【作者】 左后娟；

【导师】 刘正湘；

【作者基本信息】 华中科技大学 ， 内科学， 2009， 博士

【摘要】 研究背景和目的血管再生性治疗已成为当前冠心病治疗基础和临床研究的热点。如能在心肌缺血发生后的恰当时间内使血管再生，形成或开放冠状动脉侧支循环，就可以挽救顿抑心肌，减轻心肌缺血，减少心肌细胞的死亡数量，从而保护心脏功能，改善病人的临床症状和预后。四跨膜蛋白超家族（transmembrane-4 superfamily，TM4SF）成员CD151能与多种整合素亚型特异性结合形成CD151-整合素复合体，是整合素信号转导的跨膜连接器，也是多种整合素信号转导的交汇点。随着整合素与血管生成方面研究的不断深入，CD151也日益受到关注。研究证实CD151参与调节细胞多种功能，包括调节细胞形态、黏附、迁移、半桥粒结构的形成，及与整合素相互作用介导血管生成等。前期研究中，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；进一步的实验中，利用裸质粒注射的方法在心肌梗死模型大鼠心肌中转染CD151，发现CD151可明显促进梗死后心肌中微血管的生成。这一系列的实验结果表明增强CD151的表达能够促进缺血组织的血管生成。但新的研究提示仅促进微血管形成并不能改善血流灌注，血液的流通更倾向于依靠动脉血管而不是微血管。那么，通过增强CD151的表达促进缺血组织的血管生成，是否能够促进有效的血运重建并改善缺血组织器官功能以及其促血管生成的机制如何，均还需进一步研究。我们设计本课题是建立在CD151的研究成果上，观察CD151促血管生成的有效性，即是否促进了功能性的血运重建，并进一步探讨其促血管生成的机制。研究方法1．构建pAAV-CD151、pAAV-antiCD151重组质粒及pAAV-GFP对照质粒。质粒扩增、提取，氯化铯梯度离心法纯化质粒。采用三质粒共转染法包装CD151、antiCD151和GFP重组腺相关病毒（recombinant adeno-associated virus，rAAV）。2．1月龄小型猪22头，随机分为4组，分别为正常对照组4头（不予冠脉结扎和注射病毒，正常喂养），rAAV-GFP组6头(结扎冠状动脉左前降支，心肌内分10点注射1×10¹²viron particles的rAAV-GFP病毒)，rAAV-CD151组6头(结扎冠状动脉左前降支，心肌内分10点注射1×10¹²viron particles的rAAV-CD151病毒)，rAAV-antiCD151组6头(结扎冠状动脉左前降支，心肌内分10点注射1×10¹²vironparticles的rAAV-antiCD151病毒)。转染8周后，RT-PCR、Western blot免疫印迹和免疫组化法检测心肌中CD151的蛋白表达及mRNA水平，免疫组化法检测微血管密度和小动脉密度，左冠状脉造影评价侧支循环的建立，¹³N-NH₃ PET显像评价心肌灌注，超声心动图进行小型猪心脏射血分数（EF）、短轴缩短率（FS）及室壁厚度的测定，评价心功能。此外，Western blot检测FAK、PI3K、Akt、eNOS、ERK、p38MAPK等信号通路蛋白表达，亚硝酸还原酶法测定心肌组织NO含量。3．将构建含有正反义CD151基因的重组腺相关病毒转染ECV304细胞，MTT法测定细胞增殖能力，Boyden小室法测定细胞的迁移能力，将转染后的细胞种植在Matrigel上，观察其类微血管结构的形成情况。Western blot检测CD151及ERK、p38MAPK的蛋白表达。同时，进一步观察ERK、p38MAPK信号抑制剂对CD151诱导细胞增殖、迁移、类微血管形成的影响。实验结果1．成功建立了小型猪心肌梗死模型。病毒转染8周后，心肌组织CD151 mRNA水平及蛋白表达明显高于正常对照组和rAAV-GFP组（P＜0．05）；rAAV-CD151组微血管密度和小动脉密度均显著高于正常对照组和rAAV-GFP组（P＜0．05）。rAAV-antiCD151组CD151mRNA水平及蛋白表达则降低，微血管密度和小动脉密度均减少（P＜0．05）。冠脉造影显示rAAV-GFP组有少量的侧支血管形成，rAAV-CD151组侧支血管水平不一，0～3级都有，而rAAV-antiCD151组几乎无侧支血管形成。¹³N-NH₃ PET心肌灌注显示，与rAAV-GFP组相比，rAAV-CD151组血流灌注显著改善，缺损面积明显减少（P＜0．05），而rAAV-antiCD151组缺血更严重、缺损面积显著变大（P＜0．05）。超声心动图显示，rAAV-CD151组各项心功能参数（LVEF％、LVFS％、△ALWT和△IVST）、室壁厚度（ALwTd、IVSTd）均较rAAV-GFP组明显增高（P＜0．05），而rAAV-antiCD151组小型猪心功能参数和室壁厚度显著低于rAAV-CD151组（P＜0．05）。此外，Western blot结果显示，高表达的CD151促进磷酸化FAK、PI3K、磷酸化Akt、磷酸化eNOS、磷酸化ERK的蛋白表达，心肌组织NO含量也明显增加。2．高表达的CD151能够促进ECV304细胞的增殖、迁移和类微血管结构形成，高表达的CD151能够上调磷酸化ERK的表达，对磷酸化p38MAPK的蛋白表达影响则不大。ERK抑制剂（PD098059）能显著减弱CD151诱导的ECV304细胞的增殖、迁移和类微血管结构的形成，但p38 MAPK抑制剂（SB203580）则对CD151诱导的ECV304细胞的增殖、迁移和类微血管结构的形成无显著影响。实验结论1．高表达的CD151能促进缺血心肌微血管和小动脉的生成、促进侧支循环的建立、增加缺血心肌的血流灌注、明显改善心功能。2．高表达的CD151能促进FAK、PI3K/Akt／eNOS通路、ERK通路的激活。其促血管生成的机制可能与上述通路的激活有关。3．高表达的CD151能够促进ECV304细胞的增殖、迁移和类微血管结构形成。其作用与ERK通路的激活有关。更多还原

【Abstract】 Background & ObjectiveTherapeutic angiogenesis has been identified as a good potential way in the studies of coronary heart disease. The benefit of therapeutic angiogenesis, which makes the formation of compensatory circulation and restoration of myocardial blood flow become possible after myocardial ischemia at right time, lies in retrieving more myocardial cell and reducing myocardial ischemia, so as to protect the heart function and improve the clinical symptom and prognosis of patients.CD151, a tetraspanin superfamily protein, is reported to form a structural and functional complex with various integrins. As "transmembrane linker", CD151 regulates the signal transduction through integrins. While more researches concernes about the relationship of integrins and angiogenesis, more and more studies become focus on CD151. Although the physiological function of CD151 is largely unknown, in vitro functional studies show that CD151 involves in the cell morphology, adhesion, migration, spreading, hemidesmosome structure formation, and the angiogenesis related to integrins.We have reported that in vivo CD151 gene delivery increases the number of microvessels and improved exercise tolerance in a rat hind-limb ischemia model, and increases myocardial capillaries densities of a rat myocardial ischemia model. These researches have identified CD151 as a potential target for therapeutic angiogenesis. However, increasing evidence indicates that angiogenesis response unlikely contributes to improve the regional blood flow, which tends to depend more on arterioles than on capillaries. Whether CD151-induced neovascularization can effectively promote the blood flow is still unclear. Further, the explicit signaling mechanism by which CD151 regulates blood vessel formation and maintenance has not been well elucidated. The purpose of this study was to evaluate whether CD151 induces functional neovascularization and coronary collateralization, and to determine the signaling pathways involved.Methods and Results1. pAAV-CD151 and pAAV-antiCD151 were constructed. Then, we produced the rAAV particles by transfections of 293 cells, and the titer of virus was determined by Northern blot. Twenty-two pigs, 1 month of age, were randomized into four groups. Four normal pigs underwent no operation were served as the control group. The remaining eighteen pigs, served as three viral administered groups, underwent coronary artery ligation and received intramyocardial viral injection. rAAV- GFP group （n=6）, rAAV-CD151 group （n=6） and rAAV-antiCD151 group （n=6） animals respectively received direct intra- myocardial injection of rAAV-GFP, rAAV-CD151 and rAAV-antiCD151 (1×10¹² viron particles per pig, at 10 sites) correspondingly. Eight weeks after viral administration, the expression of CD151 protein was measured by western blot and immunohistochemistry, and CD151 mRNA was detected by RT-PCR. The densities of capillaries and arterioles were determined using immunohistochemistry. Coronary angiography was done to evaluate collateral circulation of the occluded artery. ¹³N-NH₃ PET and echocardiography were applied to evaluate the regional myocardial perfusion and other myocardial functions. Western blot was performed for assessing the signaling mechanisms. In addition, myocardium NO concentration was assayed. We have established the acute myocardial infarction model in pigs successfully. rAAV-mediated CD151 gene delivery promotes the CD151 protein expression in myocardial tissue. Overexpression of CD151 markedly increased the densities of capillaries and arterioles, showed better collateral circulation, significantly enhanced the regional myocardial perfusion, reduced myocardial ischemia and improved the myocardial contraction, wall motion and wall thickness. Conversely, antiCD151 gene delivery reversed these changes above. In addition, CD151 activated FAK, ERK, PI3K, Akt and eNOS, and increased NO level. 2. rAAV-CD151 mediated CD151 gene delivery into ECV304. After transfection, the expression of CD151 was measured by western blot. Proliferation assay was evaluated using the 3-[4,5-dimethylthiazol-2-yl]-2,5, diphenyltetrazolium bromide （MTT） method; Cell migration assay was performed using Boyden transwell; and tubule formation was determined by matrigel tests. In addtion, the potential involvement of MAPKs signaling pathways was explored. Here, we showed that CD151 promoted ECV304 proliferation, migration and tubule fomation in vitro, accompanied by phosphorylation of ERK, leading the activation of ERK. By contrast, overexpression of CD151 did not affect the activity of p38MAPK protein. Moreover, inhibitors of ERK （PD098059） can attenuate CD151-induced cell proliferation, cell migration and tubule formation in vitro, which suggests that ERK mediate the effects of CD151. However, inhibitors of p38MAPK （SB203580） had no effect on the CD151 -induced cell proliferation, cell migration and tubule formation.Conclusions1 . Intramuscular transduction of rAAV-CD151 actually promotes functional neovascularization responses in a pig myocardial infarction model, leading to a greater enhancement of blood flow restoration. The mechanism may be that CD151 can activate FAK, PI3K, MAPKs pathways and promote neovascularization via the MAPKs and PI3K pathways.2. Overexpression of CD151 promotes ECV304 proliferation, migration and tubule formation. The mechanism is that ERK signaling pathway is involved in the angiogenic effects of CD151.更多还原