【作者】 王丽；

【导师】 刘正湘；

【作者基本信息】 华中科技大学 ， 内科心血管， 2006， 博士

【摘要】 研究背景和目的冠心病是临床常见的心血管疾病,其发病在我国呈明显上升趋势。心肌缺血后的血运重建对保护心脏功能、改善远期预后起着关键作用。尽管PTCA及CABG在临床被广泛应用,但大部分患者不能实施或无法恢复有效血运,血管生成的基因治疗则为缺血性心血管病的治疗开辟了新的途径。血管内皮细胞的粘附和迁移是血管形成的重要生理步骤之一,在心肌缺血后的血运重建中起着举足轻重的作用。以往的研究大都集中在血管内皮细胞生长因子(vascular endothelial growthfactor,VEGF)在血管形成中的作用,但二期临床试验证明VEGF及FGF的效果不理想,他们并不能促进功能血管的形成。因此,必须寻找新的靶点能够促进有功能血管的形成,才能达到真正意义上的血管重建。我们将着眼于血管内皮细胞粘附和迁移在体内血管形成中的作用,这一研究对缺血性心脏病的治疗具有重要的理论及实践意义。整合素家族在细胞的分化、增殖及移行等一系列生物活动中起着重要作用,主要通过与众多跨膜蛋白相互作用从而激活细胞内信号转导得以完成。与其作用密切的主要是四跨膜超家族蛋白(transmembrane-4 superfamily,TM4SF proteins,alsocalled tetraspanins),它在细胞增殖、融合、迁移及体外血管生成中的作用已被认识。Tetraspanins在哺乳动物的细胞或组织中广泛表达,有27个家族成员,这些蛋白彼此相连也可与其他蛋白相连,组成一个"Tetraspanin网络”。对细胞粘附和迁移的调节是这一家族最重要的特性,其中与整合素作用最为密切的Tetraspanins成员为CD151,其高表达于上皮细胞、内皮细胞、血小板、巨核细胞及一些未成熟造血细胞。CD151在血管内皮细胞形成及体内外血管生成中起重要作用,目前大多数研究均表明启动内皮细胞粘附、迁移和血管形成的信号从细胞外向细胞内传递是通过integrin-Tetraspanin-PKC复合体这一模式来完成的。大量体外研究利用Matrigel模型观察CD151在维持细胞形态及体外血管生成中的作用,发现在抗CD151及抗a6131抗体分别干预下,血管内皮细胞在Matrigel上的网格状形成能力明显受抑。CDl51通过与整合素的特异性结合介导着内皮细胞的粘附和迁移,在血管形成(angiogenesis)与重塑中起着关键作用。上述研究证实CD151在内皮细胞间及与基质间的粘附及迁移、血管生成中起重要作用。但这些研究多集中在CD151与整合素结合的信号转导模式,且促血管形成研究均以细胞系为材料,CD151在整体水平促血管形成的效应又是怎样的呢?前期研究中,我们证实了CD151在原代血管内皮细胞中也具有明显的促毛细管状形成作用,并建立了高表达CD151的血管内皮细胞系,观察到它与普通内皮细胞在粘附迁移、血管形成中存在显著差异。我们将携带CD151基因的重组腺相关病毒转染大鼠后肢缺血模型,发现后肢微血管密度及运动耐力明显高于未转染CD151组,初步确定了CD151在体内促血管生成的作用。在此,我们构建CD151重组质粒,用于心肌缺血(或心肌梗死)大鼠模型,使CDl51在缺血大鼠心肌中表达上调,观察CD151的表达对心肌缺血(或心肌梗死)后心肌梗死面积、心肌血管再生及心功能的影响,以证实CD151在心肌缺血后血运重建中的作用,且对CD151在血管形成的机制方面进行探讨,为冠心病及其他缺血性疾病的治疗探索出一新的途径。研究方法首先通过PCR方法将CD151基因末端连接HA尾(HA-tag)克隆到双链腺相关病毒表达载体pAAV上,构建pAAV-CD151重组质粒,按同法构建对照质粒pAAV-GFP。质粒扩增、提取,氯化铯梯度离心法纯化质粒。心肌梗死模型制备、实验分组及CD151基因的心肌转染雄性Sprague-Dawley成年大鼠48只,随机分为4组:击假手术组:开胸后不结扎左前降支,;2心肌梗死组:结扎左前降支,在结扎点以下左室前壁分点注射生理盐水为对照;3心肌梗死+心肌转染对照质粒pAAV-GFP组:结扎左前降支,左室前壁分点注射质粒pAAV-GFP;4心肌梗死+心肌转染pAAV-CD151组:结扎左前降支,分5点注射质粒pAAV-CD151。转染四周后进行如下几个方面的研究:(1)二维超声心动图进行大鼠心脏射血分数(EF)、短轴缩短率(FS)及室壁厚度的测定,评价心功能的变化;(2)左心室血流动力学监测左室舒张末压(LVEDP)、左室收缩压峰值(LVPSP)及左室内压最大上升和下降速率(±dp/dtmax);(3)Western blot免疫印迹检测心肌中CD151的表达水平,评价pAAV-CD151在大鼠心肌中的转染效果;(4)血管内皮细胞特异性抗原FactorⅧ相关抗原抗体SP免疫组化方法标记血管内皮细胞进行心肌微血管密度(microvessel density,MVD)的测定,评价CDl51对心肌梗死后血运重建的影响。实验结果成功建立了大鼠心肌梗死模型,术后四周病理切片可见正常心肌细胞排列有序,梗死心肌为大量结构紊乱纤维组织取代。(1)pAAV-CD151在大鼠心肌中的转染效果:Western blot免疫印迹检测四组大鼠心肌中CD151蛋白表达相对吸光度值依次为0.98±0.14、0.91±0.09、1.07±0.13和1.98±0.23,可见转染CD151基因后大鼠心肌中CDl51的表达水平明显提高(P<0.01)。(2)CD151表达上调对大鼠心肌梗死后心功能的影响:二维超声及左室心导管测定结果显示转染CDl51基因组EF、FS及LVPSP及±dp/dtmax等反映左室收缩功能指标分别为(64.0±8.7)%、(32.5±5.2)%、6620.2±884.6(mmHg/s)和5311.6±584.0(mmHg/s),明显高于未转染CD151的GFP对照心肌梗死组(41.8±6.5)%、(18.8±2.6)%、5102.1±715(mmHg/s.)4418.8±542.9(mmHg/s),各指标均P<0.01。(3)CD151对梗死后心肌微血管密度的影响:免疫组化染色分析结果显示转染CD151组心肌微血管密度385.4±79.9(n/mm2)明显高于转染GFP心梗对照组240.8±40.3(n/mm2)(P<0.01),可见CD151可明显促进梗死后心肌中微血管的生成,有利于心肌缺血后的血运重建。结论上述实验结果证实:1.pAAV-CD151直接大鼠心肌内注射进行CD151基因的转染可明显上调心肌中CD151的表达。2.CD151表达的增强可明显促进梗死后大鼠心肌微血管的生成,有利于缺血后的血运重建。3.上调大鼠心肌中CD151的表达,可明显改善大鼠梗死后心脏功能。更多还原

【Abstract】 BACKGROUND & OBJECTIVENovel angiogenesis-inducing therapies have been developed recently to stimulate collateral vessel formation and to improve muscles function in the treatment of occlusive vascular diseases. Angiogenesis requires signals that are transduced from growth factor receptors and integrins. Therapeutic angiogenesis has focus on the growth factors, such as vessel endothelial growth factors, hepatic growth factors, for a long time, but the clinical trials have showed limited clinical efficacy. In response to an obstruction or occlusion of a major artery, compensation can only come from low-resistance connections between a donor artery in a non-ischemic regions and the postocclusion arterial system of the recipient ischemic regions （arteriogenesis）. For getting the satisfactory result, it is necessary to explore more powerful pro-neovascularizing factors, and use them in combination rationally.The Tetraspanin membrane protein CD151 is expressed, especially high in endothelial cells, epithelial cells, and platelets. In vitro functional studies have demonstrated an important role of CD151 in cell migration and integrin-initiated morphogenesis. CD151 is a cell surface protein that belongs to the tetrapanin superfamily. It forms complexes with many laminin-binding integrins a3β1, a6β1, a6β4 et al. and is codistributed with these integrins in many tissues at sites of cell-matrix interactions. CD151-inegrin complex may contribute to the cell biological processes, including cell adhesion, cell motility, metastasis and formation and stability of hemidesmosomes. It is not known that promoting CD151 expression in ischemic tissues could stimulate neovascularization, improve recovery of blood flow and function of the ischemic organ.We thus hypothesize that an increased expression of CD151 may stimulate neovascularization in vivo. Our study is aimed to evaluate the efficiency of pAAV-mediated CD151 gene delivery in promoting neovascularization and improving heart function after myocardial infarction in rats, and explore a new target of the neovascularization after myocardial ischemia.METHODS AND RESULTSFirstly, Functional fragment of CD151 gene with HAtag was amplified by RT-PCR, and inserted into the vector pAAV. So, the recombined plasmid pAAV-CD151 was packed successfully. The pAAV-GFP plasmid was selected as control.The rat Acute myocardial infarction （AMI） model was established by ligation of the left anterior descending coronary artery in male Spague-Dawley（SD） rats. The surviving rats were grouped randomly as MI control, pAAV-CD151 group and pAAV-GFP group（n=12/group）. pAAV-CD151 and pAAV-GFP plasmid were delivered by direct injection in myocardium. Sham-operated group （n=12） was taken randomly as non-infarction control. Four weeks after AMI, the following endpoints were measured : （1）Rats heart function and left ventricular remodeling were assessed by echocardiographic evaluation and LV catherization. （2） The expression of CD151 and MMP-9 （matrix metalloproteinase-9） were detected by Western blot. （3）Micro vessels density （MVD） counting of infarct myocardium in rats observed by factor VIII related antigen immunochemical staining was conducted to value the pro-angiogenesis effect of CD151 delivered by gene transfection in vivo.Our immunoblot analysis showed that transfection with pAAV-CD151 could apparently lead to CD151 overexpression in rat myocardium. In comparison with the MI control （OD value 1.07±0.13）, the expression of CD151（OD value 1.98±0.23） in pAAV-CD151 group was significantly increased （P<0.01）. The micro vessels density （MV） counting in pAAV-CD151-AMI group（385.4±79.9n/mm²） was significantly higher than the MI control（240.8±40.3n/mm²） （P<0.01）. All indexes reflecting left ventricular contraction function such as left ventricular ejection fraction, LVPSP and ±dp/dt_max in CD151 group were statistically increased than MI control group（P<0.01）.CONCLUSIONpAAV-CD151 mediated CD151 gene delivery into rat myocardium induced an efficient and stable expression. pAAV-mediated CD151 stable expression stimulates neovascularization, and thereby improves blood perfusion in a rat myocardial infarction modeLAs a result, the rat heart function after ischemic injury was reformed. All these findings suggest that CD151 could be a new insight for the neovascularization therapy in the ischemic disease, and pAAV-mediated CD151 gene transfer may be useful for treatment of ischemic disease. CD151 has an effective role in pro-angiogenesis in infarct myocardium in vivo, thereby improves left ventricular function through valid revascularization.This study demonstrates that1) pAAV-CD151 promotes the CD151 protein expression in rat myocardium by direct injection.2) CD151 stable expression stimulates neovascularization in rat ischemic myocardium, and thereby improves heart function in a rat myocardial infarction model.更多还原