-mediated acidic fibroblast growth factor gene transfer induces angiogenesis in the nonischemic rabbit heart. Microvasc Res, 1999; 58:238-249.
6．张端珍，盖鲁粤，陈以旺，等．血管内皮生长因子对猪心肌侧枝血管生成的作用．生理学报，2001：3．
7. Rosengart TK, Lee LY, Patel SR, et al. Angiogenesis gene therapy: Phase Ⅰ assessment of direct intramyocardial administration of an adenovirus vector expressing VEGF121 cDNA to individuals with clinically significant coronary artery disease. Circulation, 1999; 100:468-474.
8. Giordano F, Ping P, McKiman MD, et al. Intracoronary gene transfer of fibroblast growth factor-5 increases blood flow and contractile function in an ischemic region of the heart. Nat Med, 1996; 2:534-539.
9. McKirnan MD, Lai NC, Waldman L, et al. Intracoronary gene transfer of fibroblast growth factor-4 increases regional contractile function and responsiveness to adrenergic stimulation in heart failure. Cardiac Vasc Regen, 2000; 1:11-21.
10. Padua RR, Sethi R, Dhalla NS, et al. Basic fibroblast growth factor is cardioprotective in ischemia-reperfusion injury. Mol Cell Biochem, 1995; 143:129-135.
11. Hammond HK, McKirnan. Angiogenic gene therapy for heart disease: a review of animal studies and clinical trials. Cardiovascular Research, 2001: 49: 561-567.
12. Grines CL, Watkins MW, Helmer G, et al. Angiogenic Gene Therapy (AGENT) trial in patients with stable angina pectoris. Circulation, 2002; 105(11): 1291-7.
13. Simons M, Annex BH, Laham RJ, et al. Pharmacological treatment of coronary artery disease with recombinant fibroblast growth factor-2: double-blind, randomized, controlled clinical trial. Circulation, 2002; 105(7):788-93.
14. Hedman M, Hartikainen J, Syvanne M, et al. Safety and feasibility of catheter-based local intracoronary vascular endothelial growth factor gene transfer in the prevention of postangioplasty and in-stent restenosis and in the treatment of chronic myocardial ischemia: phase II results of the Kuopio Angiogenesis Trial (KAT).Circulation, 2003; 107(21):2677-83.
15. Lazarous DF, Shou M, Stiber JA, et al. Adenoviral-mediated gene transfer induces sustained pericardial VEGF expression in dogs: effect on myocardial angiogenesis. Cardiovasc Res, 1999; 44:294-302.
16. Lamping KG, Rios CD, Chun JA, et al. Intrapericardial administration of adenovirus for gene transfer. Am J Physiol, 1997 ; 272:H310-7.44.
17. Aoki K, Barker C, Danthinne X, et al. Efficient in vivo gene transfer into the heart in the rat. J Mol Cell Cardiol, 1997; 29: 949-959.45.
18. Rutanen J, Rissanen TT, Markkanen JE, et al. Adenoviral catheter-mediated intramyocardial gene transfer using the mature form of vascular endothelial growth factor-D induces transmural angiogenesis in porcine heart. Circulation, 2004; 109(8):1029-35.
19. Tio RA, Grandjean JG, Suurmeijer AJ, et al. monitoring for pericardial application of local drug or gene therapy. Int J Cardiol, 2002 ; 82(2): 117-21.
1. Bosaris T, Lee AK, Dekellei RA, et al. Microvessel quantitation and prognosis in invasive breast carcinoma. N Engl J Med, 1991; 342: 1-8.
2. Mack CA, Patel SR, Schwartz EA, et al. Biologic bypass with the use of adenovirus-mediated gene transfer of the complementary deoxyribonucleic acid for vascular endothelial growth factor 121 improves myocardial perfusion and function in the ischemic porcine heart. J Cardiovasc Surg, 1998; 115:168-177.
3. Patel SR, Lee LY, Mack CA, et al. Safety of direct myocardial administration of an adenovirus vector encoding vascular endothelial growth factor 121. Hum Gene Ther, 1999; 10:1331-1348.
4. Kornowski R, Leon MB, Fuchs S, et al. Electromagnetic guidance for catheter-based transendocardial injection: a platform for intramyocardial angiogenesis therapy. J Am Coll Cardiol, 2000; 35:1031-1039.
5. Safi J, DiPaula AF, Riccioni T, et al. Adenovirus-mediated acidic fibroblast growth factor gene transfer induces angiogenesis in the nonischemic rabbit heart. Microvasc Res, 1999; 58:238-249.
6．张端珍，盖鲁粤，陈以旺，等．血管内皮生长因子对猪心肌侧枝血管生成的作用．生理学报，2001；3．
7. Rosengart TK, Lee LY, Patel SR, et al. Angiogenesis gene therapy: Phase Ⅰ assessment of direct intramyocardial administration of an adenovirus vector expressing VEGF121 cDNA to individuals with clinically significant coronary artery disease. Circulation, 1999; 100:468-474.
8. Giordano F, Ping P, McKirnan MD, et al. Intracoronary gene transfer of fibroblast growth factor-5 increases blood flow and contractile function in an ischemic region of the heart. Nat Med, 1996; 2:534-539.
9. McKirnan MD, Lai NC, Waldrnan L, et al. Intracoronary gene transfer of fibroblast growth factor-4 increases regional contractile function and responsiveness to adrenergic stimulation in heart failure. Cardiac Vasc Regen, 2000; 1:11-21.
10. Padua RR, Sethi R, Dhalla NS, et al. Basic fibroblast growth factor is cardioprotective in ischemia-reperfusion injury. Mol Cell Biochem, 1995; 143:129-135.
11. Hammond HK, McKirnan. Angiogenic gene therapy for heart disease: a review of animal studies and clinical trials. Cardiovascular Research, 2001: 49: 561-567.
12. Grines CL, Watkins MW, Helmer G, et al. Angiogenic Gene Therapy (AGENT) trial in patients with stable angina pectoris. Circulation, 2002; 105(11): 1291-7.
13. Simons M, Annex BH, Laham RJ, et al. Pharmacological treatment of coronary artery disease with recombinant fibroblast growth factor-2: double-blind, randomized, controlled clinical trial. Circulation, 2002; 105(7):788-93.
14. Hedman M, Hartikainen J, Syvanne M, et al. Safety and feasibility of catheter-based local intracoronary vascular endothelial growth factor gene transfer in the prevention of postangioplasty and in-stent restenosis and in the treatment of chronic myocardial ischemia: phase II results of the Kuopio Angiogenesis Trial (KAT).Circulation, 2003; 107(21):2677-83.
15.Rutanen J, Rissanen TT, Markkanen JE, et al. Adenoviral catheter-mediated intramyocardial gene transfer using the mature form of vascular endothelial growth factor-D induces transmural angiogenesis in porcine heart. Circulation, 2004; 109(8):1029-35.
16. Tio RA, Grandjean JG, Suurmeijer AJ, et al. monitoring for pericardial application of local drug or gene therapy. Int J Cardiol, 2002 ; 82(2): 117-21.
17. Lazarous DF, Shou M, Stiber JA et al. Adenoviral-mediated gene transfer induces sustained pericardial VEGF expression in dogs: effect on myocardial angiogenesis. Cardiovasc Res, 1999; 44:294-302.
18. Lamping KG, Rios CD, Chun JA, et al. Intrapericardial administration of adenovirus for gene transfer. Am J Physiol, 1997; 272:H310-7.44.
19. Aoki K, Barker C, Danthinne X, et al. Efficient in vivo gene transfer into the heart in the rat. J Mol Cell Cardiol, 1997; 29: 949-959.45.
20. Fromes Y, Salmom A, Wang X, et al. Gene delivery to the myocardium by intrapericardial injection. Gene Therapy, 1999; 6:683-688.
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2．董书强；梅举；张宝仁等．复制缺陷的重组腺病毒载体直接转染猪慢性缺血心肌的研究．中国胸心血管外科临床杂志，2002，9(2)：103-106．
3. Dimmeler S, Zeiher AM, Schneider MD. Unchain my heart: the scientific foundations of cardiac repair. J. Clin Invest, 2005; 115(3):572-583.
4. Hammond HK, McKirman MD. Angiogenic gene therapy for heart disease: a review of animal studies and clinical trials. Cardiovascular Research, 2001, 49(3): 561-567.
5. Tio RA, Grandjean JG, Suurmeijer AJ, et al. Monitoring for pericardial application of local drug or gene therapy. Int J Cardiol, 2002, 82(2):117-21.
6. Lazarous DF, Shou M, Stiber JA et al. Adenoviral-mediated gene transfer induces sustained pericardial VEGF expression in dogs: effect on myocardial angiogenesis. Cardiovasc Res, 1999, 44(2):294-302.
7. Lamping KG, Rios CD, Chun JA, et al. Intrapericardial administration of adenovirus for gene transfer. Am J Physiol, 1997 Jan, 272(1 Pt 2):H310-7.
8. Aoki K, Barker C, Danthinne X, et al. Efficient in vivo gene transfer into the heart in the rat. J Mol Cell Cardiol, 1997, 29(3): 949-959.
9. Fromes Y, Salmom A, Wang X, et al. Gene delivery to the myocardium by intrapericardial injection. Gene Therapy, 1999, 6(4):683-688.
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34. McKirnan MD, Lai NC, Waldman L, et al. Intracoronary gene transfer of fibroblast growth factor-4 increases regional contractile function and responsiveness to adrenergic stimulation in heart failure. Cardiac Vasc Regen, 2000; 1:11-21.
35. Padua RR, Sethi R, Dhalla NS, et al. Basic fibroblast growth factor is cardioprotective in ischemia-reperfusion injury. Mol Cell Biochem, 1995; 143: 129-135.
36. Hammond HK, McKiman. Angiogenic gene therapy for heart disease: a review of animal studies and clinical trials. Cardiovascular Research. 2001:49:561-567.
37. Grines CL, Watkins MW. Helmet G, et al. Angiogenic Gene Therapy (AGENT) trial in patients with stable angina pectoris. Circulation, 2002; 105(11): 1291-7.
38. Simons M, Annex BH, Laham RJ, et al. Pharmacological treatment of coronary artery disease with recombinant fibroblast growth factor-2: double-blind. randomized, controlled clinical trial. Circulation, 2002; 105(7):788-93.
39. Hedman M, Hartikainen J, Syvanne M, et al. Safety and feasibility of catheter-based local intracoronary vascular endothelial growth factor gene transfer in the prevention of postangioplasty and in-stent restenosis and in lbe treatment of chronic myocardial ischemia: phase Ⅱ results of the Kuopio Angiogenesis Trial (KAT). Circulation, 2003; 107(21):2677-83.
40. Rutanen J, Rissanen TT, Markkanen JE. et al. Adenoviral catheter-mediated intramyocardial gene transfer using the mature form of vascular endothelial growth factor-D induces transmural angiogenesis in porcine heart. Circulation, 2004; 109(8):1029-35.
41. Tio RA, Grandjean JG, Suurmeijer AJ, et al. Monitoring for pericardial application of local drug or gene therapy. Int J Cardiol, 2002; 82(2):117-21.
42. Lazarous DF, Shou M, Stiber JA et al. Adenoviral-mediated gene transfer induces sustained pericardial VEGF expression in dogs: effect on myocardial angiogenesis. Cardiovasc Res, 1999; 44:294-302.
43. Lamping KG, Rios CD, Chun JA, et al. Intrapericardial administration of adenovirus for gene transfer. Am J Physiol, 1997; 272:H310-7.
44. Aoki K, Barker C, Danthinne X, et al. Efficient in vivo gene transfer into the heart in the rat. J Mol Cell Cardiol, 1997; 29: 949-959.
45. Fromes Y. Salmom A, Wang X, et al. Gene delivery to the myocardium by intrapericardial injection. Gene Therapy, 1999; 6:683-688.
46. Li J, Li G, Huang C, et al. Comparative study of catheter-mediated gene transfer into heart. Chin Med J (Engl), 2002; 115(4):612-3.
47. Andreason GL, Evans GA: Optimization of electroporation for transfection of mammalian celt lines. Anal Biochem, 1989; 180:269-275.
48. Vicar JM, Boisseau S, 3ourdes P, et al. Muscle transfection by electroporation with high-voltage and short-pulse currents provides high-level and long-lasting gene expression. Hum Gene Ther, 2000:11:909-916.
49. Kawaguchi HK, Slain VS, Wang YP, et al. In vivo gene transduction of human endothelial cell myocytes causes apoptosis-like cell death identification using sendaivirus-coated liposme. Circulation, 1997, 95:2441-2447.
50. Seppo YH, John FM. Cardiovascular gene therapy. Lancet, 2000, 355: 213-219.
51. Svenssn EC, Deborau G, Marsha LL. Efficient and stable-transduction of cardiomyocyte after intra-myocardio injection or intracoronary perfusion recombinant adeno-assioated virus vectors. Circulation, 1999, 99: 201-205.
52．心血管外科中基因转移技术的研究．梁宏立．国外医学：心血管疾病分册，2000，27(1)：7-10．
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54. Fuchs S, Baffour R, Schwartz B, et al.: Could plasmid mediated gene transfer into the myocardium be augmented by left- ventricular guided laser myocardial injury. J Am Coll Cardiol. 2001; 34:7A.
55. Holash J, Wiegand SJ, Yancopoulos GD: New model of tumor angiogenesis: dynamic balance between vessel regression and growth mediated by angiopoietins and VEGF. Oncogene. 1999; 18:5356-5362.
56. Carmeliet P: Mechanisms of angiogenesis and arteriogenesis. Nat Med. 2000; 6:389-395.
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2. Dimmeler S, Zeiher AM, Schneider MD. Unchain my heart: the scientific foundations of cardiac repair. J. Clin. Invest, 2005; 115:572-583.
3. Hammond HK, McKirman MD. Angiogenic gene therapy for heart disease: a review of animal studies and clinical trials. Cardiovascular Research, 2001, 49: 561-567.
4. Tio RA, Grandjean JG, Suurmeijer AJ, et al. Monitoring for pericardial application of local drug or gene therapy. Int J Cardiol, 2002, 82(2): 117-21.
5. Lazarous DF, Shou M, Stiber JA et al. Adenoviral-mediated gene transfer induces sustained pericardial VEGF expression in dogs: effect on myocardial angiogenesis. Cardiovasc Res, 1999, 44:294-302.
6. Lamping KG, Rios CD, Chun JA, et al. Intrapericardial administration of adenovirus for gene transfer. Am J Physiol, 1997 Jan, 272(1 Pt 2):H310-7.
7. Aoki K, Barker C, Danthinne X, et al. Efficient in vivo gene transfer into the heart in the rat. J Mol Cell Cardiol, 1997, 29: 949-959.
8. Fromes Y, Salmom A, Wang X, et al. Gene delivery to the myocardium by intrapericardial injection. Gene Therapy, 1999, 6:683-688.
9. Li J, Li G, Huang C, et al. Comparative study of catheter-mediated gene transfer into heart. Chin Med J (Engl), 2002, 115(4):612-3.
1. Bosaris T, Lee AK, Dekellei RA, et al. Microvessel quantitation and prognosis in invasive breast carcinoma. N Engl J Med. 1991; 342:1-8.
2. Mack CA, Patel SR, Schwartz EA et al. Biologic bypass with the use of adenovirus-mediated gene transfer of the complementary deoxyribonucleic acid for vascular endothelial growth factor 121 improves myocardial perfusion and function in the ischemic porcine heart. J Cardiovasc Surg. 1998; 115:168-177.
3. Patel SR, Lee LY, Mack CA, et al. Safety of direct myocardial administration of an adenovirus vector encoding vascular endothelial growth factor 121. Hum Gene Ther, 1999; 10:1331-1348.
4. Kornowski R, Leon MB, Fuchs S, et al. Electromagnetic guidance for catheter-based transendocardial injection: a platform for intramyocardial angiogenesis therapy. J Am Coil Cardiol, 2000; 35:1031-1039.
5. Safi J, DiPaula AF, Riccioni T et al. Adenovirus-mediated acidic fibroblast growth factor gene transfer induces angiogenesis in the nonischemic rabbit heart. Microvasc Res, 1999; 58:238-249.
6．张端珍，盖鲁粤，陈以旺，等．血管内皮生长因子对猪心肌侧枝血管生成的作用．生理学报，2001：3．
7. Rosengart TK, Lee LY, Patel SR, et al. Angiogenesis gene therapy: Phase Ⅰ assessment of direct intramyocardial administration of an adenovirus vector expressing VEGF121 cDNA to individuals with clinically significant coronary artery disease. Circulation, 1999; 100:468-474.
8. Giordano F, Ping P, McKiman MD, et al. Intracoronary gene transfer of fibroblast growth factor-5 increases blood flow and contractile function in an ischemic region of the heart. Nat Med, 1996; 2:534-539.
9. McKirnan MD, Lai NC, Waldman L, et al. Intracoronary gene transfer of fibroblast growth factor-4 increases regional contractile function and responsiveness to adrenergic stimulation in heart failure. Cardiac Vasc Regen, 2000; 1:11-21.
10. Padua RR, Sethi R, Dhalla NS, et al. Basic fibroblast growth factor is cardioprotective in ischemia-reperfusion injury. Mol Cell Biochem, 1995; 143:129-135.
11. Hammond HK, McKirnan. Angiogenic gene therapy for heart disease: a review of animal studies and clinical trials. Cardiovascular Research, 2001: 49: 561-567.
12. Grines CL, Watkins MW, Helmer G, et al. Angiogenic Gene Therapy (AGENT) trial in patients with stable angina pectoris. Circulation, 2002; 105(11): 1291-7.
13. Simons M, Annex BH, Laham RJ, et al. Pharmacological treatment of coronary artery disease with recombinant fibroblast growth factor-2: double-blind, randomized, controlled clinical trial. Circulation, 2002; 105(7):788-93.
14. Hedman M, Hartikainen J, Syvanne M, et al. Safety and feasibility of catheter-based local intracoronary vascular endothelial growth factor gene transfer in the prevention of postangioplasty and in-stent restenosis and in the treatment of chronic myocardial ischemia: phase II results of the Kuopio Angiogenesis Trial (KAT).Circulation, 2003; 107(21):2677-83.
15. Lazarous DF, Shou M, Stiber JA, et al. Adenoviral-mediated gene transfer induces sustained pericardial VEGF expression in dogs: effect on myocardial angiogenesis. Cardiovasc Res, 1999; 44:294-302.
16. Lamping KG, Rios CD, Chun JA, et al. Intrapericardial administration of adenovirus for gene transfer. Am J Physiol, 1997 ; 272:H310-7.44.
17. Aoki K, Barker C, Danthinne X, et al. Efficient in vivo gene transfer into the heart in the rat. J Mol Cell Cardiol, 1997; 29: 949-959.45.
18. Rutanen J, Rissanen TT, Markkanen JE, et al. Adenoviral catheter-mediated intramyocardial gene transfer using the mature form of vascular endothelial growth factor-D induces transmural angiogenesis in porcine heart. Circulation, 2004; 109(8):1029-35.
19. Tio RA, Grandjean JG, Suurmeijer AJ, et al. monitoring for pericardial application of local drug or gene therapy. Int J Cardiol, 2002 ; 82(2): 117-21.
1. Bosaris T, Lee AK, Dekellei RA, et al. Microvessel quantitation and prognosis in invasive breast carcinoma. N Engl J Med, 1991; 342: 1-8.
2. Mack CA, Patel SR, Schwartz EA, et al. Biologic bypass with the use of adenovirus-mediated gene transfer of the complementary deoxyribonucleic acid for vascular endothelial growth factor 121 improves myocardial perfusion and function in the ischemic porcine heart. J Cardiovasc Surg, 1998; 115:168-177.
3. Patel SR, Lee LY, Mack CA, et al. Safety of direct myocardial administration of an adenovirus vector encoding vascular endothelial growth factor 121. Hum Gene Ther, 1999; 10:1331-1348.
4. Kornowski R, Leon MB, Fuchs S, et al. Electromagnetic guidance for catheter-based transendocardial injection: a platform for intramyocardial angiogenesis therapy. J Am Coll Cardiol, 2000; 35:1031-1039.
5. Safi J, DiPaula AF, Riccioni T, et al. Adenovirus-mediated acidic fibroblast growth factor gene transfer induces angiogenesis in the nonischemic rabbit heart. Microvasc Res, 1999; 58:238-249.
6．张端珍，盖鲁粤，陈以旺，等．血管内皮生长因子对猪心肌侧枝血管生成的作用．生理学报，2001；3．
7. Rosengart TK, Lee LY, Patel SR, et al. Angiogenesis gene therapy: Phase Ⅰ assessment of direct intramyocardial administration of an adenovirus vector expressing VEGF121 cDNA to individuals with clinically significant coronary artery disease. Circulation, 1999; 100:468-474.
8. Giordano F, Ping P, McKirnan MD, et al. Intracoronary gene transfer of fibroblast growth factor-5 increases blood flow and contractile function in an ischemic region of the heart. Nat Med, 1996; 2:534-539.
9. McKirnan MD, Lai NC, Waldrnan L, et al. Intracoronary gene transfer of fibroblast growth factor-4 increases regional contractile function and responsiveness to adrenergic stimulation in heart failure. Cardiac Vasc Regen, 2000; 1:11-21.
10. Padua RR, Sethi R, Dhalla NS, et al. Basic fibroblast growth factor is cardioprotective in ischemia-reperfusion injury. Mol Cell Biochem, 1995; 143:129-135.
11. Hammond HK, McKirnan. Angiogenic gene therapy for heart disease: a review of animal studies and clinical trials. Cardiovascular Research, 2001: 49: 561-567.
12. Grines CL, Watkins MW, Helmer G, et al. Angiogenic Gene Therapy (AGENT) trial in patients with stable angina pectoris. Circulation, 2002; 105(11): 1291-7.
13. Simons M, Annex BH, Laham RJ, et al. Pharmacological treatment of coronary artery disease with recombinant fibroblast growth factor-2: double-blind, randomized, controlled clinical trial. Circulation, 2002; 105(7):788-93.
14. Hedman M, Hartikainen J, Syvanne M, et al. Safety and feasibility of catheter-based local intracoronary vascular endothelial growth factor gene transfer in the prevention of postangioplasty and in-stent restenosis and in the treatment of chronic myocardial ischemia: phase II results of the Kuopio Angiogenesis Trial (KAT).Circulation, 2003; 107(21):2677-83.
15.Rutanen J, Rissanen TT, Markkanen JE, et al. Adenoviral catheter-mediated intramyocardial gene transfer using the mature form of vascular endothelial growth factor-D induces transmural angiogenesis in porcine heart. Circulation, 2004; 109(8):1029-35.
16. Tio RA, Grandjean JG, Suurmeijer AJ, et al. monitoring for pericardial application of local drug or gene therapy. Int J Cardiol, 2002 ; 82(2): 117-21.
17. Lazarous DF, Shou M, Stiber JA et al. Adenoviral-mediated gene transfer induces sustained pericardial VEGF expression in dogs: effect on myocardial angiogenesis. Cardiovasc Res, 1999; 44:294-302.
18. Lamping KG, Rios CD, Chun JA, et al. Intrapericardial administration of adenovirus for gene transfer. Am J Physiol, 1997; 272:H310-7.44.
19. Aoki K, Barker C, Danthinne X, et al. Efficient in vivo gene transfer into the heart in the rat. J Mol Cell Cardiol, 1997; 29: 949-959.45.
20. Fromes Y, Salmom A, Wang X, et al. Gene delivery to the myocardium by intrapericardial injection. Gene Therapy, 1999; 6:683-688.
1．关怀敏，刘鹏，解金红，等．复制缺陷的重组腺病毒经心包腔转染急性心肌梗死猪观察．郑州大学学报(医学版)，2006，41(1)：77-80．
2．董书强；梅举；张宝仁等．复制缺陷的重组腺病毒载体直接转染猪慢性缺血心肌的研究．中国胸心血管外科临床杂志，2002，9(2)：103-106．
3. Dimmeler S, Zeiher AM, Schneider MD. Unchain my heart: the scientific foundations of cardiac repair. J. Clin Invest, 2005; 115(3):572-583.
4. Hammond HK, McKirman MD. Angiogenic gene therapy for heart disease: a review of animal studies and clinical trials. Cardiovascular Research, 2001, 49(3): 561-567.
5. Tio RA, Grandjean JG, Suurmeijer AJ, et al. Monitoring for pericardial application of local drug or gene therapy. Int J Cardiol, 2002, 82(2):117-21.
6. Lazarous DF, Shou M, Stiber JA et al. Adenoviral-mediated gene transfer induces sustained pericardial VEGF expression in dogs: effect on myocardial angiogenesis. Cardiovasc Res, 1999, 44(2):294-302.
7. Lamping KG, Rios CD, Chun JA, et al. Intrapericardial administration of adenovirus for gene transfer. Am J Physiol, 1997 Jan, 272(1 Pt 2):H310-7.
8. Aoki K, Barker C, Danthinne X, et al. Efficient in vivo gene transfer into the heart in the rat. J Mol Cell Cardiol, 1997, 29(3): 949-959.
9. Fromes Y, Salmom A, Wang X, et al. Gene delivery to the myocardium by intrapericardial injection. Gene Therapy, 1999, 6(4):683-688.
10. Li J, Li G, Huang C, et al. Comparative study of catheter-mediated gene transfer into heart. Chin Med J (Engl), 2002, 115(4):612-3.
11. Quantin B, Penicaudet LD, Jajbaksh S. et al. Adenovirus as an expression vector in muscle cells in vivo. Proc. Nail Acad Sci USA, 1992, 990: 2581-2592.
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12．孙秀玲．心血管疾病的基因治疗．生命的化学．2001；4：341-344．
13. Mack CA, Patel SR, Schwartz EA, et al. Biologic bypass with the use of adenovirus-mediated gene transfer of the complementary deoxyribonucleic acid for vascular endothelial growth factor 121 improves myocardial perfusion and function in the ischemic porcine heart. J Cardiovasc Surg, 1998; 115:168-177.
14. Patel SR, Lee LY, Mack CA, et al. Safety of direct myocardial administration of an adenovirus vector encoding vascular endothelial growth factor 121. Hum Gene Ther, 1999; 10:1331-1348.
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22. Safi J, DiPaula AF, Riccioni T. et al. Adenovirus-mediated acidic fibroblast growth factor gene transfer induces angiogenesis in the nonischemic rabbit heart. Microvasc Res, 1999; 58:238-249.
23．张端珍，盖鲁粤，陈以旺，等．血管内皮生长因子对猪心肌侧枝血管生成的作用．生理学报，2001；3．
24. Rosengart TK, Lee LY, Patel SR, et al. Angiogenesis gene therapy: Phase I assessment of direct intramyocardial administration of an adenovirus vector expressing VEGF121 cDNA to individuals with clinically significant coronary artery disease. Circulations, 1999; 100:468-474.
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28. Aoki M, Morishita R, Taniyame Y. et al. Angiogenesis induced by hepatocyte growth factor in non-infarcted myocardium and infarcted placebomyocardium: up-regulation of essential transcription factor for angiogenesis, ets. Gene Ther, 2000; 7:417-427.
29. Losordo DW, Vale PR, Symes JF, et al.: Gene therapy for myocardial angiogenesis: initial clinical results with direct myocardial injection of phVEGF165 as sole therapy for myocardial ischemia. Circulation, 1998, 98:2800-2804.
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31. Vale PR, Losordo DW, Milleiken MA, et al. Left ventricular electromechanical mapping to assess efficacy of phVEGF via a 165 minithoracotomy. Circulation, 2000; 102:965-974.
32. Vale PR, Losordo DW, Milliken CE, et al. Randomized, single-blind, placebo-controlled pilot study of catheter-based myocardial gene transfer for therapeutic angiogenesis using left ventricular electromechanical mapping in patients with chronic myocardial ischemia. Circulation, 2001; 103 (17): 138-43.
33. Giordano F, Ping P, McKirnan MD, et al. Intracoronary gene transfer of fibroblast growth factor-5 increases blood flow and contractile function in an ischemic region of the heart. Nat Med, 1996; 2:534-539.
34. McKirnan MD, Lai NC, Waldman L, et al. Intracoronary gene transfer of fibroblast growth factor-4 increases regional contractile function and responsiveness to adrenergic stimulation in heart failure. Cardiac Vasc Regen, 2000; 1:11-21.
35. Padua RR, Sethi R, Dhalla NS, et al. Basic fibroblast growth factor is cardioprotective in ischemia-reperfusion injury. Mol Cell Biochem, 1995; 143: 129-135.
36. Hammond HK, McKiman. Angiogenic gene therapy for heart disease: a review of animal studies and clinical trials. Cardiovascular Research. 2001:49:561-567.
37. Grines CL, Watkins MW. Helmet G, et al. Angiogenic Gene Therapy (AGENT) trial in patients with stable angina pectoris. Circulation, 2002; 105(11): 1291-7.
38. Simons M, Annex BH, Laham RJ, et al. Pharmacological treatment of coronary artery disease with recombinant fibroblast growth factor-2: double-blind. randomized, controlled clinical trial. Circulation, 2002; 105(7):788-93.
39. Hedman M, Hartikainen J, Syvanne M, et al. Safety and feasibility of catheter-based local intracoronary vascular endothelial growth factor gene transfer in the prevention of postangioplasty and in-stent restenosis and in lbe treatment of chronic myocardial ischemia: phase Ⅱ results of the Kuopio Angiogenesis Trial (KAT). Circulation, 2003; 107(21):2677-83.
40. Rutanen J, Rissanen TT, Markkanen JE. et al. Adenoviral catheter-mediated intramyocardial gene transfer using the mature form of vascular endothelial growth factor-D induces transmural angiogenesis in porcine heart. Circulation, 2004; 109(8):1029-35.
41. Tio RA, Grandjean JG, Suurmeijer AJ, et al. Monitoring for pericardial application of local drug or gene therapy. Int J Cardiol, 2002; 82(2):117-21.
42. Lazarous DF, Shou M, Stiber JA et al. Adenoviral-mediated gene transfer induces sustained pericardial VEGF expression in dogs: effect on myocardial angiogenesis. Cardiovasc Res, 1999; 44:294-302.
43. Lamping KG, Rios CD, Chun JA, et al. Intrapericardial administration of adenovirus for gene transfer. Am J Physiol, 1997; 272:H310-7.
44. Aoki K, Barker C, Danthinne X, et al. Efficient in vivo gene transfer into the heart in the rat. J Mol Cell Cardiol, 1997; 29: 949-959.
45. Fromes Y. Salmom A, Wang X, et al. Gene delivery to the myocardium by intrapericardial injection. Gene Therapy, 1999; 6:683-688.
46. Li J, Li G, Huang C, et al. Comparative study of catheter-mediated gene transfer into heart. Chin Med J (Engl), 2002; 115(4):612-3.
47. Andreason GL, Evans GA: Optimization of electroporation for transfection of mammalian celt lines. Anal Biochem, 1989; 180:269-275.
48. Vicar JM, Boisseau S, 3ourdes P, et al. Muscle transfection by electroporation with high-voltage and short-pulse currents provides high-level and long-lasting gene expression. Hum Gene Ther, 2000:11:909-916.
49. Kawaguchi HK, Slain VS, Wang YP, et al. In vivo gene transduction of human endothelial cell myocytes causes apoptosis-like cell death identification using sendaivirus-coated liposme. Circulation, 1997, 95:2441-2447.
50. Seppo YH, John FM. Cardiovascular gene therapy. Lancet, 2000, 355: 213-219.
51. Svenssn EC, Deborau G, Marsha LL. Efficient and stable-transduction of cardiomyocyte after intra-myocardio injection or intracoronary perfusion recombinant adeno-assioated virus vectors. Circulation, 1999, 99: 201-205.
52．心血管外科中基因转移技术的研究．梁宏立．国外医学：心血管疾病分册，2000，27(1)：7-10．
53. Sayeed-Shah U, Mann MJ, Martin J el al. Complete reversal of ischemic wall motion abnormalities by combined use of gene therapy with transmyocardial laser revascularization. J Thorac Cardiovasc Surg. 1998; 116:763-769.
54. Fuchs S, Baffour R, Schwartz B, et al.: Could plasmid mediated gene transfer into the myocardium be augmented by left- ventricular guided laser myocardial injury. J Am Coll Cardiol. 2001; 34:7A.
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