【作者】 张飞；

【导师】 尹邦良；

【作者基本信息】 中南大学 ， 外科学， 2009， 博士

【摘要】 第一部分AP-2α在小鼠血管内皮细胞中上调VEGF的表达研究背景动脉粥样硬化（atherosclerosis,As）的发生、发展和演变过程是多种因素综合致病的病理过程,血管内皮在此过程发挥着很重要的作用,内皮损伤被认为是引起As的始动环节,减少内皮损伤和恢复内皮功能在动脉粥样硬化的发生和发展中的作用已逐步得到重视。鉴别与As疾病相关的易感基因,发现新的以及有效的针对As疾病的治疗方案,一直是医学工作者关注的焦点。研究表明,AP-2（activatorprotein-2,AP-2）蛋白通过蛋白-DNA相互作用直接调控血管内皮生长因子（vaseular endothelial growth factor,VEGF）,参与了AS疾病的发生发展。在HaCaT角化细胞中,AP-2α以影响VEGF的表达。血管内皮细胞（endothelial cell,EC）为覆盖在血管内表面的单层扁平细胞,具有物质转运、自分泌、旁分泌等多种功能,通过研究血管内皮细胞来探究血管的生物学作用极为重要。AP-2α是否在主动脉血管内皮细胞中调节VEGF的表达目前未见报道,由于在VEGF启动子包含有AP-2的结合位点,本研究将探索AP-2α在血管内皮细胞中对VEGF表达的影响,进而研究其参与As形成的相关机制。方法1.颈椎脱臼处死C57BL/6J小鼠（体重20～25g）,无菌条件下彻底清除外膜结缔组织,完整取出其胸主动脉,植块法进行小鼠主动脉血管内皮细胞原代（含20%胎牛血清DMEM培养液）和传代培养（含10%胎牛血清DMEM培养液）。倒置相差显微镜下观察内皮细胞的形态和生长特性,Ⅷ因子相关抗原检测鉴定血管内皮细胞。2.以小鼠脑cDNA文库作为模板,以AP-2αFP、AP-2αRP为引物,PCR扩增约1300 bp的AP-2α全长编码序列,然后通过胶回收PCR扩增产物,连接到pMD18-T载体,得到pMD18-T-AP-2α重组表达载体。其PCR产物扩增质粒用EcoRⅠ/XhoⅠ酶切并回收AP-2α全长基因序列,然后与同样酶切的表达载体pCMV-Myc连接构建表达载体pCMV-Myc-AP-2α,用EcoRⅠ/XhoⅠ进行酶切鉴定。3.取2～4代细胞分别以转染质粒pCMV-Myc,pCMV-Myc-AP-2α,以及SiRNA-AP-2α,Control-RNAi-AP-2α,然后采用实时定量RT-PCR和Western Blot分别检测AP-2α和VEGF mRNA与蛋白的表达水平。结果1.血管植块在含有20%FBS的DMEM培养液中培养4 d左右,将血管植块除去并换液,12 d左右细胞开始融合成片,倒置显微镜下观察呈铺路石样。传代培养的细胞和原代形态相似,生长较快,4-5天可以融合成单层。血管内皮细胞Ⅷ因子相关抗原的检测,显示:细胞质均呈黄褐色着色,胞核不着色,而阴性对照用PBS代替一抗,没有加入抗细胞Ⅷ因子相关抗原兔多抗工作液,无特殊显色。2.通过连接反应将AP-2α片段正确插入载体pCMV-Myc中,酶切电泳分析和测序证明正确构建重组质粒pCMV-Myc-AP-2α。3.小鼠血管内皮细胞被分别短暂转染AP-2α表达质粒（pCMV-Myc-AP-2α）和空质粒（pCMV-Myc）,采用实时定量RT-PCR检测mRNA的表达水平,与对照组比较,转染pCMV-Myc-AP-2α后,AP-2α的表达水平显著上升,干扰AP-2α后,其表达水平显著下降,说明系统工作正常。当转染pCMV-Myc-AP-2α、pCMV-Myc、AP-2αRNAi和Control-RNAi后,分别检测VEGF的mRNA表达水平,实验结果显示,当过表达AP-2α时,VEGF mRNA的表达水平明显上升,干扰AP-2α后VEGF mRNA的表达水平明显下降（P<0.05,差异具有显著性）。同时Western blot检测也显示了相似的结果,即过表达AP-2α时,蛋白AP-2α的表达量增加,干扰AP-2α后,蛋白AP-2α的表达量降低。当过表达AP-2α时,可明显增加VEGF的蛋白表达水平;而干扰AP-2α后,则可以降低VEGF的蛋白表达水平。结论1.通过血管植块法可以实现小鼠动脉血管内皮细胞的原代和传代培养,完全可用于基础和临床科研。2.正确构建pCMV-Myc-AP-2α真核表达载体,为下一步进行VEGF及AP-2α基因的实验打下良好基础。3.VEGF为AP-2α的下游基因,AP-2α可以上调VEGF的表达。第二部分VEGF iRNA及AP-2α对动脉粥样硬化斑块的影响目的:探讨血管内皮生长因子（VEGF）及小干扰片段RNA（siRNA）对不同时期ApoE^-/-小鼠动脉粥样硬化斑块形成的影响,同时研究在干扰作用下,不同时期ApoE^-/-小鼠体内主动脉动脉粥样硬化斑块中VEGF和AP-2α量的变化及相互作用。方法1.首先在GeneBank上检索C57BL/6J小鼠VEGFA siRNA序列（NM₀09505）,由公司设计并体外化学合成。实验分5组,用Lipofectamine^TM2000试剂盒将小干扰片段（VEGFA-siRNA1,VEGFA-siRNA2,VEGFA-siRNA3,VEGFA-siRNA4）和对照序列（NC-VEGFA）短暂转染小鼠主动脉血管内皮细胞,利用RT-PCR法检测小鼠主动脉血管内皮细胞VEGF基因表达水平,筛选最有效抑制VEGF基因表达的siRNA。2.6周龄,16周龄,28周龄雄性ApoE^-/-小鼠各20只,均给予高脂高胆固醇喂养4周后,各周龄分成4组,每组5只小鼠,分为:VEGFA-siRNA组;siRNA-Negative组;阴性对照组;空白对照组。分别自小鼠尾静脉隔日注射1mg/kg/d VEGFA-siRNA;1mg/kg/dsiRNA-Negative;0.2ml的生理盐水;空白对照组不注射任何药物。共计2周,期间继续高脂高胆固醇喂养。3.血脂水平测定:检测各组ApoE^-/-小鼠血清总胆固醇（Tc）、甘油三醋（TG）、低密度脂蛋白胆固醇（LDL-C）、高密度脂蛋白胆固醇（HDL-C）的含量。4.小鼠主动脉斑块免疫组化染色:根据Envision^TM两步法免疫组化染色试剂盒说明书操作,检测VEGFA与AP-2α在小鼠动脉粥样硬化斑块内的表达情况。5.定量RT-PCR检测基因表达:每组20只ApoE^-/-小鼠主动脉窦部及胸主动脉,每2只混合提取新鲜粥样斑块组织RNA,实时定量PCR检测VEGFA、AP-2αmRNA表达水平。6.免疫印迹技术（Westemblot）检测蛋白表达:每组20只ApoE^-/-小鼠主动脉窦部及胸主动脉,每3只混合提取新鲜粥样斑块组织蛋白,Westemblot检测VEGF、AP-2α的蛋白表达水平。结果:1.siRNA1、siRNA2、siRNA3、siRNA4均抑制小鼠主动脉血管内皮细胞VEGFA mRNA和蛋白的表达水平,特别是VEGFA-siRNA4抑制作用最强,VEGFA mRNA和蛋白表达下降最明显。VEGFA-siRNA-negative对小鼠主动脉血管内皮细胞VEGFA mRNA和蛋白表达水平无抑制作用。因此,VEGFA-siRNA4基因沉默效果最好。2.同组ApoE^-/-小鼠血脂含量及体重比较差异无显著性（P>0.05）。各组间血脂含量及体重比较差异有显著性（P<0.05）。3.小鼠主动脉动脉粥样硬化斑块内均可见AP-2α及VEGF的高表达。4.实时定量RT-PCR和Western blot:实时荧光定量RT-PCR和Western blot显示:不同周龄组之间,随着ApoE^-/-小鼠周龄的增加,粥样斑块AP-2αmRNA及AP-2α蛋白和VEGFA mRNA及VEGF蛋白表达逐渐增加。VEGFA-SiRNA4干扰效果比较:不同周龄组中VEGFA-SiRNA4.组粥样斑块VEGFA mRNA含量和VEGFA蛋白表达水平最弱,差异具有显著性（P<0.05）。28周龄组干扰效果最明显（P<0.01）。但均对AP-2αmRNA含量及AP-2α蛋白表达水平无明显影响,差异无显著性（P>0.05）。结论:1.通过RNA干扰技术,成功筛选针对小鼠VEGFA基因沉默的特异性siRNA,为动物试验奠定基础。2.AP-2α及VEGF均参与了小鼠主动脉动脉粥样硬化斑块的形成。3.VEGFA及AP-2α对斑块的进展有促进作用,VEGFA基因沉默显著抑制ApoE^-/-小鼠胸主动脉粥样斑块进展期新生血管的形成,但并不影响AP-2αmRNA及AP-2α蛋白的表达。进一步说明AP-2α是VEGF上游调控基因。更多还原

【Abstract】 PartⅠAP-2αstimulate expression of vaseular endothelial growth factor in primary culture of mouse vascular endothelial cellsBackground:The occurrence,development and evolution of Atherosclerosis （atherosclerosis,As） are general pathological process induced by a variety of mediators.Vascular endothelial appears to play a key role,and endothelial dysfunction marks the beginning of an insidious disease process,which silently progresses to a point where it can only be slowed but not reversed.Reducing endothelial damage and restoring endothelial function in atherosclerosis of the occurrence and development of the role has gradually been given attention.An identification of susceptibility gene should provide novel insights into improved effective prevention and treatment of acute cardiovascular syndromes.Recent studies have showed that AP-2αcould affect the expression of VEGF in HaCaT keratinocytes through protein-DNA interaction,and AP-2αinvolved in the development of AS disease.Vascular endothelial cells layed over the inner surface of blood vessels at the simple squamous cells,and possessed the material transfer, autocrine,paracrine and many other functions.Therefore,It is extremely important to investigate the role of vascular biology through the study of vascular endothelial cells.It has not been reported that AP-2αcould regulate the expression of VEGF in aortic endothelial cells,but we known VEGF promoter contains AP-2 binding site.This study will explore how AP-2αregulated the expression of VEGF and the mechanisms of its involved in the formation of As.Methods:1.The aorta was removed from C57BIV6J mice（weight 20-25g） executed by cervical dislocation mice after thoroughly eliminate adventitial connective tissue in bacteria free condition.The vessel was opened longitudinally and cut into 1 mm×1 mm pieces,then placed on culture plates with the intima side down.Mouse aortic endothelial cells were primary cultrued（containing 20%FBS DMEM culture medium） by explant method and subcultred（containing 10%FBS DMEM culture medium）.Morphological（growth characteristic） and immunological（VDI factor-related antigen） criteria were identified under inverted phase contrast microscope.2.Full length cDNA of AP-2αgene was amplified using PCR from cDNA library of mouse,the PCR production was ligated to pMD18-T vector,and obtained pMD18-T-AP-2αrecombinant expression vector, and then the pMD18-T-AP-2αwas digested with EcoRⅠand XhoⅠ.After electrophoresis,the segments of 1320 bp were recovered and ligated to the pCMV-Myc vector digested also with EcoRⅠand XhoⅠto obtain the pCMV-Myc-AP-2αvector.3.From 2 to 4 generation culture cells were transfected with plasmid pCMV-Myc,pCMV-Myc-AP-2α,AP-2α-SiRNA and Control-Si-AP-2αto the primary culture cells,respectively,and then investigated the expression level of mRNA and protein of VEGF using quantitative real-time RT-PCR and western blot.Results:1.After explanting culture for 4 days in DMEM culture medium containing 20%FBS,the vascular explants were removed and formed confluent monolayers,it showed a cobblestone shape under inverted phase contrast microscope approximately 12 days.The growth velocity of passaged cells were more fast than that of the primary cells,the former would form confluent monolayers after 4-5 days.Characteristic granular cytoplasmic staining pattern were revealed after immunohistochemical labeling for von Willebrand factor.Nuclei did not stain with special color for the negative control stained with PBS.2.Mouse vascular endothelial cells were transfected transiently with pCMV- Myc-AP-2αand pCMV-Myc,respectively,and the mRNA levels of AP-2αwere detected.The quantitative real-time RT-PCR analysis showed that AP-2αexpression levels increased significantly when the cells were transfected with pCMV-Myc-AP-2α,while decreased significantly after suppressing AP-2a by RNA interference. This indicated that the system worked correctly.When transfected with pCMV-Myc-AP-2α,pCMV-Myc,AP-2α-SiRNA and the Control-SiRNA,the mRNA expression levels of VEGF were significantly increase after over-expressing Ap-2αand decreased significantly after suppressing AP-2a by RNA interference.Western blot detection also showed similar results that the protein expression level of VEGF increased markedly after over-expressing AP-2α,while decreased significantly after suppressing AP-2a by RNA interference.Conclusion:1.We successfully achieved the primary culture cells of mouse aorta vascular endothelial cells using explantation method,it was satisfied to the research of basic or clinical.2.The recombinant plasmid vector pCMV-Myc-AP-2αwas successfully constructed for the next step to carry out genetic experiments of the relationship between AP-2αand VEGF.3.AP-2αcan up-regulate the expression of VEGFA,which implied that VEGF was a downstream gene of AP-2α. PartⅡ.The effect of VEGF-SiRNA and AP-2αon the atherosclerotic plaqueobjectives:To investigate the effect of Vascular endothelial growth factor（VEGF） and small interfering fragment RNA（siRNA） on the formation of atherosclerotic plaque and the interactions of VEGF and AP-2αby interference effect on aortic atherosclerotic plaque in vivo in different periods of apoE^-/- mice.Methods:1.Mouse vascular endothelial cells were transiently transfected with small interfering fragment（VEGFA-siRNA1,VEGFA-siRNA2, VEGFA-siRNA3,VEGFA-siRNA4） and the control sequence （Control-SiVEGF A）.The mice was divided into 5 groups,mouse aortic endothelial cells were transfected with lipofectamine^TM2000.The mRNA expression level of VEGF gene was detected using RT-PCR analysis.2.Males apoE^-/- mice of 6-week-old,16-week-old,28-week-old （twenty mice of each age） separately were raised with a western-type diet for 4 weeks.And then the same age mice were divided to 4 groups randomly,each age group was randomly divided into VEGFA-siRNA4 group,contro1-siRNA,negative control group and blank control group, the first three groups were injected with 1mg/kg/d VEGFA-siRNA4, 1mg/kg/d control-siRNA,0.2ml of normal saline on mouse tail vein on alternate days,respectively,and the fourth group was injected nothing. The mice continued being raised on a western-type diet for two weeks.3.Serum lipid and lipoprotein detectionBefore perfusion-fixation,blood samples were collected,and serum total cholesterol,LDL cholesterol,HDL cholesterol,and triglycerides concentrations were detected,respectively.4.immunohistochemical staining in mice aortic atherosclerotic plaqueAccording to the instructions of Envision^TM two-step immunohistochemical staining kit,atherosclerotic plaques were stained with Monoclonal antibody of VEGFA and AP-2αto detect the expression levels of them.5.Real-time Quantitative RT-PCR AnalysisFor each group of animals（n=20）,department of aortic sinus and thoracic aorta from 2 mice were pooled and total RNA was extracted.The mRNA level of VEGFA and AP-2αwas performed by real-time RT-PCR using SYBR green PCR Master Mix,and the data were analyzed with the LightCyeler software version3.5.6.Western BlottingFor each group of animals,department of aortic sinus and thoracic aorta from 3 mice were pooled and total protein was extracted,protein expression levels of VEGFA and AP-2αwere detected using western blot,and the data were analyzed with the LightCyeler software version3.5.Results:1.Expression levels of VEGFA protein and mRNA can be inhibited by siRNA1,siRNA2,siRNA3,siRNA4 in mouse aortic endothelial cells. The interference effect of VEGFA-siRNA4 worked best.2.There were no significant differences（P＞0.05） in serum lipid levels and body weight among experimental groups of mice and we can get the opposite results in comparison with each group.3.Immunohistochemical staining indicated immunoreactive cells were stained brown in atherosclerotic plaque.3.Real-time quantitative RT-PCR and Western blottingThe expression level of VEGF mRNA and VEGF protein in plaque content were the weakest while injected with VEGF-siRNA among the parallel group.In comparison with each group,the expression level of VEGF mRNA,VEGF protein,AP-2αmRNA and AP-2αprotein increase gradually with the increase of the age and achieve the highest level in 28-week-old group.However,the expression level of AP-2αmRNA content and AP-2αprotein had no significant effect of interference suppression with VEGF-siRNA.Conclusions:1.The specific siRNA sequences of VEGFA were successfully screened for gene silencing in mice through RNA interference technology to lay a foundation for animal experiments.2.Both VEGF and AP-2αwere participate in the formation of aortic atherosclerotic plaque.3.Both VEGF and AP-2αcould progress neovascularization in atherosclerotic plaque.RNAi targeting VEGF significantly suppresses expression of VEGF in progress of thoracic aortic atherosclerotic plaque in the ApoE-/- mice but does not affect the expression of AP-2αmRNA and AP-2αprotein.更多还原