背景与目的 近年来，随着分子生物学技术的发展，基因转染体系日益完善，基因治疗作为传统药物治疗外的新治疗方法在心血管疾病领域显示出广阔的前景。整合素连接激酶(integrin-linked kinase，ILK)是一种能够与整合素β₂胞内区结合的Ser/Thr蛋白激酶。诸多研究显示，ILK在细胞的整合素通路中扮演主要角色，参与调控细胞的存活、增殖、肥大、迁移和血管新生。研究表明，ILK是心脏机械牵拉感受器的关键性组成，介导心肌细胞的机械牵拉—应答反应。同时，ILK也是募集内皮祖细胞(EPCs)至缺血组织的重要分子。本研究在建立动物心肌梗死模型的基础上给予外源性ILK基因转染，旨在探讨ILK基因治疗能否缓解心肌梗死后的左室重构，并进一步探讨其可能机制。 方法 I.采用AdEasy系统构建含ILK基因和人源化绿色荧光蛋白(humanized recombinant green fluorescent protein，hrGFP)报告基因的重组腺病毒，经293细胞扩增和进一步纯化得到实验用重组腺病毒载体。2.SPF级雄性成年SD (Sprague-Dawley)大鼠在呼吸机辅助呼吸支持下开胸行左前降支结扎术，建立动物心肌梗死模型。通过术中观察结扎区心肌变苍白、室壁运动减弱，以及术后1周采用红四氮唑(2，3，4-triphenyltetrazolium chloride dye，TTC)染色法确定造模成功。3.评估 ILK 重组腺病毒在体转染后表达情况。采用蛋白免疫印迹法检测ILK重组腺病毒在体转染大鼠心脏后各时间点表达水平：采用免疫荧光法从形态学上检测重组腺病毒在心梗大鼠心脏内表达情况。4.探讨急性心肌梗死后即刻转染ILK重组腺病毒是否能缓解心梗的左室重构、改善心功能及其可能机制：在大鼠结扎左前降支后立即在梗死周围区注射ILK重组腺病毒或空载腺病毒，在注射后4周后行超声心动图、血流动力学检查评估心功能，苏木素—伊红(hematoxylin & eosin，H&E)染色法检测心脏形态学，masson三染色法检测胶原沉积情况，CD31免疫组化染色评估心脏血管新生情况，TUNEL法检测心肌细胞凋亡水平，Phospho-histone H3 免疫突光染色、增殖细胞核抗原(proliferating cell nuclear antigen，PCNA)和Ki-67免疫组化染色评估心肌细胞增殖情况。5.观察ILK重组腺病毒治疗效果持续时间：制作前述的大鼠心梗周围区转染模型并延长观察期至9周，行超声心动图、血流动力学、病理形态学检测。6.探讨心肌梗死1周后转染ILK重组腺病毒是否能缓解心梗的左室重构并改善心功能：在大鼠左前降支结扎1周后于梗死周围区注射1LK重组腺病毒或空载病毒，4周后行超声心动图、血流动力学、病理形态学检 结果 1.在正常心脏转染模型中，与空载腺病毒转染组(对照组)相比，ILK腺病毒转染组(ILK组)的心肌组织内ILK和hrGFP表达明显增加，外源基因表达在约2周时达峰，随后逐渐减少，5周时完全消失。2.在心肌梗死转染模型中，转染3天后ILK组心肌组织内的外源基因表达依然高于对照组，同时免疫荧光提示外源基因主要在心肌细胞内表达，部分在心脏成纤维细胞内表达。3.转染4周后，ILK组的心功能、血流动力学、心脏形态学指标均优于对照组。ILK组心肌细胞凋亡率、梗死周围区胶原容积分数均低于对照组。与对照组比较，ILK组的微血管密度、心肌细胞增殖情况明显增加。 4.转染7周后，ILK组的心功能仍优于对照组，但9周后ILK组心功能和血流动力学参数与对照组相比无明显差异。5.在心梗1周后转染模型中，转染4周后，ILK组心功能和部分血流动力学参数较对照组有所改善，左室内径和梗死周围区室壁厚度均优于对照组，而梗死面积、梗死远区、变薄率与对照组比较无明显差异。 结论 1.急性心肌梗死后立即行ILK基因治疗可以缓解心肌梗死后的左室重构，这一作用可能与ILK降低梗死后心肌凋亡率、增加血管新生和促进心肌增殖等多种因素有关。2. ILK重组腺病毒的治疗效应持续约7周左右，不超过9周。3.心梗1周后再转染ILK基因可以部分缓解心梗的左室重构并改善心功能。 关键词 心肌梗死；左室重构；基因治疗；整合素连接激酶(ILK)；凋亡；血管新生；增殖

 Backgroud and Objective - Left ventricular (LV) remodeling is associated with the development of heart failure post-myocardial infarction. After myocardial infarction, cardiomyocyte loss and increased load trigger a cascade of biochemical intracellular signaling processes that modulate cardiac remodeling. Prevention or attenuation of these signaling processes is an important goal of anti-remodeling therapy. Integrin-linked kinase (ILK), a widely expressed serine/threonine protein kinase, plays an important role in transducing cell-matrix interaction-induced biomechanical signals which regulate cytoskeletal remodeling, angiogenesis, cell growth, proliferation, survival and differentiation. Recently, ILK has been reported to be an critical component of the cardiac stretch sensor which regulates cardiac contractility, compensatory hypertrophy, survival and repair. In the present study, we investigated whether integrin-linked kinase (ILK) could regulate LV remodeling and function after myocardial infarction. Additionally, long-term follow-up and delayed ILK administration were made to see whether the effect of ILK-overexpression is sustained or temporary and whether it have a rescue effect on the remodeling process. Methods - Recombinant adenoviral vector containing both human wild-type ILK and humanized recombinant green fluorescent protein (hrGFP), as well as null-content adenoviral vector, was prepared. Male Sprague-Dawley rats were intramyocardially injected with adenoviral vector expressing ILK or empty ad-null in the peri-infarct area following left anterior descending coronary artery (LAD) ligation. ILK and report gene (hrGFP) expression were confirmed by western blotting and immunohistochemistry in both noninfarcted and infarcted hearts. Echocardiographic and hemodynamic analyses were performed 4 weeks after transfection. Then myocardial tissues were harvested and fixed for subsequent histological, immunohistochemical and TUNEL examination. Histological (morphological) analysis of left ventricle was performed using H&E (hematoxylin & eosin) staining and fibrosis was analyzed by Masson's trichrome. Angiogenesis was evaluated by microvessel density using CD31 staining. Apoptosis was measured by TUNEL analysis. Cardiomyocyte proliferation was estimated by Proliferating Cell Nuclear Antigen, Ki-67, and phosphohistone-H3 staining. Moreover, a subset of rats was intramyocardially injected with ad-ILK or ad-null vector in the border zone immediately after LAD ligation to investigate whether the effect of ILK-overexpression following myocardial infarct is sustained or temporary and examined by echocardiography 7 weeks and 9 weeks after adenoviral delivery. Hemodynamics and histology were evaluated 9 weeks after delivery. In order to ascertain if ILK-overexpression can also have a rescue effect on the remodeling process, a separate cohort of rats was intramyocardially injected with ad-ILK or ad-null vector one week after LAD ligation. Echocardiographic, hemodynamic and histologic analyses were performed 4 weeks later. Results - Western blotting analysis revealed higher expression of ILK and hrGFP in border area of ad-ILK heart as compared with ad-null controls, from 3 to 28 days. In ad-ILK group, ILK and hrGFP protein levels rose progressively between 3-14 days after adenoviral delivery, following which ILK and hrGFP levels gradually declined and tapered off by 35 days. Elevated expression of ILK and hrGFP were also seen in infarcted hearts 3 days after ad-ILK injection in border area. Border and non-infarcted immunohistochemistry showed exotic gene expression in the border and non-infarcted areas around the injection sites in ad-ILK animals. The exotic gene was mainly expressed in cardiomyocytes and partly in cardiac fibroblasts. Four weeks after transfection, echocardiographic and hemodynamic analysis demonstrated relatively preserved cardiac function in the ILK group. ILK treatment was associated with reduced infarct scar size, increased scar thinning ratio and preserved LV geometry (including LV diameter, LV wall thickness, cardiomyocyte size and myofilament density). Enhanced angiogenesis and reduced fibrosis were observed in ad-ILK animals. TUNEL analysis also revealed a reduction in apoptosis in the ILK group. Moreover, increased cardiomyocyte proliferation was found in ad-ILK animals through PCNA, Ki-67 and phospho-histone H₃ staining. Our results also demonstrate that the effect of ILK treatment persists for approximately 7 weeks. Additionally, ILK over-expression was found to exert a rescue effect on the remodeling process, when administered in a delayed fashion 1 week following coronary artery ligation. Conclusions - ILK gene therapy improves cardiac remodeling and function in rats following myocardial infarction, and is associated with increased angiogenesis, reduced apoptosis and increased cardiomyocyte proliferation. The therapeutic effect of ILK treatment persists for approximately 7 weeks. ILK over-expression can also have a rescue effect on the remodeling process. These results may represent a new approach to the treatment of post-infarct remodeling and subsequent heart failure. Keywords: myocardial infarction; remodeling; gene therapy; Integrin-linked kinase; apoptosis; angiogenesis; proliferation