【作者】 郑振中；

【导师】 刘正湘；

【作者基本信息】 华中科技大学 ， 内科学， 2007， 博士

【摘要】 研究目的CD151具有促进细胞增殖、分化及血管新生的作用。有关CD151的研究主要集中在CD151与整合素的关系上,而其促血管新生信号机制的研究方面则较少。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。CD151能与多种整合素亚型结合形成整合素-CD151复合体,是整合素信号转导的跨膜连接器,其在细胞黏附、迁移以及体外血管形成中均发挥着重要的作用。前期研究中,我们建立了高表达CD151的血管内皮细胞系,发现CD151在血管内皮细胞中具有明显的促细胞管形形成作用,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。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。同时,提高CD151基因的表达,能够促进细胞迁移、体外血管形成以及增强缺血组织血管生成和血运重建。在缺血性疾病中增强CD151的表达,能够促进缺血组织的血管生成,进而达到改善血运重建及恢复缺血组织器官功能的效果。血管新生是原有血管芽生新血管的过程,包括内皮细胞增生和迁移、蛋白溶解酶表达调控、细胞外基质破裂重建和内皮管腔形成。血管新生过程涉及一系列信号转导途径的激活,PI3K/Akt/eNOS转导途径是其中重要的通路之一,参与多种血管新生因子的促血管新生作用。CD151是否通过PI3K/Akt/eNOS信号途径促进血管新生及PI3K/Akt/eNOS信号途径在CD151促血管新生作用中所扮演的角色及其地位仍不清楚。我们设计本课题是建立在CD151的研究成果上,推测CD151通过PI3K/Akt/eNOS途径发挥作用,对CD151促血管新生作用进行深入研究,试图从分子水平阐明其作用机制。研究方法构建pAAV-CD151、pAAV-anti-CD151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。首先通过脂质体将pAAV-CD151和pAAV-anti-CD 151转染ECV304细胞系,经Western blot检测CD151蛋白质的表达时间梯度,观察转染pAAV-CD151和pAAV-anti-CD151后细胞CD151表达时间和表达水平的变化,同时观察eNOS活力的变化和细胞增殖、迁移、和管形形成的变化,同时观察eNOS拮抗剂L-NAME对CD151诱导的内皮细胞的增殖、迁移、和管形形成的影响。然后将构建含有正反义CD151基因的重组腺相关病毒转染人脐静脉内皮细胞,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,观察其条索状微血管结构的形成情况。Western Blot检测CD151及PI3K、Akt、eNOS的表达,亚硝酸盐法测定细胞培养液NO浓度;根据[3H]-L-精氨酸转变为[3H]-L-瓜氨酸的量来测量eNOS活力。进一步观察PI3K、Akt、eNOS信号拮抗剂对CD151诱导的细胞增殖、迁移、管形形成的影响。此外,制备心肌梗死模型并行CD151基因转染大鼠心肌。雄性Sprague-Dawley成年大鼠48只,随机分为4组:⑴假手术组:开胸后不结扎左前降支;⑵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作为对照;⑶GFP组:结扎左前降支,左室前壁分5点注射注射病毒rAAV-GFP;⑷CD151组:结扎左前降支,分5点注射rAAV-CD151。转染四周后进行如下几个方面的研究:⑴RT-PCR检测外源基因导入情况;⑵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);⑶血管内皮细胞特异性抗原Factor VIII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CD151对心肌梗死后血运重建的影响;⑷Western blot免疫印迹检测心肌中CD151的表达水平,评价rAAV-CD151在大鼠心肌中的转染效果;检测PI3K、Akt、eNOS的表达情况,了解高表达CD151对PI3K/Akt/eNOS信号通路的影响。本研究共分四部分:病毒的制备;正反义CD151基因转染对ECV304增殖、迁移、管形形成及eNOS活力的影响;腺相关病毒介导的CD151基因转移对HUCEC增殖、迁移、管形形成及PI3K/Akt/eNOS信号途径的影响;腺相关病毒介导的CD151基因转移对大鼠急性心肌梗死缺血心肌血管新生的影响和信号机制的研究。实验结果结果发现高表达的CD151能够促进内皮细胞的增殖、迁移和管形形成;高表达的CD151能够上调PI3K、Akt、eNOS的表达,增加eNOS活力和NO产量;CD151低表达下调PI3K、Akt、eNOS的表达。说明CD151能够激活PI3K/Akt/eNOS信号通路,进而参与促血管生成作用。PI3K拮抗剂LY-294002、eNOS拮抗剂L-NAME抑制CD151诱导的内皮细胞增殖、迁移和管形形成,表明PI3K/Akt/eNOS信号通路在CD151诱导的血管生成中具有重要作用。成功建立了大鼠心肌梗死模型,rAAV-CD151经注射转染大鼠心肌梗死模型术后4周, CD151组心肌组织检测到外源性CD151mRNA的表达,假手术组、对照组、GFP组未检测到外源性CD151mRNA的表达,且CD151组心肌组织CD151蛋白表达水平高于假手术组、对照组、GFP组(P<0.05);CD151组血管密度与对照组、GFP组比较明显增加(P<0.05)。CD151组大鼠的血流动力学参数与对照组、GFP组比较有一定程度的改善(P<0.05)。说明rAAV-CD151可以有效地转染大鼠心肌组织,促进缺血心肌的血管生成,改善左室功能。取注射部位心肌组织,Western blot分别检测PI3K、Akt、磷酸化Akt、eNOS、磷酸化eNOS及VEGF的表达情况,发现PI3K的表达上调,Akt/p-Akt、eNOS/p-eNOS比值增加;VEGF的表达无显著性改变。实验结论1.高表达的CD151能够促进ECV304的增殖、迁移、管形形成。其作用可能是高表达的CD151增加eNOS活力有关。2.高表达的CD151能够促进HUVEC的增殖、迁移、管形形成。其作用与PI3K/Akt/eNOS信号通路激活有关。3.rAAV-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,激活PI3K/Akt/eNOS信号通路。CD151促进缺血心肌血管生成和改善左室心功能可能与PI3K/Akt/eNOS信号通路的激活有关。更多还原

【Abstract】 Background & Objective: Tetraspanins have been shown to associate with each other and with other integral membrane proteins, including integrins,the major receptors for extracellular matrix (ECM) adhesive proteins. Although the physiological function of tetraspanin CD151 is largely unknown,in vitro functional studies showed that CD151 is involved in cell adhesion,motility,and polarity. High CD151 expression was found to be associated with a poor prognosis in lung,colon,and prostate cancer. Monoclonal antibodies to CD151 inhibited in vivo metastasis of human cancer cells and transfection of CD151 cDNA into different tumor cell lines resulted in enhanced cell motility and metastasis. This implies that CD151 does not only play an important role in normal physiological processes, but also in pathological events,such as tumor cell invasion and metastasis. Recent research has identified CD151 as a potential target for therapeutic angiogenesis. However, little is known about mechanism(s) of CD151-induced cell proliferation,migration and angiogene- sis.Therapeutic angiogenesis that may be beneficial in the treatment of ischemia has recently been substantiated by a large amount of experimental data. Ischemic heart diseases develop as a consequence of coronary atherosclerotic lesion formation. Coronary collateral vessels and microvascular angiogenesis develop as an adaptive response to myocardial ischemia,which ameliorate the function of the damaged heart. Endothelial cell proliferation and migration play critical roles in angiogenesis. Angiogenesis is the formation of new blood vessels from pre-existing vessels,a physiological or pathological neovascularization process in response to tissue ischemia and tumor growth or metastasis,which is complex and involves several discrete steps such as extracellular matrix degradation,proliferation and migration of endothelial cells,and morphological differentiation of endothelial cells to form tubes.Many growth factors and hormones have been shown to regulate cell proliferation, migration and angiogenesis,including the activation of eNOS activity,via the PI3K/Akt signaling pathway.We hypothesized that CD151 may activate PI3K/Akt pathway and exert affects angiogenesis. To test whether CD151 promotes neovascularization and angiogenesis after myocardial infarction,we used rAAV for direct delivery of the human CD151 gene into the rat myocardium,evaluating the effect of CD151 on PI3K pathway and neovascularization. Therefore,the purpose of this study was to determine the mechanisms by which CD151 induces angiogenesis and promotes neovascularization after myocardial infarction.Methods and results: pAAV-CD151 and pAAV-anti-CD151 were constructed, and used to transiently transfect to ECV304 mediated with LipofectamineTM 2000. After transfection,the expression of CD151 was measured by western blot. Cell migration assay was performed using boyden transwell,proliferation assay was evaluated using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) method and tube formation test was examined on Matrigel. eNOS activity was assayed by L-[3H] citrulline production from L-[3H] arginine. The involvement of eNOS was explored using eNOS inhibitor (L-NAME) and observed the effects in the process. We found that CD151 promotes cell migration,proliferation and tube formation. In addition, CD151 increases eNOS activity. Moreover,cell migration,proliferation and tube formation induced by CD151 are inhibited when L-NAME is used,which indicated that an involvement of eNOS in CD151-induced cell migration,cell proliferation,tube formation.Then,we produced the rAAV particles by transfections of 293 cells,and the titer of virus was determined by quantitative real-time PCR. Here we showed that CD151 promotes human umbilical vein endothelial cell (HUVEC) proliferation,migration and tube formation in vitro, accompanied by increased phosphorylation of Akt and eNOS, leading to increased eNOS activity and nitric oxide (NO) levels after rAAV-CD151 infection,whereas infection with rAAV-anti-CD151 attenuated the effects of CD151,which suggested that CD151 can activate PI3K/Akt pathway. Moreover, inhibitors of PI3K (LY294002) and eNOS (L-NAME) can attenuate CD151-induced cell proliferation and cell migration,which suggests that PI3K/Akt pathway mediates the effects of CD151.In order to know whether rAAV-CD151 promotes neovascularization and the mechanism involved,rats were subjected to sham surgery or coronary artery ligation. We used rAAV for direct delivery of the human CD151 gene into the rat myocardium. At 4 weeks after coronary artery ligation,human CD151 mRNA was detected using RT-PCR. Measurement of capillary density was evaluated using immunostaining for von Willebrand factor. Hemodynamic variables and physiological parameters were monitored,too. Western blot analysis for CD151,PI3K,phosphor-Akt,total Akt, phosphor-eNOS and total eNOS was performed. In addition,we also observed the effect of CD151 on the expression of VEGF using western blot analysis. We found that CD151 gene delivery could increase the expression of CD151 at gene level and protein level. Overexpression of CD151 could increase the number of microvessels in the ischemic myocardium and significantly improved the hemodynamic variables after myocardial infarction. In addition, CD151 could activate PI3K pathway, including activation of Akt and eNOS, but not affect the expression of VEGF.Conclusions: 1.CD151 promotes ECV 304 migration,proliferation and tube formation. The mechanism is that overexpression of CD151 increases eNOS activity. This result also suggests that eNOS is involved in the angiogenic effecs of CD151. 2.Activation of PI3K/Akt signaling pathway mediates CD151-induced HUVECs proliferation and migration. 3.CD151 could promote neovascularization and improve ventricular function after myocardial infarction in rats. The mechanism may be that CD151 can activate PI3K pathway and promote neovascularization via PI3K pathway,without affecting ischemia- induced VEGF expression.更多还原