【作者】 丁士芳；

【导师】 张运；

【作者基本信息】 山东大学 ， 内科学， 2007， 博士

【摘要】 目的:构建携带人VEGF₁₆₅基因的重组腺病毒。方法:应用EcoRⅠ和XbaⅠ分别对质粒PDC-VEGF和PDC315进行酶切,凝胶电泳鉴定、回收纯化目的DNA片段,连接目的片段并转化大肠杆菌DH5α构建重组质粒PDC315-VEGF,对重组质粒进行EcoRⅠ和XbaⅠ、EcoRⅠ和BamHⅠ酶切和测序鉴定。通过脂质体将质粒PDC315-VEGF与含有腺病毒右臂的质粒pBHGE3共转染293细胞,9～12天出现病毒空斑效应。提取重组腺病毒DNA,通过PCR扩增法鉴定构建的腺病毒。大量扩增、氯化铯梯度离心纯化、浓缩重组腺病毒,微量滴定法测定腺病毒滴度。结果:通过连接反应将3973bp VEGF₁₆₅片段正确插入538bp PDC315载体,酶切电泳分析和测序证明正确构建重组质粒PDC315-VEGF。经细胞内同源重组构建携带人VEGF₁₆₅基因的重组腺病毒,以重组腺病毒DNA为模板,扩增出421bp VEGF₁₆₅基因片段,证实携带人VEGF₁₆₅基因成功克隆到复制缺陷型腺病毒载体,腺病毒滴度为5.0×10⁹pfu/ml。结论:通过双质粒细胞同源重组,生产携带人VEGF₁₆₅基因的重组腺病毒ad.VEGF,为动物试验奠定基础。目的:利用RNA干扰（RNA interference,RNAi）技术,以小鼠VEGFa基因（NM₀01025257）靶基因,构建携带短发夹状干扰RNA（siRNA）的慢病毒载体。方法:1.根据小鼠VEGFa mRNA序列（NM₀01025257）,选择针对VEGF cds区3个19nts靶序列,设计并合成三对包含正反义靶序列的互补DNA链（siRNA1、siRNA2、siRNA3）,同时设计并合成一对针对无关序列的互补DNA链为阴性对照（Negative）。退火与线性化pRNAT-U6.2/Lenti质粒连接、转化感受态细胞DH5α,扩增、纯化质粒,通过PCR扩增以及基因测序鉴定片段大小及插入片段序列。2.钓取小鼠NIH/3T3细胞总RNA,利用RT-PCR方法大量扩增、纯化小鼠NIH/3T3细胞VEGFa cDNA,将BamHⅠ和EcoRⅠ酶切VEGFa基因片段与线性化处理pCDH1质粒进行连接、转化感受态细胞DH5α,构建携带小鼠VEGFa基因表达质粒pCDNA-VEGF。扩增、纯化质粒,通过PCR扩增后进行琼脂糖电泳以及基因测序鉴定片段大小及插入片段序列。3.实验分6组,用脂质体将pCDNA-VEGF质粒或/和siRNA慢病毒质粒共转染NIH/3T3细胞,通过荧光Real time PCR和Western blot检测NIH/3T3细胞VEGF基因表达水平,筛选最有效抑制VEGF基因表达siRNA慢病毒表达质粒。4.通过脂质体分别将最有效抑制小鼠VEGFa基因表达的siRNA慢病毒表达质粒和pRNAT-Negative慢病毒表达质粒与慢病毒包装质粒混合物混合,共转染293T细胞,24h后观察绿色荧光表达情况,转染48h后收集慢病毒上清。用慢病毒液转染96孔板培养的293T细胞,荧光显微镜计数GFP阳性细胞法测定慢病毒滴度。结果:1.特异性合成DNA链退火后定向克隆到线性化pRNAT-U6.2/Lenti质粒,PCR扩增出316bp或317bp目的基因片断,重组质粒测序结果与设计siRNA序列完全一致,成功构建针对小鼠VEGFa基因的特异性siRNA慢病毒表达质粒。2.自NIH/3T3细胞获取高质量总RNA,通过RT-PCR扩增VEGFa cDNA,克隆VEGFa cDNA长885bp。构建pCDNA-VEGF质粒为1077bp（含有864bpVEGFa基因片断和213bp pCDNA载体片断）,插入小鼠VEGFa寡核苷酸序列与设计序列完全相符。3.pCDNA-VEGF质粒增加NIH/3T3细胞VEGFa mRNA和蛋白表达水平,siRNA1、siRNA2、siRNA3慢病毒表达质粒抑制pCDNA-VEGF质粒增加NIH/3T3细胞VEGFa mRNA和蛋白的表达水平,特别是pRNAT-siRNA3抑制作用最明显,VEGFa mRNA和蛋白表达均下降70%以上,pRNAT-negative慢病毒表达质粒对pCDNA-VEGF质粒增加NIH/3T3细胞VEGFa mRNA和蛋白表达水平无抑制作用。因此,pRNAT-siRNA3慢病毒表达质粒基因沉默效果最好。4.通过脂质体分别将pRNAT-siRNA3质粒和pRNAT-negative质粒与慢病毒包装质粒混合物共转染293T细胞,24h后观察293T细胞有绿色荧光表达,成功建立慢病毒载体的包装细胞。转染48h收集慢病毒上清,浓缩、纯化后的慢病毒滴度均为l×10⁸ ifu/ml。结论:通过RNA干扰技术,成功设计、筛选、生产针对小鼠VEGFa基因沉默的特异性siRNA慢病毒,为动物试验奠定基础。背景动脉粥样斑块内新生血管内皮细胞连接不紧密、内皮下缺乏基底膜、管壁无平滑肌细胞,具有通透性高和脆性大的特点,易于破裂出血。VEGF是高度特异性血管内皮细胞有丝分裂素,在血管新生发挥重要作用,是促进或抑制新生血管形成的靶点。目前,动脉粥样斑块破裂与粥样斑块内新生血管之间的关系有争议。因此,建立apoE^-/-。小鼠颈动脉粥样斑块模型,干预粥样斑块内新生血管形成,有助于阐明粥样斑块内血管新生、斑块内出血、粥样斑块破裂的相互关系。目的建立apoE^-/-小鼠颈动脉粥样斑块模型,将携带目的基因病毒经动脉外膜转染动脉粥样斑块,通过促进或抑制粥样斑块内新生血管形成,明确粥样斑块内新生血管形成、斑块内出血、粥样斑块破裂的相互关系及其机制。方法1.动物模型:164只8w雄性apoE^-/-小鼠,均给予高脂高胆固醇喂养。通过缩窄性颈动脉套管建立apoE^-/-小鼠左颈总动脉粥样斑块模型。按20%浓度（V/V）将pluronic-127 gel加入Ad.VEGF（5×10⁹pfu/ml）、Ad.lacZ（5×10⁹pfu/ml）、siRNA.VEGF（1×1O⁸ifu/ml）、siRNA.Negative（1×10⁸ifu/ml）,制成转染用腺病毒或慢病毒溶液。缩窄性颈动脉套管4w后,去除缩窄性硅胶套管。将100ul病毒液均匀涂抹左颈总动脉外膜,室温孵育20min。术毕,继续高脂饲养4w。局部基因转染2w时,自Ad.lacZ组、siRNA.Negative组分别随机处死2只apoE^-/-小鼠,观察粥样斑块内β-半乳糖苷酶和GFP表达,判断局部基因转染的可行性和有效性。2.血脂水平测定:检测各组apoE^-/-小鼠血清总胆固醇（TC）、甘油三酯（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。3.病理学检测:每组20只apoE^-/-小鼠,对左颈总动脉粥样斑块分别进行HE染色、油红O染色、Masson染色、天狼猩红染色、Perl’s染色（含铁血黄素染色）,通过免疫组织化学方法检测斑块内巨噬细胞（MOMA-2）、平滑肌肌动蛋白（α-actin）、血管内皮生长因子（VEGF）、vWF、纤维蛋白原、基质金属蛋白酶2（MMP2）、基质金属蛋白酶9（MMP9）的蛋白表达。统计粥样斑块破裂（斑块内出血和血栓形成）发生率,计算易损指数=（巨噬细胞+脂质）阳性面积百分比/（平滑肌细胞+胶原）阳性面积百分比。4.实时荧光定量RT-PCR检测基因表达:每组12只apoE^-/-小鼠左颈总动脉,每3只混合提取新鲜粥样斑块组织RNA,实时荧光定量PCR检测VEGF、MMP2、MMP9 mRNA表达水平。5.免疫印迹技术（Western blot）检测蛋白表达:每组8只左颈总动脉,混合后提取粥样斑块组织蛋白,Western blot检测VEGF、MMP9的蛋白表达水平。结果1.ApoE^-/-小鼠基本特征:缩窄性颈动脉套管、去除套管以及颈动脉外膜局部转染病毒的手术顺利,实验期间无apoE^-/-小鼠意外死亡。各组apoE^-/-小鼠血脂含量及体重比较差异无统计学意义（p＞0.05）。2.颈动脉外膜局部有效转染腺病毒或慢病毒液:颈动脉局部转染ad.LacZ腺病毒2w,颈动脉外膜、中膜、新生内膜均分布β-半乳糖苷酶蓝色颗粒;携带GFP基因的慢病毒转染2w,颈总动脉粥样斑块有强GFP表达,极少分布于其他脏器。腺病毒和慢病毒可将携带目的基因,通过血管外膜成功转染粥样斑块。局部转染基因影响粥样斑块内VEGF合成,免疫组化示Ad.VEGF组粥样斑块内VEGF呈弥漫性强阳性表达（69.97±2.74%）,荧光实时定量RT-PCR和Western blot示粥样斑块VEGF mRNA和VEGF蛋白（1.76±0.32）表达最高;siRNA.VEGF组粥样斑块内VEGF表达最弱（14.33±1.37%）,荧光实时定量PCR和Western blot均显示siRNA.VEGF组粥样斑块VEGF mMRNA和VEGF蛋白（0.22±0.02）表达最弱。3.局部基因转染对粥样斑块大小及斑块成分的影响:颈动脉局部转染Ad.VEGF组粥样斑块面积最大,siRNA.VEGF组粥样斑块面积最小,Ad.LacZ组和siRNA.Negative组粥样斑块面积差异无统计学意义。各组均形成以脂质沉积为主粥样斑块,脂质含量差异有统计学意义。Ad.VEGF组斑块α-SM actin含量最少（8.35±0.3%）,siRNA.VEGF组粥样斑块内α-SM actin含量最多（19.01±1.17%）。Ad.VEGF组粥样斑块纤维帽最薄（6.07±0.25um）,siRNA.VEGF组粥样斑块纤维帽最厚（15.40±1.46um）。粥样斑块内胶原分布呈类似表现,Ad.VEGF组粥样斑块胶原含量最少（10.11±1.05%）,siRNA.VEGF组粥样斑块胶原含量最多（25.61±1.69%）。Ad.VEGF组粥样斑块内巨噬细胞含量最多（22.18±1.87%）,siRNA.VEGF组粥样斑块巨噬细胞含量最少（8.25±0.86%）。Ad.VEGF组粥样斑块内MMP2、MMP9呈弥漫性强阳性分布,荧光实时定量RT-PCR显示Ad.VEGF组粥样斑块内MMP2 mRNA、MMP9 mRNA表达含量最高,Western blot显示Ad.VEGF组粥样斑块内MMP9以具有酶活性蛋白表达为主:siRNA.VEGF组粥样斑块内MMP9呈局限性弱阳性分布,粥样斑块内MMP2 mRNA、MMP9 mRNA表达含量最弱,斑块内以不具有酶活性的MMP9蛋白前体为主。Ad.VEGF组粥样斑块易损指数显著升高（3.59±0.42）,高于Ad.LacZ组（2.08±0.17）、siNRA.Negative组（2.03±0.18）、siRNA.VEGF组（1.12±0.08）（P均＜0.001）。siNRA.Negative组和siRNA.VEGF组粥样斑块易损指数差异无统计学意义（p＞0.05）。4.基因转染对斑块内新生血管形成及斑块稳定性的影响:根据vWF染色结果,粥样斑块内新生血管形成,新生血管管壁薄,多由单个或数个内皮细胞构成,无SMC组成。Ad.VEGF组斑块内新生血管密度最高（24.0±1.9）,siRNA.VEGF组斑块内新生血管密度最低（6.6±1.2）。Ad.VEGF组粥样斑块破裂发生率最高,分别有8例（40%）发生斑块内出血和6例（30%）血栓形成,其中3例直视下可见粥样斑块近心端附壁血栓形成。Ad.LacZ组分别有2例（10%）斑块内出血和1例（5%）血栓形成,siRNA.VEGF组和siRNA.Negative组分别有2例（10%）斑块内出血,Pearson Chi-Square检验差异有统计学意义。结论（1）通过颈动脉外膜局部转染方式,可将目的基因高效转染粥样斑块。（2）粥样斑块局部转染Ad.VEGF₁₆₅,促进斑块内新生血管形成,增加粥样斑块破裂发生率。（3）VEGFa基因沉默显著抑制粥样斑块进展,降低粥样斑块易损指数,增加粥样斑块稳定性。目的人类动脉粥样硬化病变主要发生在大、中动脉弯曲、分又及狭窄部位,如主动脉弓、颈动脉分支、冠状动脉、腹主动脉分支和股动脉分支等血流缓慢或涡流部位,即低管壁剪切力区。ApoE^-/-小鼠是研究动脉粥样硬化较理想动物模型,但小鼠主动脉管壁剪切力约为人主动脉管壁剪切力的20倍。利用Visualsonics Vevo770显微超声仪实时无创性观察小鼠血流动力学变化,研究管壁剪切力在apoE^-/-小鼠动脉粥样斑块形成过程中的作用。方法高脂高胆固醇饲料饲养apoE^-/-小鼠,将内径0.3mm缩窄性或内径0.6mm非缩窄性硅胶管套扎apoE^-/-小鼠左颈总动脉。静脉注射HRP（50mg/kg体重）观察缩窄性套管和非缩窄性套管对apoE^-/-小鼠动脉管壁通透性的影响。应用显微超声仪于相应观察时间点进行颈动脉超声检查,根据τ（dyne/cm²）=4·V·η/ID公式计算动脉管壁剪切力。将颈动脉标本进行拍照和免疫组织化学分析。结果apoE^-/-小鼠颈总动脉内经0.51±0.02mm,管壁剪切力33.4±2.5dynes/cm²,内经0.3mm缩窄性套管导致颈总动脉狭窄≈64%,缩窄性套管近端颈总动脉管壁剪切力降至12.2±0.8dynes/cm²,而缩窄性套管段颈总动脉管壁剪切力显著升高至98.5±7.7dynes/cm²。静脉注射HRP显示缩窄性套管段和缩窄性套管近端颈总动脉管壁通透性均增加。缩窄性套管近端颈总动脉形成以脂质沉积和单核/巨噬细胞黏附浸润为主粥样斑块,缩窄性套管段颈总动脉形成以收缩型平滑肌细胞为主的中膜增生,但无脂质沉积和单核/巨噬细胞黏附浸润。结论①在高脂喂养基础上,通过缩窄性套管加速apoE^-/-小鼠颈总动脉粥样斑块形成;②apoE^-/-小鼠颈总动脉管壁剪切力较人类高;③低管壁剪切力有促动脉粥样斑块形成作用;④高管壁剪切力抑制动脉粥样斑块形成;⑤显微超声仪可以实时无创性检测apoE^-/-小鼠血流动力学在动脉粥样斑块形成中的作用。更多还原

【Abstract】 Objective:To construction of recombinant adenoviruses encoding human vascular endothelial growth factor165(VEGF₁₆₅)gene.Methods:The PDC315 plasmid and PDC-VEGF plasmid were digested with EcoR I and Xba I,then 538bp of CMV and 3973bp VEGF₁₆₅cDNA fragment were recovered and ligated by T4 DNA ligase.The ligation products were transformed into DH5a competent cells,resulting in the recombinant plasmid PDC315-VEGF which was confirmed by restriction enzyme digestion and DNA sequencing.The plasmid PDC315-VEGF was cotransfected together with pBHGE3 into 293 cells,where a near-complete cytopathic effect appeared 9～12 days later.Then 293 cells were exposed to 3 freeze-thaw cycles, and centrifuged.Three cycles of plaque purification and virus expansion were performed to ensure a single viral clone.The DNA of the adenovirus was extracted and then verified by PCR.The PCR primer sequences for VEGF₁₆₅are as follows: 5’-CCTTGC TGCTCTACCTCC-3’（sense）,5’-AAATGCTTTCTCCGCTCTG-3’ （antisense）.The adenoviruses were purified using a double cesium chloride banding procedure according to standard techniques,dialyzed extensively against TBS buffer at 4℃and sterilized using a 0.22μm filter.The functional PFU titers were determined by plaque assays in 293 cells.Results:The recombinant plasmid PDC315-VEGF was correctly constructed and confirmed by restriction enzyme digestion analysis and DNA sequencing for full length of human VEGF₁₆₅cDNA fragment.The replication deficient recombinant adenoviruses vector was correctly constructed by homologous recombination in 293 cells and confirmed by PCR which showed that VEGF₁₆₅ mRNA was transcripted from the VEGF gene.The virus concentration reach to 5.0×10⁹ pfu/ml.Conclusion:The recombinant adenoviruses vector encoding human VEGF₁₆₅gene was successfully constructed. Objective:To design the small interference RNA（siRNA）specific to mouse endothelial vascular growth factor A（VEGFa）gene by RNA interfering technique, and construct its recombinant lentiviral expression vector.Methods:1.According to Tuschl’s principle,three target sequences of mouse VEGFa gene were selected,and an irrelevant siRNA with a random combination of the mouse VEGFa gene was used as negative control sequence.Then four couples of complementary oligonucleotides of each sequence with hairpin loop of siRNA were synthesized.After annealing of the complementary strands,the DNA fragments were ligated into linearized plasmid pRNAT-U6.2/Lenti which linearized by restriction endonucleases BamH I and Xho I.The recombinant plasmid was transformed into competent E.coli.DH5a cells to amply and then purified.The purified plasmids were identified by PCR amplification and DNA sequencing.2.According to mouse vascular endothelial growth factor A（VEGFa）cDNA sequence,a pair of specific primers which contained respectively digestion site of EcoR I and BamH I on the 5’ end were designed and constructed.Then revert transcript polymerase chain reaction（RT-PCR）was employed to clone VEGFa cDNA from mouse cells NIH/3T3 strain.After being purified,the VEGFa fragment was subcloned into linearized plasmid pKCDNA-EF1-Puro which linearized by restriction endonucleases BamH I and EcoR I.The recombinant plasmid pCDNA-VEGF was transformed into competent E.coli.DH5a cells to amply and then purified.The purified plasmids were identified by PCR amplification and DNA sequencing.3.Six groups were assigned.After plasmid pCDNA-VEGF and siRNA lentiviral plasmids were cotransfected into NIH/3T3 cells with the liposome mediation,then the cells were collected 48h late.The effect of RNAi on the protein and mRNA expression of VEGFa was examined with western blot and real time fluorescence quantitative reverse transcriptase PCR,respectively.4.With the help of lipofectamine^TM2000,recombinant lentiviruses were produced by 293T cells following the cotransfection of plasmid pRNAT-siRNA3 or pRNAT-negative with three package plasmid compound which consists of pKCPACK-GAG、pKCPACK-REV、pKCPACK-VSV-G.The expression of GFP was examined under fluorescent microscope 24h after transfection.After 48h transfection, the lentivirus supernatant on 293T cells was collected.The titers of the recombinant lentiviruses were determined by scoring GFP expression following serial dilutions of the viral supernatant.Results:1.Three recombinant lentiviral plasmids of siRNA specific to mouse VEGFa gene and one negative were constructed successfully.The results of the gel electrophoresis and their DNA sequence analysis completely coincided with their designed sequences.2.The product of RT-PCR contained the mouse VEGFa cDNA.The recombinant plasmid pCDNA-VEGF contained correct nucleotide sequence for full length of mouse VEGFa cDNA fragment by DNA sequence analysis.3.After plasmid pCDNA-VEGF was transferred into NIH/3T3 ceils,the VEGFa expression effectively increased at the level of mRNA and protein.VEGFa siRNA knocked down VEGFa expreesion in NIH/3T3 cells obviously.However,the RNA interference effects showed a significant disparity.Compared with pRNAT-negative, recombinated siRNA lentiviral plasmids（pRNAT-siRNA1,pRNAT-siRNA2 and pRNAT-siRNA3）inhibited the VEGFa expression at the different levels of mRNA and protein.Among them,pRNAT-siRNA3 could causes more efficient down regulation of VEGFa expression,resulting in down regulation of VEGFa mRNA and protein by approximately 80.0%and 66.3%respectively.4.The transfected 293T cells were found containing strong expression of GFP 24h after transfection,confirming that the four plasmid system of the lentiviral vector and its packaging cell line were successfully constructed.After 48h of transfection, the lentivirus supernatant was collected and the titer of the recombinant lentiviruses reached 1×10⁸ ifu/ml.Conclusion:The recombinant lentiviruses,which can express siRNA hairpin aimed at VEGFa gene,have been constructed successfully. BackgroundIntraplaque hemorrhage（IPH）is believed to arise from the disruption of thin-walled microvessels that are lined by a discontinuous endothelium without supporting smooth-muscle cells.Therefore,the immature neovascular may be more fragile and probably contribute to intraplaque hemorrhage and cause plaque rupture.However, the role of angiogenesis in plaque destabilization and rupture has emerged as a major unresolved issue.Vascular endothelial growth factor-A（VEGF-A）,which is the most important and dominant proangiogenic cytokines,plays a major role in neovascularization.This study aims to assess the effect of neoangiogenesis on plaque stability in shear-induced advanced atherosclerotic plaques in apoE^-/-mice.ObjectivesIn this study we analyzed the effects of VEGF overexpression in advanced atherosclerotic plaques in the carotid artery of apoE^-/-mice.On the mean while,we also sought to assess the ability of RNA interference targeting VEGF to inhibit plaque neovascularization.Then we analyzed the effects of neoangiogenesis on plaque stability.Methods1.Animal modelMale apoE-deficient mice（n=164）,8 to 10weeks of age,were placed on a Western-type diet.Atherosclerotic lesions were induced by perivascular constrictive collar placement on the left common carotid artery.The collar was removed after four weeks and 100μl of Ad.VEGF（5.0×10⁹ pfu/ml）,or Ad.LacZ（5.0x 10⁹ pfu/ml）, siRNA.VEGF（1.0×10⁸ ifu/ml）,siRNA.Negative（1.0×10⁸ ifu/ml）suspension in 20% pluronic-127 gel was added locally on the adventitia surface of carotid artery and incubated at room temperature for 20 minutes.Four weeks late,atherosclerotic lesions from carotids were analyzed.2.Recombinant adenoviral and lentiviruses expression patternTo evaluate the efficiency and distribution of adenoviral vascular transduction,the gene transfer efficiency was examined using 5-bromo-4-chloro-3-indolyl-β-D-galactopyransidase（X-gal）staining assay. To evaluate the efficiency and distribution of recombinant lentiviruses vascular transduction,the expression of green fluorescence protein was observed which auto-fluorescence was suppressed with 0.5%Chicago Sky Blue.3.Serum lipid and lipoprotein measurementBefore perfusion-fixation,blood samples were collected and serum total cholesterol, LDL cholesterol,HDL cholesterol,and triglycerides concentrations measured.4.Histological and morphometric analysisFor each group of animals（n=20）,cross cryosections were stained with hematoxylin and eosin（H & E）,oil red O,picrosirius red,Masson’ s trichrome,Perl’s staining. Immunohistochemical staining were performed for detecting macrophage,smooth muscle cells,von Willebrand factor（vWF）,Fibrinogen,VEGF,MMP2,MMP9.The plaque component was expressed as a percentage of the total intimal area.5.Real-time Quantitative RT-PCR AnalysisFor each group of animals（n=12）,carotids from 3 mice were pooled and total RNA was extracted.The gene expression analysis was performed by real-time RT-PCR using SYBR green PCR Master Mix and data were analyzed with the LightCycler software version 3.5.7.Western BlottingFor each group of animals,carotids from 8 mice were pooled and total protein was extracted.Equal amounts of protein lysate was loaded onto SDS-PAGE,and Western blot analyses using antibodies and visualization by ECL were performed.Results1.General characteristicThroughout the experiments,the mice remained in good health and gene transfer was well-tolerated.There were no significant differences in serum lipid levels and body weight among experimental groups of mice.2.Recombinant adenoviral and lentiviruses expression pattern Abundant strongly positive staining forβ-galactosidase activity was revealed in the neointimal compartment 2 week after periadventitial transduction with Ad.LacZ. Two weeks after lentiviruses transfection,the expression of green fluresence protein was observed in RNAi plaque. The relative area of VEGF staining,VEGF mRNA and VEGF protein in Ad.VEGF transferred atherosclerotic lesions was significantly increased which determined by immunostaining,real-time PCR,and Western blot,whereas the plaques transferred with VEGF-targeted siRNA expression lentiviruses significantly inhibited VEGF expression.3.Histological and morphometric analysis Morphometric analysis showed that Ad.VEGF delivery to shear-induced plaques significantly increased plaque size and led to an almost complete occlusion of the lumen,but there were considerably smaller plaques in siRNA.VEGF transferred carotid.Lipids were abundantly present in plaques and no difference in lipid content among groups.In Ad.VEGF transferred lesions,the collagen staining andα-SM-actin staining were decreased,macrophage positive areas were significantly larger,the increased expression of endogenous MMP-2 and MMP-9 were consistent with the distribution of macrophages,MMP2 mRNA and MMP9 mRNA synthesis and protein secretion were increased.However,in siRNA.VEGF transduction lesions,the collagen staining andα-SM-actin staining were increased,macrophage positive areas were significantly lower,the decreased expression of endogenous MMP-2 and MMP-9 were consistent with the distribution of macrophages,MMP2 mRNA and MMP9 mRNA synthesis and protein secretion were decreased.Subsequent analysis revealed that MMP9 and MMP2 protein expression was predominantly in the proteolytically activated form in Ad.VEGF transferred lesions,whereas MMP9 and MMP2 protein expression was predominantly as a proform in siRNA.VEGF transferred lesions. Vulnerable index from Ad.VEGF group was 3.59±0.42,significantly greater than other three group（Ad.LacZ:2.08±0.17,P＜0.001;siNRA.Negative:2.03±0.18, P＜0.001;siRNA.VEGF:1.12±0.08,P＜0.001）.4.Angiogenesis and plaque stability Thin-walled,capillary-like vessels were observed in atherosclerotic lesions.The Ad.VEGF transferred lesions contained extensive areas of neovascularization （24.0±1.9）,whereas the siRNA.VEGF transferred lesions contained local areas of neovascularization（6.6±1.2）.Subsequently,the Ad.VEGF transduction led to a considerable increase in the incidence of IPH and fibrous cap rupture accompanying the thrombus formation in 14 mice（14 of 20）,whereas in Ad.LacZ group, siRNA.Negative group and siRNA.VEGF group,a mere 15%（3/20）,10%（2/20）and 10%（2/20）of such adverse events were observed.The hemorrhage mostly located in neovascularized areas.Conclusions1.The mean for gene transduction through local periadventitial is effective.2.VEGF overexpression promote angiogenesis in advanced plaques and induce plaque vulnerability.3.RNAi targeting VEGF significantly suppresses expression of VEGE, neovascularization and stability in advanced plaques. ObjectiveIt is well known that atherosclerotic plaques are preferentially present at lesion-prone sites and shear stresses play a key role in the pathogenesis of atherosclerosis in humans.However,artery shear stress is much higher in mice than in humans.The effect of shear stress on atherosclerosis in apoE^-/-mice is little to known because of technical difficulties.The aim of this study was to determine in vivo shear stress values in the development of rapid,site-controlled atherogenesis in apoE^-/-mice.MethodsMale apoE^-/-mice,age 8 weeks,were raised on Western-type diet two weeks before operation and continued after operation.A constricting silastic tube（0.30mm inner diameter）or a nonconstrictive silastic tube（0.60mm inner diameter）was placed on left carotid artery.Horseradish peroxidase（HRP,TypeⅡ,50 mg/kg body wt）, which as a marker of vascular permeability to proteins,intravenously into apoE^-/-mice and allowed to circulate for 10 minutes.At several time points,atherosclerotic lesions and local hemodynamic environment in the collar treated artery was analyzed using Vevo 770 ultrasound biomicroscopy（UBM）,which with a transducer frequency of 40 MHz with B-scan imaging and Doppler flow measurement capabilities.Peak wall shear stress was calculated using the following formula:τ（dyne/cm²）=4·V·η/ID.The collared carotid artery was photographed under a stereomicroscope connected to a standard CCD.Then each vessel was assessed throughout the entire length of the carotid artery for histological analysis.Results Before placement of collar.common carotid diameter was 0.51±0.02mm and peak blood velocity was 1217.5±92.54mm/s.Placement of constrictive collar resulted in≈64%axisymmetric stenosis in collar region of right carotid artery and peak blood velocity decreased to 443.00±28.94 mm/s in the proximal to the collar region. Whereas peak blood velocity within the collar region was accelerate,high-velocity jet formed（2109.41±165.05mm/s,2w;2665.13±289.66 mm/s,4w）.Accordingly,shear stress in the proximal to the constrictive collar region was decreased,on the other hand,shear stress within the constrictive collar region was sharply increased.After 2w placement of constrictive collar,the endothelial integrity was confirmed by staining for VWF throughout the entire length of carotid artery.However,the permeability of endothelium to macromolecules increased diffusely both in the region proximal to constrictive collar and intra constrictive collar region.At 2 weeks,the early atherosclerotic lesions contained diffuse deposition of monocyte/macrophages and extensive lipid deposits in proximal to the constrictive collar.At 8 weeks,the atherosclerotic plaques had grown markedly,with near-total occlusion of the lumen in the low shear-stress regions.A gradual thickening of the intimal lesion developed, which consisted predominantly ofα-actin positive SMCs within the constrictive collar region beginning at week 8.However,almost no lipid deposition and macrophage accumulation were observed in the elevated shear-stress regions at all time points.The UBM-observed lesions were highly correlated to those found on en face staining and histology.ConclusionsThis study provides in vivo noninvasive evidence for a causal relationship between shear stress and atherosclerosis in hypercholesterolemic apoE^-/-mice by placement of constrictive collar around carotid artery,even though actual carotid shear stress is much higher in mice than in humans.Then,the atherosclerotic plaque formed in the relatively lowered shear stress region,whereas higher shear stress caused an atheroprotective phenotype.更多还原