冠状动脉再狭窄基因治疗中靶基因的选择

二十余年来，冠心病介入治疗取得了巨大进展。但是冠状动脉成形术(PTCA)后有约30～50％的病人可发生再狭窄，其机理涉及血管回缩、血栓形成、血管收缩性重构和内膜增生。将外源基因导入病变血管，从不同环节抑制平滑肌细胞的迁移、增殖及其他病理生理过程为再狭窄的治疗提供了新的选择。目前在基因治疗冠状动脉再狭窄的研究中，靶基因的选择有以下几个方面。     

血管内皮生长因子与心肌缺血的血管重建

心肌缺血时血管内皮生长因子及其mRNA表达增加可促进心肌新生血管的形成和侧枝循环的建立;用血管内皮生长因子基因治疗心肌缺血可促进心肌的血管重建,改善心肌供血,明显减轻临床症状,为冠心病患者提供了一种有效的治疗方法.     

血管内皮生长因子复合人工骨材料修复骨缺损

背景：由创伤、感染、肿瘤切除以及某些先天性疾病等所导致的四肢骨缺损的治疗仍是骨科临床最常见、最棘手的一大难题。 目的：文章综述了血管内皮生长因子的生物学特性、及其复合人工骨的作用机制、局部基因治疗在治疗骨缺损中的应用。 方法：应用计算机检索CNKI和PubMed数据库中1999-01/2009-12关于血管内皮生长因子和骨缺损修复相关的内容的文章,在标题和摘要中以“血管内皮生长因子,骨缺损,人工骨,基因治疗”或“VEGF,Bone defect,Bone substitute,Gene therapy”为检索词进行检索。选择文章内容与血管内皮生长因子和骨缺损修复相关,同一领域文献则选择近期发表或发表在权威杂志文章。 结果与结论：初检得到285篇文献,根据纳入标准选择45篇文章进行综述。血管内皮生长因子是增加血管渗透性和血管发生的特殊生长因子,在骨的生长、发育和重建中密切协调血管发生和骨发生的关系,而目前的人工骨材料只具有传导成骨的特性,所以改善人工骨材料中的血管生成可促进血运丰富骨组织的形成。     

勃起功能障碍基因治疗研究进展

ED发病率较高，现有多种治疗手段存在诸多弊端，分子生物学技术的应用使ED基因治疗初显成效。本文综述了在腺病毒、腺相关病毒、逆转录病毒载体与非病毒载体等介导下，转导一氧化氮合酶基因、血管内皮细胞生长因子基因、反义核酸基因、maxi-k+基因、Ca2+通道相关调节基因、降钙素基因相关肽基因、SOD基因等治疗ED的研究进展。     

rhbFGF基因及其蛋白促兔缺血心肌血管新生的对比研究

目的 探讨通过心肌内注射重组人碱性成纤维细胞生长因子(rhbFGF)基因及其蛋白促兔缺血心肌血管新生的“基因血管搭桥”的效果。方法 无菌条件下开胸结扎兔冠状动脉左前降支(LAD)，建立急性心肌梗死动物模型。将成功构建的真核表达质粒pcDNA3-bFGF、rhbFGF蛋白、生理盐水，直接四点注射人兔缺血心肌内。实验兔饲养6周、12周以后，通过病理切片光镜观察、图像分析对比研究三组间血管新生情况。结果 1)成功构建pcDNA3-bFGF真核表达质粒并制备rhbFGF蛋白；(2)pcDNA3-bFCF真核表达质粒在心肌中成功表达为rhbFGF蛋白；(3)血管新生的观察：通过对心肌内注射基因及其蛋白进行对比研究，发现心肌内注射蛋白和基因均有促兔缺血心肌血管新生的作用，而基因的促血管新生作用更强。结论 rhbFGF蛋白及基因心肌内注射均有促兔缺血心肌血管新生的作用，而基因的促血管新生效果显著。“基因治疗促血管新生”法是另一种有效的冠心病治疗方法，rhbFGF基因有重要的推广应用价值。     

VEGF与肿瘤血管生成及抗血管生成基因治疗

ＶＥＧＦ是一种重要的血管生成因子，已发现许多生长因子如ＥＧＦ，ＰＤＧＦＢＢ，ｂＦＧＦ，ＴＧＦβ等都是通过诱导ＶＥＧＦ的表达而起作用的。ＶＥＧＦ 可改变内皮细胞基因的活化形式，增强血管通透性，诱导血管生成。目前抗血管生成基因治疗是肿瘤治疗研究的热点，大多数肿瘤表达高水平ＶＥＧＦ，且其表达与肿瘤的恶性程度呈正相关。     

人类生长因子诱导缺血性心肌的血管生成:一种冠心病治疗新方法的首次临床报告

该研究旨在评价动物实验中血管生长因子生物学效应以及人类生长因子 (HGF)治疗冠心病的潜在疗效。研究涉及 1通过基因工程对 HGF的提取、分离、定性及纯化。 2应用动物实验证实 HGF的新生血管作用 ,以及排除任何可能的致病作用。 3临床应用碱性成纤维细胞生长因子 (FGF-I)作为冠脉搭桥术后的辅助治疗并证实其新生血管作用。方法与结果　选择 2 0例患冠状动脉多支病变的患者接受研究 (男性 14例 ,女性 6例 ) ,最小年龄 5 0岁 ,均无心肌梗塞和手术史。FGF- I将在搭桥手术中使用。另选 2 0例情况相同的患者作为对照组。两组患者在临床症…     

β—地中海贫血基因治疗研究进展

基因治疗是根治β地贫的唯一途径。本就目前对β地贫基因治疗研究的β珠蛋白基因转移治疗，药物遥基因治疗，反义核酸技术的治疗作了较为系统的综述，着重介绍了ＲＶ、ＡＡＶ载体介导β基因导入β地贫患造血干／祖细胸以获得细胞和红系组织的稳定整合和正常     

原发性高血压的基因治疗

基因治疗为高血压的长效（数周，数月或数年）治疗提供了可能，高血压病基因治疗的临床前研究运用了两种策略：1，将功能基因导入体内增加扩血管蛋白物质。2，将反义脱氧寡核苷酸（AS－ODN）或反义DNA导入体内，抑制缩血管基因的功能。为达到此目的，已建立了两种基因导入方法：1，将AS－ODN直接注射入人体或附着于阳离子脂质体注入。2，利用含完整DNA的病毒载体。目前，临床前研究资料提示高血压病的基因治疗是可行的，但在临床用于人的基因治疗前还需进行深入研究，一些理论问题还尚待解决。     

十二烷基化壳聚糖基因支架血管内基因转运的实验研究

目的目前基因治疗方面存在的重要问题是缺乏有效的基因转运体系可以足量、安全地将治疗基因(无论病毒载体或非病毒载体)运送至体内靶细胞并提高其转染效率,以得到基因的高效表达。心血管内基因治疗的特殊性还在于很难把基因专一递送至血管组织的病灶处而不进入血液循环系统。实验研究提出了一种携带质粒DNA的烷基化壳聚糖纳米粒的血管内支架,可以有效地将质粒DNA递送至血管壁靶细胞并达到了高效转染的效果。     

New Approaches to Coronary Heart Disease

Currently available approaches for treating human coronary heart disease aim to relieve symptoms and the risk of myocardial infarction by reducing myocardial oxygen demand (drugs), preventing further disease progression (drugs), restoring coronary blood flow either pharmacologically (thrombolysis) or mechanically (angioplasty), or bypassing the stenotic lesions and obstructed coronary artery segments (surgery). Direct gene therapy, as well as gene-derived therapy, especially by angiogenic growth factors, is emerging as a potential new treatment for cardiovascular disease. After extensive experimental research on angiogenic growth factors, the first clinical studies on patients with coronary heart disease or peripheral vascular lesions are being performed. The polypeptides fibroblast growth factor (FGF) and vascular endothelial growth factor seem to be effective in initiating neovascularisation (neo-angiogenesis) in hypoxic or ischaemic tissues. The first clinical study on patients with coronary heart disease treated by local injection of FGF-1 into the compromised underperfused myocardial tissue showed a 3-fold increase of capillary density mediated by the growth factor. Angiogenic therapy of the human myocardium introduces a new modality of treatment for coronary heart disease in terms of regulation of blood vessel growth. Beyond drug therapy, angioplasty and bypass surgery, this therapy may evolve to be a fourth principle of treatment of atherosclerotic cardiovascular disease.     

FGF-4 Gene Therapy GENERX — Collateral Therapeutics

Collateral Therapeutics and Schering AG in Germany are developing a gene therapy product, GENERX for coronary artery disease. Based on the terms of the agreement, Schering or its affliates will be responsible for conducting and financing phase II/III clinical trials which are currently underway in the US and Europe. In particular, Berlex Labs (the US subsidiary of Schering AG), is involved in developing the gene therapy in the US. GENERX is an angiogenic gene therapy which triggers the production of a protein that stimulates new blood vessel growth providing an alternative route for blood to bypass clogged and blocked arteries in the heart. GENERX involves a one-time, non-surgical delivery of an adenovirus vector containing the human fibroblast growth factor-4 (FGF-4) into coronary arteries via a standard catheter. The FGF-4 gene was licensed from New York University. Collateral Therapeutics has been granted a US patent for "gene transfer-mediated angiogenesis therapy" for the nonsurgical administration of angiogenic genes for coronary and peripheral vascular disease. The patented technology has been licensed from the University of California. Collateral and Berlex have initiated pivotal phase IIb/III trials with GENERX in the US and Europe. The US-based study will evaluate the safety and efficacy of GENERX in patients with stable exertional angina due to coronary artery disease. The European-based study will evaluate patients with advanced coronary artery disease who are not considered candidates for interventions such as angioplasty and bypass surgery and/or patients who are unlikely to have positive outcomes from such interventions. Both studies, of a multicentre, randomised, double-blind and placebo-controlled design, will evaluate 2 dose levels of GENERX which will be non-surgically administered to the heart via intracoronary infusion through a standard cardiac catheter. Collateral also plans to develop a non-surgical gene therapy product using the FGF-4 gene for the treatment of patients with heart failure. In a blinded placebo-controlled study in a pig model of pacing-induced heart failure, intracoronary delivery of human FGF-4 expressed in an adenovirus vector showed significant improvement in regional cardiac function and a reduction in the size of the heart over a 3-week study period. If these results translated favourably to humans, FGF-4 gene therapy may be a therapeutic option for patients with dilated heart failure. Collateral Therapeutics has also announced a research collaboration with Targeted Genetics on the use of viral vectors to deliver therapeutic genes in cardiovascular disease. Under the terms of the agreement, Targeted Genetics and Collateral Therapeutics each have the option to collaborate further to use Targeted Genetics' recombinant adeno-associated viral vector to treat congestive heart failure. In such an event, Targeted Genetics would be responsible for constructing and manufacturing the vector, and Collateral Therapeutics will fund the costs of future collaboration. Either party may terminate this agreement at any time upon 30 days prior written notice.     

Gene therapy in cardiovascular diseases

Established modalities of treatment for obstructive coronary artery disease include medical therapy, bypass surgery and percutaneous coronary intervention. Similarly, conventional treatment of congestive heart failure is also limited to medical therapy, temporary assist devices and transplantation. A significant subset of patients with severe symptomatic coronary artery disease and end stage heart failure is not eligible for these traditional methods of treatment. In spite of maximal medical and revascularization therapy, these patients may not get adequate symptomatic benefit. After a decade of investigations, gene therapy has emerged as a promising therapeutic option for this group of patients. This review discusses newer modalities of therapy for this subset, including therapeutic angiogenesis with growth factors and cell transplantation.     

Therapeutic angiogenesis in ischemic heart disease: gene or recombinant vascular growth factor protein therapy?

In the last decennium the challenge to research has been to find methods of inducing new vascular growth in ischemic myocardium due to atherosclerotic coronary artery disease, which could not be treated with balloon angioplasty or coronary artery by-pass grafting. Therapeutic angiogenesis with recombinant vascular endothelial growth factor proteins or gene encoding for the proteins is a new potential treatment for cardiovascular disease. The greatest interest and research has been concentrated on basic Fibroblast Growth Factor (FGF1 and FGF2) and Vascular Endothelial Growth Factor A (VEGF-A165 and VEGF-A121). Several small clinical phase I-II safety and efficacy trials with recombinant vascular endothelial growth factor proteins or gene encoding for the proteins have demonstrated that these treatment regimes seem to be safe and the results have been encouraging. However, two large doubleblind randomized placebo-controlled studies with intracoronary infusions of the recombinant proteins FGF2 and VEGF-A165 could not detect any clinical effect. Large scaled phase II studies with gene therapy are in progress. Therapeutic angiogenesis is still a promising new treatment in patients with coronary artery disease. However, more research including large scaled clinical trials is needed before deciding whether the vascular endothelial growth factor therapy either as a gene or a recombinant slow-release protein formulation therapy can be offered to patients with severe coronary artery disease, which cannot be treated with conventional revascularization.     

Does gene therapy become pharmacotherapy?

Recent progress in molecular and cellular biology has led to the development of numerous effective cardiovascular drugs. However, there are still a number of diseases for which no known effective therapy exists, such as peripheral arterial disease, ischaemic heart disease, restenosis after angioplasty, and vascular bypass graft occlusion. Currently, gene therapy is emerging as a potential strategy for the treatment of cardiovascular disease despite its limitations. The first human trial in gene therapy for cardiovascular disease was started at 1994 to treat peripheral vascular disease using vascular endothelial growth factor (VEGF). Then, many different potent angiogenic growth factors were tested in clinical trials to treat peripheral arterial disease and ischaemic heart disease. Improvement of clinical symptoms in peripheral arterial disease and ischaemic heart disease has been reported. This review focuses on the future potential of gene therapy for the treatment of cardiovascular disease. In the future, gene therapy might become a real pharmacotherapy to treat cardiovascular disease.     

Clinical use of intracoronary gene transfer of fibroblast growth factor for coronary artery disease

Anginal symptoms due to myocardial ischemia continue to affect millions of patients despite ongoing improvements in the diagnosis and treatment of coronary artery disease. Revascularization therapy with percutaneous coronary interventions and coronary artery bypass graft surgery can be highly effective in eligible subjects, but many patients are suboptimal candidates due to various factors, which include diffuse vascular disease, poor ventricular function and failure of prior procedures. Introduction of vascular growth factors to the heart to promote angiogenesis and collateral vessel formation has emerged as an alternative strategy for the relief of myocardial ischemia in these patients. Early preclinical work demonstrated that gene transfer of fibroblast growth factor using an E1-deleted adenovirus vector via intracoronary injection could safely reverse stress-induced ischemic ventricular dysfunction with no discernible evidence of inflammatory response. The AGENT trial established that intracoronary administration of Ad5FGF-4 could be performed with reasonable safety to patients with coronary artery disease, and that a one-time dose could provide an anti-ischemic effect out to 12 weeks of evaluation. Further evaluation of the efficacy and safety of Ad5FGF-4 is now being conducted in two simultaneous multicenter, randomized, double-blind, placebo-controlled pivotal trials in the United States and the European Union, with planned enrollment of approximately 1000 treated subjects. The primary efficacy variable in the trial will be changed in treadmill exercise duration at 12 weeks compared to baseline. Secondary efficacy variables include the rate of all-cause mortality and coronary events (non-fatal myocardial infarction, and unplanned hospitalization and revascularization due to myocardial ischemia) up to 1 year.     

Functional characteristics of coronary vasomotor function following intramyocardial gene therapy with naked DNA encoding for vascular endothelial growth factor165.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor. VEGF gene therapy improves perfusion of ischemic myocardium in experimental models and possibly in patients with end-stage coronary artery disease. In addition to its proliferative and migratory effect on endothelial cells, it also activates and up-regulates endothelial nitric oxide synthase (eNOS). Therefore, the authors investigated coronary endothelium-dependent vasodilatation in patients before and after VEGF gene therapy. The effect of intracoronary acetylcholine infusion on coronary diameter was assessed at baseline and after 3 months follow-up in patients with end-stage coronary artery disease treated with VEGF gene and in controls scheduled for elective percutaneous transluminal coronary angioplasty (PTCA) (acetylcholine test at diagnostic angiography and before a subsequently scheduled PTCA). Five out of six VEGF patients experienced a reduction in anginal complaints. Angiographic evidence for improved collateral filling was evident in two out of six patients. The vasoconstrictive response to acetylcholine was partly converted into dilatation. In contrast, the acetylcholine response in control patients remained vasoconstrictive. In conclusion, VEGF gene therapy has an important beneficial effect on the functional characteristics of the myocardial vascular network. Therefore, this therapy can potentially play an important role in all stages of the atherosclerotic process.     

浅析心理治疗和抗焦虑剂对冠心病疗效的影响

为探讨心理治疗和抗焦虑剂在冠心病治疗中的作用，对冠心病患者进行常规药物治疗加服抗焦虑剂佳静安定，并予以支持性心理治疗、认识治疗、行为放松训练；对照组采用常规药物治疗。结果显示，研究组HAMA降分显著，疗效明显提高，治疗时间缩短。提示心理治疗和抗焦虑剂能够提高冠心病的疗效，经常性心理治疗对冠心病患者的康复有重要意义。     

Gene therapy approaches for cardiovascular diseases

Cardiovascular diseases are one of the main causes of mortality in Western countries. Gene therapy is emerging as a potential strategy for the treatment of cardiovascular diseases, such as peripheral arterial disease, ischemic heart disease, restenosis after angioplasty, vascular bypass graft occlusion and transplant-associated coronary artery disease. Since the initial experiments more than one decade ago, remarkable progress has been made in the field of gene transfer and human clinical trials are underway. In here we give an overview of available gene transfer strategies describing several delivery routes and currently used vectors in animal studies and clinical trials. Hereby we want to focus on new approaches including the potential combination of gene therapy with cell therapy and tissue engineering, gene silencing and recently developed techniques for targeting genes to the vascular wall and the myocardium.     

血浆活化凝血因子Ⅶ及其基因多态性与老年人冠状动脉粥样硬化性心脏病的相关分析

目的探讨血浆活化因子Ⅶ(activated coagubtionfactorⅦ,F7a)水平及其基因的MspⅠ多态性与老年人冠状动脉粥样硬化性心脏病的关系。方法采用候选基因及病例-对照的方法,以聚合酶链反应及限制性片段长度多态性分析技术,对108例老年冠状动脉粥样硬化性心脏病患者(冠状动脉粥样硬化性心脏病组)及120名年龄、性别匹配的健康人(正常对照组)行F7基因的MspⅠ多态性分析并确定基因型,同时采用重组可溶性组织因子法(rSTF2-219)测定血浆F7a水平。结果(1)老年冠状动脉粥样硬化性心脏病组血浆F7a水平显著高于正常对照组(2.88±0.62vs2.58±0.60μg/L,P<0.05),Logistic回归分析显示,血浆F7a水平与老年人冠状动脉粥样硬化性心脏病的危险性独立相关(OR1.21,P<0.05)。(2)F7基因型频率分布符合Hardy-Weinberg平衡,基因型及等位基因频率分布在两组间差异无统计学意义(P>0.05)。(3)两组血浆F7a水平均与F7基因多态性显著相关,RR基因型血浆F7a水平显著高于Q等位基因携带者(P<0.05)。结论血浆F7a水平增高是老年人冠状动脉粥样硬化性心脏病发病的独立危险因素,F7a水平受其基因的MspI多态性影响。