Toll样受体亚型单一和联合基因沉默稳定易损斑块的实验研究
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摘要
背景
动脉粥样硬化(atherosclerosis,AS)是心脑血管疾病的病理基础。研究表明,伴有免疫反应的炎症过程贯穿于动脉粥样硬化的始终。炎性细胞如T淋巴细胞和巨噬细胞在动脉粥样硬化病变的进程中扮演了重要角色。血管内皮细胞、平滑肌细胞及外膜的成纤维细胞和肥大细胞均可作为免疫细胞产生炎性因子。
但在动脉粥样硬化疾病的发生和发展中,其发病分子机制至今尚未阐明。近年来,Toll样受体(Toll-like receptors,TLRs)在炎症反应与天然(固有)和获得性(适应性)免疫反应中的作用日益受到人们的重视。Toll蛋白是最先在果蝇体内发现的一种信号转导蛋白,参与对细菌和真菌的识别及病原体的清除。随后.在哺乳动物体内发现的与Toll相似的跨膜蛋白被称为Toll样受体。TLRs能识别种类繁多的一病原相关分子模式(pathogen-associated molecular patterns,PAMPs),属于模式识别受体。目前为止,人类TLR家族至少有11名成员已被发现。不同的PAMP活化人类TLR家族的不同成员。TLRs通过其跨膜结构将LPS信号传递入细胞内,级联式激活转导蛋白MyD88、白介素1受体激酶(IRAK)、肿瘤坏死因子6调节蛋白(TRAF6)和NF-κB诱导激酶(NIK),进而激活核转录因子NF-κB,引起细胞因子的释放而导致炎症反应。启动炎症因子的释放,导致炎症反应的发生,在炎症反应的信号转导系统中发挥着极其重要的作用。
AS的整个过程是炎症和免疫反应的过程,而TLRs能够介导天然性和获得性免疫反应,因此,TLRs与AS的关系正逐渐引起人们的重视。大量研究证实TLR1、TLR2、TLR4、TLR6等TLRs家族成员在AS的发生和发展过程中可能起到了重要作用,在人类和动物的AS斑块中已检测出上述TLRs的mRNA和蛋白质表达。已有的实验证实:动脉粥样硬化斑块表达TLR2和TLR4,这两种受体的表达量随病变的进展而增加,并且TLRs调控炎症和免疫反应、细胞凋亡、斑块稳定性以及血管重塑等AS病理过程。
虽然近年来国内外学者在TLRs与AS斑块相互关系的研究中已取得较大的进展,但仍存在许多重大问题亟待解决:(1)既然TLR2和TLR4在AS形成和斑块破裂中扮演重要角色,能否通过封闭TLR2和TLR4基因表达阻止AS进程并降低AS斑块的易损性。这构成本课题的主导思想。(2)目前造成基因失活进行基因研究和基因治疗的方式主要基因沉默技术,又称RNA干扰(RNA interference,RNAi),RNAi可以高效特异地阻断目标基因的表达,目前已经广泛地应用于体内和体外基因功能的研究中。哺乳动物细胞实验发现,化学合成的21-23核苷酸长的小分子干扰RNA片段(small interfering RNAs,siRNAs),或是质粒或病毒载体介导的siRNA,均可引发基因沉默或者表达抑制。RNA干扰技术在细胞水平致病基因的功能研究中得到广泛应用。此外,通过高通量RNAi筛选,siRNA还被用于系统分析某些疾病的多种致病基因。借助于质粒或病毒载体的介导,siRNA已经被用于体内实验,特异性降低靶基因的表达。在进一步完善siRNA稳定性的基础上,RNAi的试剂有望应用于临床。(3)AS斑块中,TLR2和TLR4都发挥作用,那么这两者的各自作用及机制是什么?它们之间相的关系是独立的并联关系还是协同的网络联系?
因此本课题开展了应用RNAi技术,单独或联合沉默LR2和TLR4基因,阻止AS进程,稳定易损斑块的研究。
目的
1.慢病毒表达载体的构建:根据TLR2和TLR4基因设计siRNA相应的DNA片段,分子克隆到BLOCK-It Lentiviral PolⅡmiR RNAi表达载体;
2.动物实验研究TLR2i和LTR4i对于阻止AS进程,降低动脉粥样硬化斑块易损性的疗效;
3.联合应该用TLR2i和LTR4i,对比研究联合干扰和单一干扰对动脉粥样硬化的稳定作用。
方法
1.RNA干扰慢病毒载体的构建以及有效靶点的筛选:
1.1分别构建TLR2和TLR4基因的小分子干扰RNA载体的构建:
设计、合成分别特异性针对小鼠TLR2和TLR4基因的小分子干扰RNA序列(各3段序列),以BLOCK-It Lentiviral PolⅡmiR RNAi为母本,分别构建小分子干扰RNA载体BLOCK-TLR2-A、BLOCK-TLR2-B、BLOCK-TLR2-C、BLOCK-TLR4-A、BLOCK-TLR4-B、BLOCK-TLR4-C,DNA序列测定鉴定重组质粒的正确性。
1.2慢病毒干扰载体的构建及体外扩增:
将已构建好的上述6个穿梭质粒和对照载体BLOCK-mock,与携带CMV启动子的BLOCK-It Lentiviral PolⅡmiR表达载体进行rapid BP/LR重组,继而与293FT包装细胞结合形成病毒表达载体。
1.3有效封闭TLR2和TLR4基因表达的RNA干扰慢病毒载体的筛选:
将mock-LV、TLR2i-siteA、TLR2i-siteB、TLR2i-siteC、TLR4i-siteA、TLR4i-siteB、TLR4i-siteC的慢病毒载体转染小鼠巨噬细胞系RAW264.7细胞。荧光定量RT-PCR、Western blot分别检测各转染细胞TLR2和TLR4 mRNA水平、蛋白质水平的表达,筛选出有效阻断TLR2,TLR4表达的小分子干扰RNA载体。
2.动物模型的建立:
140只10-12周龄的雄性apoE-/-小鼠实验全程高脂饮食(含0.25%胆固醇和15%脂肪)喂养。全部实验鼠均于实验初行右颈总动脉套管(缩窄性硅胶管)术以诱发AS病变:0.08%戊巴比妥钠(40mg/kg)腹腔注射麻醉小鼠。颈部正中皮肤切开,剥离右侧颈部的腺体和肌肉,暴露右侧颈总动脉,小心分离与之伴行的迷走神经,固定右颈总动脉,将长度为3mm、内径为0.3mm的硅胶管(小鼠颈动脉血管直径为0.5mm)套置于血管的外周,固定套管。缝合皮肤切口,将小鼠放回笼中。
3.RNA干扰慢病毒载体体内转染实验:
颈动脉套管手术8周后,将10μL的LV-mock病毒悬液,10μL的LV-TLR2病毒悬液,10μL的LV-TLR4病毒悬液,5μL的LV-TLR2病毒悬液联合5μL的LV-TLR4病毒悬液,局部转染右颈动脉斑块,根据转染病毒不同,各组分别命名:空载体组(n=28),TLR2干扰组(n=28),TLR2干扰组(n=28)和TLR2+4联合干扰组(n=28)。另28只未转染病毒的小鼠归为对照组。我们将,TLR2干扰组,TLR4干扰组和TLR2+4联合干扰组统称为干扰组;对照组和空载体组称为非干扰组。病毒转染一周后和二周后空载体组分别处死3只动物,检测斑块内内GFP的表达。其余实验鼠于转染四周后处死,观察斑块内GFP的表达,检测斑块的病理学形态结构。
4.血液生化指标检测:
检测血清总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)的水平。
5.病理学检测:
对颈动脉斑块分别进行H&E染色、油红O染色、天狼猩红染色,并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)的表达。测量斑块面积和纤维帽的厚度,观察斑块的形态结构,检测斑块内胶原、脂质、巨噬细胞、血管平滑肌细胞的含量,计算易损指数: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。
6.动脉斑块内部超微结构的检查:
用2.5%戊二醛固定新鲜颈动脉斑块,脱水,浸透包埋,制作超薄切片,染色后投射电镜观察拍片。
7.实时定量RT-PCR检测:
取新鲜颈动脉斑块组织,提取RNA,实时荧光定量RT-PCR反应检测斑块内β-actin,TLR2,TLR4,IL-1β,IL-6,TNF-α和MCP-1 mRNA的表达水平。
8.Western blot检测:
取新鲜颈动脉斑块组织,提取蛋白质,Western blot检测斑块内β-actin,TLR2,TLR4。
结果
1.RNA干扰慢病毒载体的构建:
制备编码携带小鼠TLR2和TLR4基因的BLOCK-It Lentiviral PolⅡmiR慢病毒载体,转染RAW264.7小鼠细胞进行筛选,病毒转染五天后收集细胞进行mRNA和蛋白水平的检测。结果表明TLR2i-siteA、TLR2i-siteB、TLR2i-siteC对基因TLR2 mRNA水平的沉默效果分别是56%,81%,和62%,蛋白水平的抑制效果分别是50%,62%,和35%,筛选出有效的克隆TLR2i-siteB;TLR4i-siteA、TLR4i-siteB、TLR4i-siteC对基因TLR2 mRNA水平的沉默效果分别是61%,54%,和72%,蛋白水平的抑制效果分别是45%,32%,和61%,筛选出有效的克隆TLR4i-siteC。
2.慢病毒载体体内转染效率和基因沉默效率的检测:
慢病毒载体转染一周后颈动脉AS斑块内可见绿色荧光蛋白GFP;转染二周后斑块内GFP的表达量增加;当实验结束,转染四周后斑块内仍可见GFP的表达。说明慢病毒载体有效的转入斑块内。
TLR2mRNA水平分别下降50%和59%在TLR2干扰组和TLR2+4干扰组与对照组相比(P＜0.05);TLR4干扰组的TLR2mRNA与对照组相比没有统计学差异;TLR2蛋白水平在TLR2干扰组和TLR2+4干扰组与其他组相比明显降低。
TLR4mRNA水平分别下降48%和53%在TLR4干扰组和TLR2+4干扰组与对照组相比(P＜0.05);TLR2干扰组的TLR4mRNA与对照组相比没有统计学差异;TLR4蛋白水平在TLR4干扰组和TLR2+4干扰组与其他组相比明显降低。
3.血液生化指标检测:
RNAi组和对照组血清TC、TG、HDL-C、LDL-C未见统计学差异。
4.病理学检测:
对照组,空载体组,TLR2干扰组,TLR4干扰组和TLR2+4联合干扰组的巨噬细胞含量百分比分别为31.8%,30.8%,26.9%,26.6%和18.6%;平滑肌细胞含量百分比分别为8.7%,8.7%,9.2%,10.5%和10.2%;斑块内脂质含量百分比分别为30.0%,29.2%,24.7%,24.9%和20.9%;斑块内胶原含量百分比分别为21.9%,23.2%,29.2%,29.0%和40%;纤维帽的厚度分别为9.96±0.97μm,10.03±1.38μm,16.97±4.2μm,15.41±1.2μm和17.29±0.72μm:易损指数分别为2.09±0.17,2.06±0.14,1.40±0.27,1.38±0.19和0.99±0.25。
基因干扰各组与非干扰组相比,其巨噬细胞含量明显减少,脂质含量明显减低,胶原含量明显增加(P＜0.05);在TLR2+4联合干扰组上述指标较TLR2,TLR4单独作用变化更为明显(P＜0.05);析因分析显示,TLR2干扰,TLR4干扰对斑块内巨噬细胞数量和胶原含量的改变有协同作用。TLR4干扰在TLR4干扰组和TLR2+4联合干扰组与非干扰组相比可以增加平滑肌细胞含量,TLR2未见此作用。
TLR2干扰和TLR4干扰可以增加斑块纤维帽的厚度(P＜0.05),并且两者之间存在协同作用。
干扰各组易损指数与非干扰组相比明显降低(P＜0.05),且联合干扰组与TLR2,TLR4单独干扰组相比,降低更为明显(P＜0.05)。
5.动脉斑块内部超微结构的检查:
非干扰组电镜检查:内皮细胞胞质内有脂质颗粒,内皮连接结构疏松,基膜下有大小不等的脂质颗粒和泡沫细胞。斑块内可见大量泡沫细胞,胞质被大小不等的脂质颗粒占据。平滑肌细胞以合成型为多,合成型平滑肌细胞肌丝和密斑密体少,内质网、线粒体和脂质颗粒多。巨噬细胞内含大量颗粒和脂滴。斑块内可见破损溶解细胞,其中有大量脂质颗粒和钙化结晶。
干扰组电镜检查:内皮细胞胞质内有少量脂质颗粒,内皮连接结构基本正常。平滑肌细胞合成型与收缩型均可见,细胞内可见脂质颗粒和各种细胞器,肌丝和密斑密体增多,粗面内质网及线粒体减少。泡沫细胞减少,细胞内及外基质中脂质颗粒明显减少。
6.斑块内炎症因子的检测:
TLR2干扰组的IL-6,MCP-1的mRNA水平与非干扰组相比显著降低(P＜0.05);TLR4干扰组的IL-6,TNFα和MCP-1的mRNA水平与非干扰组相比显著降低(P＜0.05);TLR2+4联合干扰组的IL-1β,IL-6,TNFα和MCP-1的mRNA水平与其余四组相比显著降低(P＜0.05)。析因分析显示,TLR2干扰和TLR4干扰对斑块内炎症因子的表达有协同作用。
结论
1.本研究成功地应用RNA干扰技术构建了针对小鼠TLR2和TLR4基因的慢病毒干扰载体。
2.体内实验结果表明,TLR2和TLR4基因沉默能有效的抑制斑块局部TLR2和TLR4基因的表达,显著减少斑块内巨噬细胞的含量,降低斑块内炎症因子的表达,从而抑制斑块内炎症反应,增强斑块的稳定性。
3.联合应用TLR2干扰和TLR4干扰,能够更有效的增加斑块的稳定性。TLR2和TLR4之间在动脉粥样硬化进程中相互之间存在着协同增强作用。
背景
动脉粥样硬化(atherosclerosis,AS)是心脑血管疾病的病理基础。研究表明,伴有免疫反应的炎症过程贯穿于动脉粥样硬化的始终。炎性细胞如巨噬细胞和T淋巴细胞在动脉粥样硬化病变的进程中扮演了重要角色。血管内皮细胞、平滑肌细胞及外膜的成纤维细胞和肥大细胞均可作为免疫细胞产生炎性因子。
但在动脉粥样硬化疾病的发生和发展中,其发病分子机制至今尚未阐明。近年来,Toll样受体(Toll-like receptors,TLRs)在炎症反应与天然(固有)和获得性(适应性)免疫反应中的作用日益受到人们的重视。Toll蛋白是最先在果蝇体内发现的一种信号转导蛋白,参与对细菌和真菌的识别及病原体的清除。随后,在哺乳动物体内发现的与Toll相似的跨膜蛋白被称为Toll样受体。TLRs能识别种类繁多的一病原相关分子模式(pathogen-associated molecular patterns,PAMPs),属于模式识别受体。目前为止,人类TLR家族至少有11名成员已被发现。不同的PAMP活化人类TLR家族的不同成员。TLRs通过其跨膜结构将LPS信号传递入细胞内,级联式激活转导蛋白MyD88、白介素1受体激酶(IRAK)、肿瘤坏死因子6调节蛋白(TRAF6)和NF-κB诱导激酶(NIK),进而激活核转录因子NF-κB,引起细胞因子的释放而导致炎症反应。启动炎症因子的释放,导致炎症反应的发生,在炎症反应的信号转导系统中发挥着极其重要的作用。
AS的整个过程是炎症和免疫反应的过程,而TLRs能够介导天然性和获得性免疫反应,因此,TLRs与AS的关系正逐渐引起人们的重视。大量研究表明TLR1、TLR2、TLR4等TLRs家族成员在AS的发生和发展过程中可能起到了重要作用,在人类和动物的AS斑块中已检测出上述TLRs的mRNA和蛋白质表达。已有的实验证实:动脉粥样硬化斑块表达TLR2表达量随病变的进展而增加,并且TLRs调控炎症和免疫反应、细胞凋亡、斑块稳定性以及血管重塑等AS病理过程。
TLR2必须与TLR家族中的其他成员,如TLR1,或者TLR6结合形成异源二聚体才能进行下游的信号传导过程。已有研究表明,在动脉粥样硬化斑块中,TLR1也同样有表达,尽管表达量较TLR2和TLR4含量偏少。既然TLR1和TLR2都在动脉粥样硬化斑块中表达,并且TLR2通过与其它TLR家族成员如TLR1形成异源二聚体然后进行下游的信号转导,我们不禁想探究TLR1和TLR2在易损斑块进展过程中的作用是什么?于是我们提出这样的假说:TLR1和TLR2可能在动脉粥样硬化易损斑块形成过程的作用不尽相同,应用基因沉默技术联合干扰TLR1和TLR2可能会较单一干扰TLR1或TLR2表达能更好的稳定易损斑块。
因此本课题开展了应用RNAi技术,单独或联合沉默LR1和TLR2基因,阻止AS进程,稳定易损斑块的研究。
目的
1.慢病毒表达载体的构建:根据TLR1和TLR2基因设计siRNA相应的DNA片段,分子克隆到BLOCK-It Lentiviral PolⅡmiR RNAi表达载体;
2.动物实验研究TLR1i和LTR2i对于阻止AS进程,降低动脉粥样硬化斑块易损性的疗效;
3.联合应该用TLR1i和LTR2i,对比研究联合干扰和单一干扰对动脉粥样硬化的稳定作用。
方法
1.RNA干扰慢病毒载体的构建以及有效靶点的筛选:
1.2分别构建TLR1和TLR2基因的小分子干扰RNA载体的构建:
设计、合成分别特异性针对小鼠TLR1和TLR2基因的小分子干扰RNA序列(各3段序列),以BLOCK-It Lentiviral PolⅡmiR RNAi为母本,分别构建小分子干扰RNA载体BLOCK-TLR1-A、BLOCK-TLR1-B、BLOCK-TLR1-C、BLOCK-TLR2-A、BLOCK-TLR2-B、BLOCK-TLR2-C,DNA序列测定鉴定重组质粒的正确性。
1.2慢病毒干扰载体的构建及体外扩增:
将已构建好的上述6个穿梭质粒和对照载体BLOCK-mock,与携带CMV启动子的BLOCK-It Lentiviral PolⅡmiR表达载体进行rapid BP/LR重组,继而与293FT包装细胞结合形成病毒表达载体。
1.3有效封闭TLR1和TLR2基因表达的RNA干扰慢病毒载体的筛选:
将mock-LV、TLR1i-siteA、TLR1i-siteB、TLR1i-siteC、TLR2i-siteA、TLR2i-siteB、TLR2i-siteC的慢病毒载体转染小鼠巨噬细胞系RAW264.7细胞。荧光定量RT-PCR、Western blot分别检测各转染细胞TLR1和TLR2 mRNA水平、蛋白质水平的表达,筛选出有效阻断TLR1,TLR2表达的小分子干扰RNA载体。
2.动物模型的建立:
140只10-12周龄的雄性apoE-/-小鼠实验全程高脂饮食(含0.25%胆固醇和15%脂肪)喂养。全部实验鼠均于实验初行右颈总动脉套管(缩窄性硅胶管)术以诱发AS病变:0.08%戊巴比妥钠(40mg/kg)腹腔注射麻醉小鼠。颈部正中皮肤切开,剥离右侧颈部的腺体和肌肉,暴露右侧颈总动脉,小心分离与之伴行的迷走神经,固定右颈总动脉,将长度为3mm、内径为0.3mm的硅胶管(小鼠颈动脉血管直径为0.5mm)套置于血管的外周,固定套管。缝合皮肤切口,将小鼠放回笼中。
3.RNA干扰慢病毒载体体内转染实验:
颈动脉套管手术8周后,将10μL的LV-mock病毒悬液,10μL的LV-TLR1病毒悬液,10μL的LV-TLR2病毒悬液,5μL的LV-TLR1病毒悬液联合5μL的LV-TLR2病毒悬液,局部转染右颈动脉斑块,根据转染病毒不同,各组分别命名:空载体组(n=28),TLR1干扰组(n=28),TLR2干扰组(n=28)和TLR1+2联合干扰组(n=28)。另28只未转染病毒的小鼠归为对照组。我们将,TLR1干扰组,TLR2干扰组和TLR1+2联合干扰组统称为干扰组;对照组和空载体组称为非干扰组。病毒转染一周后和二周后空载体组分别处死3只动物,检测斑块内内GFP的表达。其余实验鼠于转染四周后处死,观察斑块内GFP的表达,检测斑块的病理学形态结构。
4.血液生化指标检测:
检测血清总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)的水平。
5.病理学检测:
对颈动脉斑块分别进行H&E染色、油红O染色、天狼猩红染色,并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)的表达。测量斑块面积和纤维帽的厚度,观察斑块的形态结构,检测斑块内胶原、脂质、巨噬细胞、血管平滑肌细胞的含量,计算易损指数: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。
6.实时定量RT-PCR检测:
取新鲜颈动脉斑块组织,提取RNA,实时荧光定量RT-PCR反应检测斑块内β-actin,TLR1,TLR2,IL-6和MCP-1 mRNA的表达水平。
7.Western blot检测:
取新鲜颈动脉斑块组织,提取蛋白质,Western blot检测斑块内β-actin,TLR1,TLR2。
结果
1.RNA干扰慢病毒载体的构建:
制备编码携带小鼠TLR1和TLR2基因的BLOCK-It Lentiviral PolⅡmiR慢病毒载体,转染RAW264.7小鼠细胞进行筛选,病毒转染五天后收集细胞进行mRNA和蛋白水平的检测。结果表明TLR1i-siteA、TLR1i-siteB、TLR1i-siteC对基因TLR1 mRNA水平的沉默效果分别是77%,44%,和63%,蛋白水平的抑制效果分别是70%,39%,和57%,筛选出有效的克隆TLR2i-siteA;TLR2i-siteA、TLR2i-siteB、TLR2i-siteC对基因TLR2 mRNA水平的沉默效果分别是56%,81%,和62%,蛋白水平的抑制效果分别是50%,62%,和35%,筛选出有效的克隆TLR2i-siteB。
2.慢病毒载体体内转染效率和基因沉默效率的检测:
慢病毒载体转染一周后颈动脉AS斑块内可见绿色荧光蛋白GFP;转染二周后斑块内GFP的表达量增加;当实验结束,转染四周后斑块内仍可见GFP的表达。说明慢病毒载体有效的转入斑块内。
TLR1mRNA水平分别下降57%和48%在TLR1干扰组和TLR1+2干扰组与对照组相比(P＜0.05);TLR2干扰组的TLR1mRNA与对照组相比没有统计学差异;TLR1蛋白水平在TLR1干扰组和TLR1+2干扰组与其他组相比明显降低。
TLR2mRNA水平分别下降50%和59%在TLR2干扰组和TLR1+2干扰组与对照组相比(P＜0.05);TLR1干扰组的TLR2mRNA与对照组相比没有统计学差异;TLR2蛋白水平在TLR2干扰组和TLR1+2干扰组与其他组相比明显降低。
3.血液生化指标检测:
RNAi组和对照组血清TC、TG、HDL-C、LDL-C未见统计学差异。
4.病理学检测:
对照组,空载体组,TLR1干扰组,TLR1干扰组和TLR1+2联合干扰组的巨噬细胞含量百分比分别为31.8%,30.8%,27.0%,26.9%和22.2%;平滑肌细胞含量百分比分别为8.7%,8.7%,8.5%,9.2%和10.2%;斑块内脂质含量百分比分别为30.0%,29.2%,28.4%,24.7%和21.9%;斑块内胶原含量百分比分别为21.9%,23.2%,23.4%,29.2%和29.4%;纤维帽的厚度分别为9.96±0.97μm,10.03±1.38μm,12.62±1.99μm,16.97±4.2μm和17.26±2.00μm;易损指数分别为2.09±0.17,2.06±0.14,1.94±0.20,1.40±0.27和1.22±0.15。
基因干扰各组与非干扰组相比,其巨噬细胞含量明显减少(P＜0.05);在TLR1+2联合干扰组上述指标较TLR1,TLR2单独作用变化更为明显(P＜0.05)。
联合干扰组与非干扰组及TLR1单独干扰组相比,平滑肌细胞含量明显增加(P＜0.05)。
TLR1干扰和TLR2干扰可以增加斑块纤维帽的厚度(P＜0.05)。
TLR2干扰组易损指数与非干扰组相比明显降低(P＜0.05),且联合干扰组与TLR1单独干扰组相比,降低更为明显(P＜0.05)。
5.斑块内炎症因子的检测:
TLR1干扰组的IL-6的mRNA水平与非干扰组相比显著降低(P＜0.05):TLR2干扰组的和TLR1+2联合干扰组的IL-6和MCP-1的mRNA水平与其余三组相比显著降低(P＜0.05)。
结论
1.本研究成功地应用RNA干扰技术构建了针对小鼠TLR1和TLR2基因的慢病毒干扰载体。
2.体内实验结果表明,TLR1和TLR2基因沉默能有效的抑制斑块局部TLR1和TLR2基因的表达,显著减少斑块内巨噬细胞的含量,降低斑块内炎症因子的表达,从而抑制斑块内炎症反应,增强斑块的稳定性。
3.联合应用TLR1干扰和TLR2干扰,能够更有效的增加斑块的稳定性。
Background
Atherosclerosis is a long-term chronic disease characterized by the accumulation of lipids and fibrous connective tissue in the large arteries,accompanied by a local inflammatory response.Inflammatory cells,like T lymphocytes and macrophages, play an important role in all stages of atherosclerotic lesion development.Moreover, cells from the vessel wall,like endothelial cells,smooth muscle cells,adventitial fibroblasts and mast cells,are able to act as immunological cells that produce proinflammatory cytokines.The triggers and pathways of initiation and regulation of the immune responses in atherosclerotic disease are largely unknown.In general,the human immune system has two closely related pathways to respond to potentially harmful agents:the innate and the adaptive immune recognition systems.The adaptive immune system involves dynamic adaptation to unique antigenic epitopes that are present in the environment.The innate immune response is the first line of defense in which highly conserved pathogen motifs,entitled pathogen-associated molecular patterns(PAMPs),are recognized.
That atherosclerosis is a chronic inflammatory disease,highlighting the pivotal role played by the immune system,leads inevitably to the question of which molecule and downstream signal pathway might dominate inflammatory responses within arteries.In many current discussions of inflammatory states,the subject of toll-like receptors(TLRs) is bound to arise.The response of TLRs in atherosclerosis and finding a novel potential target for intervention in these processes is important.
Toll-like receptors(TLRs) are a group of receptors that play a key role in innate immune signaling and in initiating inflammatory responses,possibly in atherosclerosis.To date,11 human and 13 mouse TLRs have been cloned.TLRs have a short intracellular TolI/IL-1R(TIR) domain,which,upon ligand binding,results in recruitment of adapter molecules and induction of downstream activation.Activation of one or more TLRs(either as homomeric or heteromeric assemblies) causes the recruitment and activation of adaptor proteins such as myeloid differentiation factor 88(MyD88),then recruitment of members of the IL-1R-associated kinase(IRAK) family and activation of tumor necrosis factor(TNF) receptor-associated factor 6 (TRAF-6) and ultimately nuclear factor-kappa B(NF-kB).
Of the 11 human TLR-subtypes,TLR4 is strongly expressed in the healthy human artery,although each of the four main TLRs(TLR 1,2,4 and 6) involved in the recognition of lipids are considerably upregulated in diseased arteries.TLR2 and TLR4 are the best studied and are generally believed to be involved in the pathogenesis of cardiovascular disease.They are expressed in most cardiovascular cells,such as endothelial cells,smooth muscle cells,macrophages and adventitial fibroblasts.Activation of these cells as a response to self- and non-self ligands appears to be mediated by TLR2 and TLR4.In atherosclerosis,apolipoprotein E (apoE)-/- mice show accelerated atherosclerosis after TLR2 or TLR4 ligand stimulation.Several lines of evidence suggest that TLRs are proatherogenic.In mice lacking TLR4,small neointimal lesions developed after vascular injury,and TLR2 has similar effects.Recent study showed both TLR2 and TLR4 expression significantly increased over time in the arterial lesions of apoE-/- mice.Recent animal studies showed that MyD88 and TLR4 play an important role in atherosclerotic plaque accumulation.As well,local TLR2 stimulation in the femoral artery in apoE-/- mice accelerated atherosclerosis.These results support those of a study demonstrating impaired atherosclerotic lesion development in TLR2~(-/-)/LDLR~(-/-) mice.
Recently,small interfering RNAs(siRNAs) have been efficacious in silencing target genes by means of RNA interference(RNAi),a natural mechanism conserved in nature from yeast to humans,and lentiviral vectors can efficiently deliver siRNA because they are capable of stably transducing both dividing and nondividing cells.In this study,we constructed two lentiviral vectors aimed at knocking down TLR2 and TLR4 to elucidate their roles in atherosclerosis in apoE-/- mice by stabilizing plaque.
Objectives
1.To construct lentiviruses targeting mouse TLR2 or TLR4 small interfering RNA (siRNA).
2.Constricting collars were placed around the carotid arteries of apolipoprotein E (apoE)-/- mice to induce formation of atherosclerosis plaque.The two lentiviruses were transfected into carotid lesions separately or together.Plaque characteristics were analyzed.
3.Combined interfering TLR2 and TLR4 gene on atherosclerosis plaque to study the synthetic effect between TLR2 and TLR4.
Methods
1.Cell culture
RAW264.7 mouse macrophage cell line purchased from the American Type Culture Collection(Rockville,MD) was routinely maintained in RPMI-1640 medium (Sigma,St.Louis,MO) supplemented with 10%fetal bovine serum,at 37℃in humidified air containing 5%CO_2.The 293FT cell line(invitrogen,Carlsbad,CA), which stably expresses SV40 large T antigen and facilitates the optimal production of viruses,was cultured in Dulbecco's modified Eagle's medium(GIBCO,Grand Island, NY).
2.Lentiviral vector construction
First,we selected three different sequences(siteA,siteB,siteC) for targeting the TLR2 and TLR4 genes using the BLOCK-R RNAi Designer(Invitrogen) (http://maidesigner.invitrogen.com).targeted sequence of TLR2-siteA was 5'-TGCTGAATAGAGGTGAAAGACCTGGAGTTTTGGCCACTGACTGACTCCA GGTCTCACCTCTATT-3';targeted sequence of TLR2-siteB was 5'-TGCTGTGTACTGGACAAATTCAGGAAGTTTTGGCCACTGACTGACTTCCT GAATGTCCAGTACA-3';targeted sequence of TLR2-siteC was 5'-TGCTGAAAGGCATCATAGCAAACGTCGTTTTGGCCACTGACTGACGACG TTTGATGATGCCTTT-3';targeted sequence of TLR4-siteA was 5'-TGCTGTATGCAGGTAACTTACAGGAAGTTTTGGCCACTGACTGACTTCCT GTATTACCTGCATA-3';targeted sequence of TLR4-siteB was 5'-TGCTGTTCACGTAGAAACTGTAAGTCGTTTTGGCCACTGACTGACGACTT ACATTCTACGTGAA-3';targeted sequence of TLR4-siteC was 5'-TGCTGTGCACATGTCATTTGTTCAACGTTTTGGCCACTGACTGACGTTGA ACATGACATGTGCA-3'.A scrambled siRNA sequence(named mock-siRNA) with no known homology to mammal genes served as a control.The preparation of lentiviral vectors expressing human TLR2 and TLR4 involved use of the BLOCK-It Lentiviral PolⅡmiR RNAi Expression System(catalog no.K4948-00;Invitrogen) following the manufacturer's instructions.In brief,three TLR2(siteA,B,C) and three TLR4(siteA,B,C) PolⅡsiRNA expression vectors containing the respective mouse TLR2 and TLR4 siRNA-expressing cassettes were constructed.A replication-incompetent lentivirus was produced by cotransfeetion of the siRNA-expression vector and ViraPower packaging mix(Invitrogen) containing an optimized mixture of three packaging plasmids--pLP1,pLP2,and pLP/VSVG--into 293FT cells.Viral supernatant was harvested 48 h after transfection,filtered through a 0.45-μm cellulose acetate filter and frozen at -70℃.The three TLR2i lentiviruses (siteA,B,C) and three TLR4i lentiviruses(siteA,B,C) were transfected into RAW264.7 cells.Five days after transfection,cells were collected for real-time PCR and western blot analysis to select the most effective targeting site for the TLR2 and TLR4 genes to amplify.The siRNA mock vector containing lentivirus only was used as a control.Finally,three lentivirus vectors were produced:TLR2i-,TLR4i-,and mock,and the terminal virus titer was 2×10~9 TU/mL.
3.Animal protocol
Male apoE-/- mice(n=140),aged 10 to 12 weeks,were purchased from Beijing Laboratory Animal Research Center.Mice were kept on a 12-h light/12-h dark cycle with food and water freely available.All animals received a high-fat diet(0.25% cholesterol and 15%cocoa butter) for 12 weeks.All animal care and procedures were approved by Shandong University Institutional Animal Care and Use Committee and complied with the Guide for the Use and Care of Laboratory Animals published by the US National Institutes of Health(NIH publication 80-23,revised 1996)
4.Carotid collar placement and lentivirus injection
All mice were anesthetized with an intraperitoneal injection of pentobarbital sodium(40 mg/kg).As described by vonder Th(u|¨)sen et al.,a constricting silastic tube (0.30-mm inner diameter,0.50-mm outer diameter,and 2.5-mm long;Shandong Key Laboratory of Medical Polymer Materials,Jinan,China) was placed on the right common carotid artery near its bifurcation.Eight weeks after surgery,the collars were removed and lentiviral suspension was instilled into the right common carotid artery via the external carotid.The suspension was left in situ for 15 min after temporary ligation of the proximal common carotid artery and the internal carotid artery,and subsequently drawn off before ligation of the external carotid and closure of the skin wound with silk sutures as described.The mice were randomly divided into five groups according to lentivirus injection:control,no lentivirus;mock,10μL mock lentiviral suspension;TLR2 interfering(TLR2i),10μL TLR2i lentiviral suspension; TLR4 interfering(TLR4i),10μL TLR4i lentiviral suspension;and TLR2+4i,5μL each of TLR2i and TLR4i lentiviral suspension.Control and mock groups were non-knockdown groups,and TLR2i,TLR4i and TLR2+4i groups were gene-knockdown groups.
5.Serum lipid measurement
Before perfusion-fixation,blood was collected from the inferior vena cava. Serum total cholesterol,triglycerides,low-density lipoprotein(LDL) cholesterol,and high-density lipoprotein(HDL) cholesterol concentrations were measured by enzymatic assay(Zhejiang Dongou Biotechnology,Wenzhou,China)
6.Tissue preparation and histological analysis
Mice were perfused with phosphate-buffered saline through the left ventricle and then underwent peffusion fixation at 100 mm Hg with 4%formaldehyde(pH 7.2) for 2 and 15 min.The right common carotid artery with bifurcation was carefully excised and immersed in 4%formaldehyde overnight(4℃),embedded in OCT compound, and stored at -20℃until use.Each vessel throughout the entire length of the carotid artery underwent histological analysis.
In the 6 mice of the mock group sacrificed at week 9 and week 10,Sky Blue 6B (Sigma C8679) was used to eliminate background autofluorescence and cryosections were viewed under fluorescence microscopy to identify GFP expression.In all of the mice,OCT-embedded carotid artery was cross-sectioned into pieces 6μm thick at 50-μm intervals.Five cross sections in each mouse were used for a particular type of staining and the whole cross section viewed in one field was used for quantitative measurements.Sections were stained with hematoxylin and eosin for histological analysis.The plaque area was calculated by subtracting the lumen area from the area circumscribed by the internal elastic lamina.The plaque was subdivided into a fibrous cap and a necrotic core on the basis of extracellular matrix staining by HE,which was further confirmed by staining for collagen and lipids using picrosirius red and Oil-red O,respectively.Corresponding sections on separate slides were immunostained with monoclonal antibodies.Smooth muscle cells(SMCs) and macrophages were immunostained with anti-α-actin antibodies(1:1000;Sigma) and MOMA-2(1:25; Serotec,Oxford,UK),respectively.After incubation with the appropriate horseradish peroxidase(HRP)-conjugated secondary antibodies(1:100;Zhongshan Biological Technology,Wenzhou,China),the sections were incubated with 3',3'-diaminobenzidine and counterstained with hematoxylin.Sections reacted with non-immune IgG,secondary antibody only and no primary and secondary antibodies were used as negative controls.An automated image analysis system(Image-Pro Plus 5.0,Media Cybernetics,USA) was used for quantitative measurements.The positive staining area of macrophages,SMCs,lipids and collagen was quantified by computer-assisted color-gated measurement,and the ratio of positive staining area to the intimal area was calculated.The vulnerability index was calculated by the following formula:positive staining area of(macrophages +lipid)/positive staining area of(α-SMCs +collagen).
7.Transmission electron microscopy
Tissues for TEM were prepared as previously described.Tissue samples were trimmed into small pieces and fixed in chilled 2.5%glutaraldehyde in 0.1 M cacodylate buffer(pH 7.4) for 2 h and then postfixed in osmium tetroxide dissolved in the same buffer for 1-2 h.They were dehydrated through a graded series of ethanol and propylene oxide and embedded in epoxy resin.The embedded blocks were cut into ultrathin sections,then stained with lead citrate and uranyl acetate and observed under a 12A electron microscope(Hitachi,Japan).
8.Quantitative real-time RT-PCR analysis
RNA was extracted with TriZol Reagent(Invitrogen) following the manufacturer's instruction.The mRNA levels of TLR2,TLR4,interleukin-1β(IL-1β), IL-6,tumor necrosis factor-α(TNF-α),and monocyte chemoattractant protein-1 (MCP-1) were quantified in in vitro RAW264.7 cells and in vivo fresh carotid plaque tissue by use of SYBR Green technology(Applied Biosystems).Quantitative values were obtained from the threshold cycle value(Ct).The housekeeping geneβ-actin was quantified as an internal RNA control.The "2~(-△△CT) method" for comparing relative expression results was applied as described.
9.Western blot analysis
Equal amounts of protein from in vitro RAW264.7 cells and in vivo fresh carotid plaque tissue were separated on 14%SDS-PAGE and transferred to nitrocellulose membrane.Following blocking with 5%non-fat milk,the blots were washed with PBS containing 0.1%Tween 20 and incubated with an appropriate primary antibody at 4℃overnight.The blots were probed with antibodies againstβ-actin(1:100, Zhongshan,China),as well as TLR2(1:500),TLR4(1:200).After overnight incubation,the blots were washed with TBST and incubated with secondary antibody conjugated to HRP(1:1000,Zhongshan),then washed again.The blots were then visualized by enhanced chemiluminescence.
10.Data Analysis
Data were expressed as mean±SD.Multiple comparisons were analyzed by LSD post hoc tests.Factorial ANOVA was used to analyze the individual and interactive effects of two independent variables,TLR2i and TLR4i.With the statistical design of 2 x 2 factorial,the main effects of TLR2i,main effects of TLR4i and the interaction between TLR2i and TLR4i were compared.In such an analysis,if a significant interaction is present,the effects between TLR2i and TLR4i should be interpreted as synergistic rather than additive.All analyses involved use of SPSS 11.5 (SPSS Inc.,Chicago,IL).P<0.05 was considered statistically significant.
Results
1.Gene silencing in vitro:screening the most effective targeting sites for the TLR2 and TLR4 genes by real-time PCR and western blot analysis.
RAW264.7 cell line was transfected with lentivirus-based vectors expressing three different TLR2-siRNAs and gene silencing analysis showed that the TLR2i-site B lentivirus was the most effective vector in blocking TLR2 expression. TLR2-knockdown clone A,clone B,and clone C exhibited 56%,81%,and 62% reduction,respectively,in the level of TLR2 mRNA expression and 50%,62%,and 35%reduction,respectively,in the level of TLR2 protein expression.
The TLR4i-site C lentivirus was found to be the most effective vector in blocking TLR4 expression.TLR4 knockdown clone A,clone B,and clone C exhibited 61%,54%,and 72%reduction,respectively,in the level of TLR4 mRNA expression and 45%,32%,and 61%reduction,respectively,in the level of TLR4 protein expression.As a result,TLR2-site B and TLR4-site C lentiviruses were selected for further in vivo studies.
2.Efficient transfection of lentivirus in atherosclerosis plaque
Previous local virus delivery to pre-collar-induced carotid artery lesions of apoE-/- mice had resulted in efficient transfection to atherosclerosis plaque.Since GFP expression provides an efficient,convenient monitor to check transfection efficiency of lentivirus,we detected GFP fluorescence in carotid artery plaques 1 week after transfection,which indicated transfection of the siRNA,and greater fluorescence at 2 weeks after transfection.When the study was terminated,four weeks after transfection,GFP was still visible,although weak.These results demonstrated the efficient in vivo transfection of lentivirus by siRNA in atheroselerotie plaque.The virus local transfection did not affect normal functioning of the animals and did not result in weight change(30.6±3.1g in transfected mice vs.29.9±3.5g in controls) or serum cholesterol level(29.6±2.6 vs.30.1±2.0 mmol/L).
3.Gene silencing in vivo:effect of knockdown of siRNA lentivirus on TLR2 and TLR4 expression in atheroselerosis plaque by real-time PCR and western blot analysis.
To evaluate the efficacy of lentivirus-mediated gene silencing in vivo,we tested atherosclerosis plaque for TLR2 and TLR4 mRNA and protein expression levels.The TLR2 mRNA levels were lower,by 50%and 59%,in the TLR2i group and TLR2+4i group,respectively,than in the control group(P<0.05 for each).The TLR4 mRNA levels were lower,by 48%and 53%,in the TLR4i and TLR2+4i groups,respectively, than in the control group(P<0.05 for each).The mock,TLR2i and TLR4i groups showed no significant change in TLR4 or TLR2 mRNA level,respectively.
TLR2 protein was lower in the TLR2i and TLR2+4i groups than in the other groups,as was TLR4 protein lower in the TLR4i group and TLR2+4i group than in the other groups.So the local application of siRNA-lentivirus can efficiently silence gene expression in vivo.
4.Effect of TLR2i and TLR4i on plaque features
The atherosclerotie plaque composition of collagen,lipid,macrophages,and SMCs was demonstrated by histological and immunohistochemieal staining.The gene-knockdown groups showed higher content of collagen and lower content of lipid and macrophages than the non-knockdown groups(P<0.05 for all).Moreover,plaques in the TLR2+4i group showed a significant change as compared with plaques in the TLR2i- and TLR4i-alone groups(P<0.05 each).ANOVA revealed TLR2i and TLR4i significantly related in terms of macrophage and collagen content(P<0.05 for each) and TLR4i was shown to increaseα-SMC content in TLR4i and TLR2+4i groups (P<0.05 for each),with no differences in SMCs among other groups.
Both TLR2i and TLR4i induced markedly increased cap thickness(P<0.05 for all).Furthermore,the combination of TLR2i and TLR4i resulted in significantly increased mean cap thickness(P<0.05).
The vulnerable index differed significantly between gene-knockdown groups and non-knockdown groups,with 1.00±0.25 for the TLR2+4i group,signifieantly smaller than that of the other groups(2.09±0.17 for control,2.06±0.14 for mock,1.40±0.27 for TLR2i,and 1.38±0.19 for TLR4i groups;P<0.05 for all).
The gene-knockdown groups showed a lower accumulation of lipids and macrophages,a higher content of collagen,thicker fibrous caps and reduced vulnerable index than non-knockdown groups,with a synergistic combination effect of TLR2i and TLR4i on maerophage and collagen content and the mean cap thickness.
5.Gene knockdown effect on the ultrastructure of atherosclerosis plaque
The non-knockdown group ultrastucture consisted of foam cells and severely discontinuous innermost lining of endothelial cells,with wide gaps between adjacent cells and more synthetic(increased rough endoplasmic reticulum) SMCs than a contractile phenotype(cytoplasmic filaments prominent).Lipid-filled macrophages, characterized by numerous variable-sized granules and lipid droplets,were also present in the intima.The extracellular matrix contained fragmented elastic tissue, myelin figures,necrotic debris,and cholesterol clefts.
In the gene-knockdown group ultrastructure,the endothelial cells were smooth with integrity.Both synthetic and contractile types of SMCs were present,and the cytoplasm had more fine filaments,with dense bodies,decreased rough endoplasmic reticulum,mitoehondrial number and cytoplasmic lipid level.Foam cells were reduced in number as were extracellular matrix lipid droplets.
6.Effect of TLR2i and TLR4i on inflammation within lesions
The mRNA expression level of IL-6 and MCP-1 in the TLR2i subgroup and that of IL-1β,IL-6,TNF-αand MCP-1 in the TLR4i subgroup were lower than the corresponding measurements in the control or mock subgroups(all P<0.05).The TLR2+4i subgroup showed a significantly lower mRNA expression level of IL-1β, IL-6,TNF-α,MCP-1 than the other four subgroups(P<0.05).Furthermore,the synergistic effect of TLR2i and TLR4i in the reduction of the mRNA expression level of these cytokines was confirmed by Factorial ANOVA analysis(P<0.05).
Conclusions
1.In the present study,we applied lentivirus-mediated RNAi methods to efficiently knock down mice TLR2 and TLR4 genes in vitro and in vivo.
2.In vivo study demonstrated that transfected lentiviruses could silence TLR2 and TLR4 expression in apoE-/- mice with collar-induced carotid plaques,then decrease the level of inflammatory cytokines,and finally postpone the progression of atherosclerosis by stabilizing plaques.
3.Moreover,we found a synergistic effect between TLR2i and TLR4i in preventing atherosclerosis progression.These results point to a potential role for knockdown of TLR2 and TLR4 in local therapy for atherosclerosis.
Background
Atherosclerosis is a complicated inflammatory process with immune reactions during the initiation and progression of the disease.Toll-like receptors(TLRs) are known to play a key role in innate immune signaling and initiating inflammatory responses and their potential roles in the pathogenesis of atherosclerosis have received great attention.To date,11 human and 13 mouse TLRs have been cloned.TLRs have a short intracellular Toll/IL-1R domain,which,upon ligands binding,results in recruitment of adapter molecules and induction of downstream activation.Activation of one or more TLRs(either as homomeric or heteromeric assemblies) causes the recruitment and activation of adaptor proteins,and ultimately,nuclear factor-kappa B (NF-κB),which leads to stimulated release of a large number of inflammatory cytokines such as interleukin-6(IL-6),and chemoattractant cytokines such as monocyte chemoattractant protein-1(MCP-1).
Of the 11 human TLR-subtypes,TLR2 and TLR4 are the best studied and are generally believed to be involved in the pathogenesis of atherosclerosis.They are highly expressed in most vascular cells,such as endothelial cells,smooth muscle cells (SMCs),macrophages and adventitial fibroblasts.Studies in mice found that TLR2 played a critical role in the progression of atherosclerosis that was independent of dietary lipids,and TLR2 has to form dimers with other receptors such as TLR1 or TLR6 for signaling.TLR1 was also detected in atherosclerotic plaques,especially in areas infiltrated with inflammatory cells although the expression of TLR1 was found to be lower than that of TLR2 and TLR4.However,whether TLR1 and TLR2 are involved in the pathogenesis of vulnerable plaques is unknown.Since both TLR1 and TLR2 are expressed in atherosclerotic plaques and TLR2 may integrate with TLR1 or other TLRs for signaling,we hypothesized that TLR1 and TLR2 may play different roles in the formation of vulnerable plaques and combinatorial knockdown of TLR1 and TLR2 genes may enhance the effects of isolated knockdown of TLRI or TLR2 gene on plaque stabilization.
Recently,small interfering RNAs(siRNAs) have proven efficacious in silencing target genes and lentivirus can efficiently deliver siRNA because they are able to transduce both dividing and nondividing cells stably.In this study,we constructed two lentiviral vectors to knockdown TLR1 and TLR2 to elucidate their individual and synergistic roles in stabilizing atherosclerotic plaques in apolipoprotein E(apoE)-/-mice.
Objectives
1.To construct lentiviruses targeting mouse TLRI or TLR2 small interfering RNA(siRNA).
2.Constricting collars were placed around the carotid arteries of apolipoprotein E(apoE)-/- mice to induce formation of atherosclerosis plaque.The two lentiviruses were transfected into carotid lesions separately or together.Plaque characteristics were analyzed.
3.Combined interfering TLR1 and TLR2 gene on atherosclerosis plaque to study the synthetic effect between TLR1 and TLR2.
Methods
1.Cell culture
RAW264.7 mouse macrophage cell line purchased from the American Type Culture Collection(Rockville,MD) was routinely maintained in RPMI-1640 medium (Sigma,St.Louis,MO) supplemented with 10%fetal bovine serum,at 37℃in humidified air containing 5%CO_2.The 293FT cell line(Invitrogen,Carlsbad,CA), which stably expresses SV40 large T antigen and facilitates the optimal production of viruses,was cultured in Dulbecco's modified Eagle's medium(GIBCO,Grand Island, NY).
2.Lentiviral vector construction
First,three different sequences(site A,B and C) of TLRI and TLR2 genes in mice were selected as the target for RNA interference(RNAi),using the BLOCK-It RNAi Designer(invitrogen)(http://maidesigner.invitrogen.com).Targeted sequence of TLR1-siteA was 5'-TGCTGATTAAAGGAGAGGTCCAAATGGTTTTGGCCACTGACTGACCATTT GGATCTCCTTTAAT-3';targeted sequence of TLR1-siteB was 5'-TGCTGTAAAGTAGTTGTTTGCAAGGGGTTTTGGCCACTGACTGACCCCTT GCACAACTACTTTA-3';targeted sequence of TLRl-siteC was 5'-TGCTGTAATGAAGGAATTCCACGTTGGTTTTGGCCACTGACTGACCAAC GTGGTTCCTTCATTA-3';targeted sequence of TLR2-siteA was 5'-TGCTGAATAGAGGTGAAAGACCTGGAGTTTTGGCCACTGACTGACTCCA GGTCTCACCTCTATT-3';targeted sequence of TLR2-siteB was 5'-TGCTGTGTACTGGACAAATTCAGGAAGTTTTGGCCACTGACTGACTTCCT GAATGTCCAGTACA-3';targeted sequence of TLR2-siteC was 5'-TGCTGAAAGGCATCATAGCAAACGTCGTTTTGGCCACTGACTGACGACG TTTGATGATGCCTTT-3'.A scrambled siRNA sequence(named moek-siRNA) with no known homology to mammal genes served as a control.The preparation of lentiviral vectors expressing human TLR1 and TLR2 involved use of the BLOCK-It Lentiviral PolⅡmiR RNAi Expression System(catalog no.K4948-00;Invitrogen) following the manufacturer's instructions.In brief,three TLR1(siteA,B,C) and three TLR2(siteA,B,C) PolⅡsiRNA expression vectors containing the respective mouse TLR1 and TLR2 siRNA-expressing cassettes were constructed.A replication-incompetent lentivirus was produced by cotransfection of the siRNA-expression vector and ViraPower packaging mix(invitrogen) containing an optimized mixture of three packaging plasmids--pLP1,pLP2,and pLP/VSVG--into 293FT ceils.Viral supernatant was harvested 48 h after transfection,filtered through a 0.45-μm cellulose acetate filter and frozen at -70℃.The three TLRIi lentiviruses (siteA,B,C) and three TLR2i lentiviruses(siteA,B,C) were transfected into RAW264.7 cells.Five days after transfection,cells were collected for real-time PCR and western blot analysis to select the most effective targeting site for the TLR1 and TLR2 genes to amplify.The siRNA mock vector containing lentivirus only was used as a control.Finally,three lentivirus vectors were produced:TLR1i-,TLR2i-,and mock,and the terminal virus titer was 2×10~9 TU/mL.
3.Animal protocol
Male apoE-/- mice(n=140),aged 10 to 12 weeks,were purchased from Beijing Laboratory Animal Research Center.Mice were kept on a 12-h light/12-h dark cycle with food and water freely available.All animals received a high-fat diet(0.25% cholesterol and 15%cocoa butter) for 12 weeks.All animal care and procedures were approved by Shandong University Institutional Animal Care and Use Committee and complied with the Guide for the Use and Care of Laboratory Animals published by the US National Institutes of Health(NIH publication 80-23,revised 1996)
4.Carotid collar placement and lentivirus injection
All mice were anesthetized with an intraperitoneal injection of pentobarbital sodium(40 mg/kg).As described by vonder Th(u|¨)sen et al.,a constricting silastic tube (0.30-mm inner diameter,0.50-mm outer diameter,and 2.5-mm long;Shandong Key Laboratory of Medical Polymer Materials,Jinan,China) was placed on the right common carotid artery near its bifurcation.Eight weeks after surgery,the collars were removed and lentiviral suspension was instilled into the right common carotid artery via the external carotid.The suspension was left in situ for 15 min after temporary ligation of the proximal common carotid artery and the internal carotid artery,and subsequently drawn off before ligation of the external carotid and closure of the skin wound with silk sutures as described.The mice were randomly divided into five groups according to lentivirus injection:control,no lentivirus;mock,10μL mock lentiviral suspension;TLR1 interfering(TLRli),10μL TLR1i lentiviral suspension; TLR2 interfering(TLR2i),10μL TLR2i lentiviral suspension;and TLR1+2i,5μL each of TLR1i and TLR2i lentiviral suspension.Control and mock groups were non-knockdown groups,and TLR1i,TLR2i and TLR1+2i groups were gene-knockdown groups.
5.Serum lipid measurement
Before perfusion-fixation,blood was collected from the inferior vena cava. Serum total cholesterol,triglycerides,low-density lipoprotein(LDL) cholesterol,and high-density lipoprotein(HDL) cholesterol concentrations were measured by enzymatic assay(Zhejiang Dongou Biotechnology,Wenzhou,China)
6.Tissue preparation and histological analysis
Mice were perfused with phosphate-buffered saline through the left ventricle and then underwent perfusion fixation at 100 mm Hg with 4%formaldehyde(pH 7.2) for 2 and 15 min.The right common carotid artery with bifurcation was carefully excised and immersed in 4%formaldehyde overnight(4℃),embedded in OCT compound, and stored at -20℃until use.Each vessel throughout the entire length of the carotid artery underwent histological analysis.
In the 6 mice of the mock group sacrificed at week 9 and week 10,Sky Blue 6B (Sigma C8679) was used to eliminate background autofluorescence and cryosections were viewed under fluorescence microscopy to identify GFP expression.In all of the mice,OCT-embedded carotid artery was cross-sectrned into pieces 6 lira thick at 50-μm intervals.Five cross sections in each mouse were used for a particular type of staining and the whole cross section viewed in one field was used for quantitative measurements.Sections were stained with hematoxylin and eosin for histological analysis.The plaque area was calculated by subtracting the lumen area from the area circumscribed by the internal elastic lamina.The plaque was subdivided into a fibrous cap and a necrotic core on the basis of extracellular matrix staining by HE,which was further confirmed by staining for collagen and lipids using picrosirius red and Oil-red O,respectively.Corresponding sections on separate slides were immunostained with monoclonal antibodies.Smooth muscle cells(SMCs) and macrophages were immunostained with anti-α-actin antibodies(1:1000;Sigma) and MOMA-2(1:25; Serotec,Oxford,UK),respectively.After incubation with the appropriate horseradish peroxidase(HRP)-conjugated secondary antibodies(1:100;Zhongshan Biological Technology,Wenzhou,China),the sections were incubated with 3',3'-diaminobenzidine and counterstained with hematoxylin.Sections reacted with non-immune IgG,secondary antibody only and no primary and secondary antibodies were used as negative controls.An automated image analysis system(Image-Pro Plus 5.0,Media Cybernetics,USA) was used for quantitative measurements.The positive staining area of macrophages,SMCs,lipids and collagen was quantified by computer-assisted color-gated measurement,and the ratio of positive staining area to the intimal area was calculated.The vulnerability index was calculated by the following formula:positive staining area of(macrophages +lipid)/positive staining area of(α-SMCs +collagen).
7.Quantitative real-time RT-PCR analysis
RNA was extracted with TriZol Reagent(Invitrogen) following the manufacturer's instruction.The mRNA levels of TLR1,TLR2,interleukin-6(IL-6), and monocyte chemoattractant protein-1(MCP-1) were quantified in in vitro RAW264.7 cells and in vivo fresh carotid plaque tissue by use of SYBR Green technology(Applied Biosystems).Quantitative values were obtained from the threshold cycle value(Ct).The housekeeping geneβ-actin was quantified as an internal RNA control.The "2~(-△△CT) method" for comparing relative expression results was applied as described.
8.Western blot analysis
Equal amounts of protein from in vitro RAW264.7 cells and in vivo fresh carotid plaque tissue were separated on 14%SDS-PAGE and t
动脉粥样硬化(atherosclerosis,AS)是心脑血管疾病的病理基础。研究表明,伴有免疫反应的炎症过程贯穿于动脉粥样硬化的始终。炎性细胞如T淋巴细胞和巨噬细胞在动脉粥样硬化病变的进程中扮演了重要角色。血管内皮细胞、平滑肌细胞及外膜的成纤维细胞和肥大细胞均可作为免疫细胞产生炎性因子。
但在动脉粥样硬化疾病的发生和发展中,其发病分子机制至今尚未阐明。近年来,Toll样受体(Toll-like receptors,TLRs)在炎症反应与天然(固有)和获得性(适应性)免疫反应中的作用日益受到人们的重视。Toll蛋白是最先在果蝇体内发现的一种信号转导蛋白,参与对细菌和真菌的识别及病原体的清除。随后.在哺乳动物体内发现的与Toll相似的跨膜蛋白被称为Toll样受体。TLRs能识别种类繁多的一病原相关分子模式(pathogen-associated molecular patterns,PAMPs),属于模式识别受体。目前为止,人类TLR家族至少有11名成员已被发现。不同的PAMP活化人类TLR家族的不同成员。TLRs通过其跨膜结构将LPS信号传递入细胞内,级联式激活转导蛋白MyD88、白介素1受体激酶(IRAK)、肿瘤坏死因子6调节蛋白(TRAF6)和NF-κB诱导激酶(NIK),进而激活核转录因子NF-κB,引起细胞因子的释放而导致炎症反应。启动炎症因子的释放,导致炎症反应的发生,在炎症反应的信号转导系统中发挥着极其重要的作用。
AS的整个过程是炎症和免疫反应的过程,而TLRs能够介导天然性和获得性免疫反应,因此,TLRs与AS的关系正逐渐引起人们的重视。大量研究证实TLR1、TLR2、TLR4、TLR6等TLRs家族成员在AS的发生和发展过程中可能起到了重要作用,在人类和动物的AS斑块中已检测出上述TLRs的mRNA和蛋白质表达。已有的实验证实:动脉粥样硬化斑块表达TLR2和TLR4,这两种受体的表达量随病变的进展而增加,并且TLRs调控炎症和免疫反应、细胞凋亡、斑块稳定性以及血管重塑等AS病理过程。
虽然近年来国内外学者在TLRs与AS斑块相互关系的研究中已取得较大的进展,但仍存在许多重大问题亟待解决:(1)既然TLR2和TLR4在AS形成和斑块破裂中扮演重要角色,能否通过封闭TLR2和TLR4基因表达阻止AS进程并降低AS斑块的易损性。这构成本课题的主导思想。(2)目前造成基因失活进行基因研究和基因治疗的方式主要基因沉默技术,又称RNA干扰(RNA interference,RNAi),RNAi可以高效特异地阻断目标基因的表达,目前已经广泛地应用于体内和体外基因功能的研究中。哺乳动物细胞实验发现,化学合成的21-23核苷酸长的小分子干扰RNA片段(small interfering RNAs,siRNAs),或是质粒或病毒载体介导的siRNA,均可引发基因沉默或者表达抑制。RNA干扰技术在细胞水平致病基因的功能研究中得到广泛应用。此外,通过高通量RNAi筛选,siRNA还被用于系统分析某些疾病的多种致病基因。借助于质粒或病毒载体的介导,siRNA已经被用于体内实验,特异性降低靶基因的表达。在进一步完善siRNA稳定性的基础上,RNAi的试剂有望应用于临床。(3)AS斑块中,TLR2和TLR4都发挥作用,那么这两者的各自作用及机制是什么?它们之间相的关系是独立的并联关系还是协同的网络联系?
因此本课题开展了应用RNAi技术,单独或联合沉默LR2和TLR4基因,阻止AS进程,稳定易损斑块的研究。
目的
1.慢病毒表达载体的构建:根据TLR2和TLR4基因设计siRNA相应的DNA片段,分子克隆到BLOCK-It Lentiviral PolⅡmiR RNAi表达载体;
2.动物实验研究TLR2i和LTR4i对于阻止AS进程,降低动脉粥样硬化斑块易损性的疗效;
3.联合应该用TLR2i和LTR4i,对比研究联合干扰和单一干扰对动脉粥样硬化的稳定作用。
方法
1.RNA干扰慢病毒载体的构建以及有效靶点的筛选:
1.1分别构建TLR2和TLR4基因的小分子干扰RNA载体的构建:
设计、合成分别特异性针对小鼠TLR2和TLR4基因的小分子干扰RNA序列(各3段序列),以BLOCK-It Lentiviral PolⅡmiR RNAi为母本,分别构建小分子干扰RNA载体BLOCK-TLR2-A、BLOCK-TLR2-B、BLOCK-TLR2-C、BLOCK-TLR4-A、BLOCK-TLR4-B、BLOCK-TLR4-C,DNA序列测定鉴定重组质粒的正确性。
1.2慢病毒干扰载体的构建及体外扩增:
将已构建好的上述6个穿梭质粒和对照载体BLOCK-mock,与携带CMV启动子的BLOCK-It Lentiviral PolⅡmiR表达载体进行rapid BP/LR重组,继而与293FT包装细胞结合形成病毒表达载体。
1.3有效封闭TLR2和TLR4基因表达的RNA干扰慢病毒载体的筛选:
将mock-LV、TLR2i-siteA、TLR2i-siteB、TLR2i-siteC、TLR4i-siteA、TLR4i-siteB、TLR4i-siteC的慢病毒载体转染小鼠巨噬细胞系RAW264.7细胞。荧光定量RT-PCR、Western blot分别检测各转染细胞TLR2和TLR4 mRNA水平、蛋白质水平的表达,筛选出有效阻断TLR2,TLR4表达的小分子干扰RNA载体。
2.动物模型的建立:
140只10-12周龄的雄性apoE-/-小鼠实验全程高脂饮食(含0.25%胆固醇和15%脂肪)喂养。全部实验鼠均于实验初行右颈总动脉套管(缩窄性硅胶管)术以诱发AS病变:0.08%戊巴比妥钠(40mg/kg)腹腔注射麻醉小鼠。颈部正中皮肤切开,剥离右侧颈部的腺体和肌肉,暴露右侧颈总动脉,小心分离与之伴行的迷走神经,固定右颈总动脉,将长度为3mm、内径为0.3mm的硅胶管(小鼠颈动脉血管直径为0.5mm)套置于血管的外周,固定套管。缝合皮肤切口,将小鼠放回笼中。
3.RNA干扰慢病毒载体体内转染实验:
颈动脉套管手术8周后,将10μL的LV-mock病毒悬液,10μL的LV-TLR2病毒悬液,10μL的LV-TLR4病毒悬液,5μL的LV-TLR2病毒悬液联合5μL的LV-TLR4病毒悬液,局部转染右颈动脉斑块,根据转染病毒不同,各组分别命名:空载体组(n=28),TLR2干扰组(n=28),TLR2干扰组(n=28)和TLR2+4联合干扰组(n=28)。另28只未转染病毒的小鼠归为对照组。我们将,TLR2干扰组,TLR4干扰组和TLR2+4联合干扰组统称为干扰组;对照组和空载体组称为非干扰组。病毒转染一周后和二周后空载体组分别处死3只动物,检测斑块内内GFP的表达。其余实验鼠于转染四周后处死,观察斑块内GFP的表达,检测斑块的病理学形态结构。
4.血液生化指标检测:
检测血清总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)的水平。
5.病理学检测:
对颈动脉斑块分别进行H&E染色、油红O染色、天狼猩红染色,并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)的表达。测量斑块面积和纤维帽的厚度,观察斑块的形态结构,检测斑块内胶原、脂质、巨噬细胞、血管平滑肌细胞的含量,计算易损指数: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。
6.动脉斑块内部超微结构的检查:
用2.5%戊二醛固定新鲜颈动脉斑块,脱水,浸透包埋,制作超薄切片,染色后投射电镜观察拍片。
7.实时定量RT-PCR检测:
取新鲜颈动脉斑块组织,提取RNA,实时荧光定量RT-PCR反应检测斑块内β-actin,TLR2,TLR4,IL-1β,IL-6,TNF-α和MCP-1 mRNA的表达水平。
8.Western blot检测:
取新鲜颈动脉斑块组织,提取蛋白质,Western blot检测斑块内β-actin,TLR2,TLR4。
结果
1.RNA干扰慢病毒载体的构建:
制备编码携带小鼠TLR2和TLR4基因的BLOCK-It Lentiviral PolⅡmiR慢病毒载体,转染RAW264.7小鼠细胞进行筛选,病毒转染五天后收集细胞进行mRNA和蛋白水平的检测。结果表明TLR2i-siteA、TLR2i-siteB、TLR2i-siteC对基因TLR2 mRNA水平的沉默效果分别是56%,81%,和62%,蛋白水平的抑制效果分别是50%,62%,和35%,筛选出有效的克隆TLR2i-siteB;TLR4i-siteA、TLR4i-siteB、TLR4i-siteC对基因TLR2 mRNA水平的沉默效果分别是61%,54%,和72%,蛋白水平的抑制效果分别是45%,32%,和61%,筛选出有效的克隆TLR4i-siteC。
2.慢病毒载体体内转染效率和基因沉默效率的检测:
慢病毒载体转染一周后颈动脉AS斑块内可见绿色荧光蛋白GFP;转染二周后斑块内GFP的表达量增加;当实验结束,转染四周后斑块内仍可见GFP的表达。说明慢病毒载体有效的转入斑块内。
TLR2mRNA水平分别下降50%和59%在TLR2干扰组和TLR2+4干扰组与对照组相比(P＜0.05);TLR4干扰组的TLR2mRNA与对照组相比没有统计学差异;TLR2蛋白水平在TLR2干扰组和TLR2+4干扰组与其他组相比明显降低。
TLR4mRNA水平分别下降48%和53%在TLR4干扰组和TLR2+4干扰组与对照组相比(P＜0.05);TLR2干扰组的TLR4mRNA与对照组相比没有统计学差异;TLR4蛋白水平在TLR4干扰组和TLR2+4干扰组与其他组相比明显降低。
3.血液生化指标检测:
RNAi组和对照组血清TC、TG、HDL-C、LDL-C未见统计学差异。
4.病理学检测:
对照组,空载体组,TLR2干扰组,TLR4干扰组和TLR2+4联合干扰组的巨噬细胞含量百分比分别为31.8%,30.8%,26.9%,26.6%和18.6%;平滑肌细胞含量百分比分别为8.7%,8.7%,9.2%,10.5%和10.2%;斑块内脂质含量百分比分别为30.0%,29.2%,24.7%,24.9%和20.9%;斑块内胶原含量百分比分别为21.9%,23.2%,29.2%,29.0%和40%;纤维帽的厚度分别为9.96±0.97μm,10.03±1.38μm,16.97±4.2μm,15.41±1.2μm和17.29±0.72μm:易损指数分别为2.09±0.17,2.06±0.14,1.40±0.27,1.38±0.19和0.99±0.25。
基因干扰各组与非干扰组相比,其巨噬细胞含量明显减少,脂质含量明显减低,胶原含量明显增加(P＜0.05);在TLR2+4联合干扰组上述指标较TLR2,TLR4单独作用变化更为明显(P＜0.05);析因分析显示,TLR2干扰,TLR4干扰对斑块内巨噬细胞数量和胶原含量的改变有协同作用。TLR4干扰在TLR4干扰组和TLR2+4联合干扰组与非干扰组相比可以增加平滑肌细胞含量,TLR2未见此作用。
TLR2干扰和TLR4干扰可以增加斑块纤维帽的厚度(P＜0.05),并且两者之间存在协同作用。
干扰各组易损指数与非干扰组相比明显降低(P＜0.05),且联合干扰组与TLR2,TLR4单独干扰组相比,降低更为明显(P＜0.05)。
5.动脉斑块内部超微结构的检查:
非干扰组电镜检查:内皮细胞胞质内有脂质颗粒,内皮连接结构疏松,基膜下有大小不等的脂质颗粒和泡沫细胞。斑块内可见大量泡沫细胞,胞质被大小不等的脂质颗粒占据。平滑肌细胞以合成型为多,合成型平滑肌细胞肌丝和密斑密体少,内质网、线粒体和脂质颗粒多。巨噬细胞内含大量颗粒和脂滴。斑块内可见破损溶解细胞,其中有大量脂质颗粒和钙化结晶。
干扰组电镜检查:内皮细胞胞质内有少量脂质颗粒,内皮连接结构基本正常。平滑肌细胞合成型与收缩型均可见,细胞内可见脂质颗粒和各种细胞器,肌丝和密斑密体增多,粗面内质网及线粒体减少。泡沫细胞减少,细胞内及外基质中脂质颗粒明显减少。
6.斑块内炎症因子的检测:
TLR2干扰组的IL-6,MCP-1的mRNA水平与非干扰组相比显著降低(P＜0.05);TLR4干扰组的IL-6,TNFα和MCP-1的mRNA水平与非干扰组相比显著降低(P＜0.05);TLR2+4联合干扰组的IL-1β,IL-6,TNFα和MCP-1的mRNA水平与其余四组相比显著降低(P＜0.05)。析因分析显示,TLR2干扰和TLR4干扰对斑块内炎症因子的表达有协同作用。
结论
1.本研究成功地应用RNA干扰技术构建了针对小鼠TLR2和TLR4基因的慢病毒干扰载体。
2.体内实验结果表明,TLR2和TLR4基因沉默能有效的抑制斑块局部TLR2和TLR4基因的表达,显著减少斑块内巨噬细胞的含量,降低斑块内炎症因子的表达,从而抑制斑块内炎症反应,增强斑块的稳定性。
3.联合应用TLR2干扰和TLR4干扰,能够更有效的增加斑块的稳定性。TLR2和TLR4之间在动脉粥样硬化进程中相互之间存在着协同增强作用。
背景
动脉粥样硬化(atherosclerosis,AS)是心脑血管疾病的病理基础。研究表明,伴有免疫反应的炎症过程贯穿于动脉粥样硬化的始终。炎性细胞如巨噬细胞和T淋巴细胞在动脉粥样硬化病变的进程中扮演了重要角色。血管内皮细胞、平滑肌细胞及外膜的成纤维细胞和肥大细胞均可作为免疫细胞产生炎性因子。
但在动脉粥样硬化疾病的发生和发展中,其发病分子机制至今尚未阐明。近年来,Toll样受体(Toll-like receptors,TLRs)在炎症反应与天然(固有)和获得性(适应性)免疫反应中的作用日益受到人们的重视。Toll蛋白是最先在果蝇体内发现的一种信号转导蛋白,参与对细菌和真菌的识别及病原体的清除。随后,在哺乳动物体内发现的与Toll相似的跨膜蛋白被称为Toll样受体。TLRs能识别种类繁多的一病原相关分子模式(pathogen-associated molecular patterns,PAMPs),属于模式识别受体。目前为止,人类TLR家族至少有11名成员已被发现。不同的PAMP活化人类TLR家族的不同成员。TLRs通过其跨膜结构将LPS信号传递入细胞内,级联式激活转导蛋白MyD88、白介素1受体激酶(IRAK)、肿瘤坏死因子6调节蛋白(TRAF6)和NF-κB诱导激酶(NIK),进而激活核转录因子NF-κB,引起细胞因子的释放而导致炎症反应。启动炎症因子的释放,导致炎症反应的发生,在炎症反应的信号转导系统中发挥着极其重要的作用。
AS的整个过程是炎症和免疫反应的过程,而TLRs能够介导天然性和获得性免疫反应,因此,TLRs与AS的关系正逐渐引起人们的重视。大量研究表明TLR1、TLR2、TLR4等TLRs家族成员在AS的发生和发展过程中可能起到了重要作用,在人类和动物的AS斑块中已检测出上述TLRs的mRNA和蛋白质表达。已有的实验证实:动脉粥样硬化斑块表达TLR2表达量随病变的进展而增加,并且TLRs调控炎症和免疫反应、细胞凋亡、斑块稳定性以及血管重塑等AS病理过程。
TLR2必须与TLR家族中的其他成员,如TLR1,或者TLR6结合形成异源二聚体才能进行下游的信号传导过程。已有研究表明,在动脉粥样硬化斑块中,TLR1也同样有表达,尽管表达量较TLR2和TLR4含量偏少。既然TLR1和TLR2都在动脉粥样硬化斑块中表达,并且TLR2通过与其它TLR家族成员如TLR1形成异源二聚体然后进行下游的信号转导,我们不禁想探究TLR1和TLR2在易损斑块进展过程中的作用是什么?于是我们提出这样的假说:TLR1和TLR2可能在动脉粥样硬化易损斑块形成过程的作用不尽相同,应用基因沉默技术联合干扰TLR1和TLR2可能会较单一干扰TLR1或TLR2表达能更好的稳定易损斑块。
因此本课题开展了应用RNAi技术,单独或联合沉默LR1和TLR2基因,阻止AS进程,稳定易损斑块的研究。
目的
1.慢病毒表达载体的构建:根据TLR1和TLR2基因设计siRNA相应的DNA片段,分子克隆到BLOCK-It Lentiviral PolⅡmiR RNAi表达载体;
2.动物实验研究TLR1i和LTR2i对于阻止AS进程,降低动脉粥样硬化斑块易损性的疗效;
3.联合应该用TLR1i和LTR2i,对比研究联合干扰和单一干扰对动脉粥样硬化的稳定作用。
方法
1.RNA干扰慢病毒载体的构建以及有效靶点的筛选:
1.2分别构建TLR1和TLR2基因的小分子干扰RNA载体的构建:
设计、合成分别特异性针对小鼠TLR1和TLR2基因的小分子干扰RNA序列(各3段序列),以BLOCK-It Lentiviral PolⅡmiR RNAi为母本,分别构建小分子干扰RNA载体BLOCK-TLR1-A、BLOCK-TLR1-B、BLOCK-TLR1-C、BLOCK-TLR2-A、BLOCK-TLR2-B、BLOCK-TLR2-C,DNA序列测定鉴定重组质粒的正确性。
1.2慢病毒干扰载体的构建及体外扩增:
将已构建好的上述6个穿梭质粒和对照载体BLOCK-mock,与携带CMV启动子的BLOCK-It Lentiviral PolⅡmiR表达载体进行rapid BP/LR重组,继而与293FT包装细胞结合形成病毒表达载体。
1.3有效封闭TLR1和TLR2基因表达的RNA干扰慢病毒载体的筛选:
将mock-LV、TLR1i-siteA、TLR1i-siteB、TLR1i-siteC、TLR2i-siteA、TLR2i-siteB、TLR2i-siteC的慢病毒载体转染小鼠巨噬细胞系RAW264.7细胞。荧光定量RT-PCR、Western blot分别检测各转染细胞TLR1和TLR2 mRNA水平、蛋白质水平的表达,筛选出有效阻断TLR1,TLR2表达的小分子干扰RNA载体。
2.动物模型的建立:
140只10-12周龄的雄性apoE-/-小鼠实验全程高脂饮食(含0.25%胆固醇和15%脂肪)喂养。全部实验鼠均于实验初行右颈总动脉套管(缩窄性硅胶管)术以诱发AS病变:0.08%戊巴比妥钠(40mg/kg)腹腔注射麻醉小鼠。颈部正中皮肤切开,剥离右侧颈部的腺体和肌肉,暴露右侧颈总动脉,小心分离与之伴行的迷走神经,固定右颈总动脉,将长度为3mm、内径为0.3mm的硅胶管(小鼠颈动脉血管直径为0.5mm)套置于血管的外周,固定套管。缝合皮肤切口,将小鼠放回笼中。
3.RNA干扰慢病毒载体体内转染实验:
颈动脉套管手术8周后,将10μL的LV-mock病毒悬液,10μL的LV-TLR1病毒悬液,10μL的LV-TLR2病毒悬液,5μL的LV-TLR1病毒悬液联合5μL的LV-TLR2病毒悬液,局部转染右颈动脉斑块,根据转染病毒不同,各组分别命名:空载体组(n=28),TLR1干扰组(n=28),TLR2干扰组(n=28)和TLR1+2联合干扰组(n=28)。另28只未转染病毒的小鼠归为对照组。我们将,TLR1干扰组,TLR2干扰组和TLR1+2联合干扰组统称为干扰组;对照组和空载体组称为非干扰组。病毒转染一周后和二周后空载体组分别处死3只动物,检测斑块内内GFP的表达。其余实验鼠于转染四周后处死,观察斑块内GFP的表达,检测斑块的病理学形态结构。
4.血液生化指标检测:
检测血清总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)的水平。
5.病理学检测:
对颈动脉斑块分别进行H&E染色、油红O染色、天狼猩红染色,并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)的表达。测量斑块面积和纤维帽的厚度,观察斑块的形态结构,检测斑块内胶原、脂质、巨噬细胞、血管平滑肌细胞的含量,计算易损指数: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。
6.实时定量RT-PCR检测:
取新鲜颈动脉斑块组织,提取RNA,实时荧光定量RT-PCR反应检测斑块内β-actin,TLR1,TLR2,IL-6和MCP-1 mRNA的表达水平。
7.Western blot检测:
取新鲜颈动脉斑块组织,提取蛋白质,Western blot检测斑块内β-actin,TLR1,TLR2。
结果
1.RNA干扰慢病毒载体的构建:
制备编码携带小鼠TLR1和TLR2基因的BLOCK-It Lentiviral PolⅡmiR慢病毒载体,转染RAW264.7小鼠细胞进行筛选,病毒转染五天后收集细胞进行mRNA和蛋白水平的检测。结果表明TLR1i-siteA、TLR1i-siteB、TLR1i-siteC对基因TLR1 mRNA水平的沉默效果分别是77%,44%,和63%,蛋白水平的抑制效果分别是70%,39%,和57%,筛选出有效的克隆TLR2i-siteA;TLR2i-siteA、TLR2i-siteB、TLR2i-siteC对基因TLR2 mRNA水平的沉默效果分别是56%,81%,和62%,蛋白水平的抑制效果分别是50%,62%,和35%,筛选出有效的克隆TLR2i-siteB。
2.慢病毒载体体内转染效率和基因沉默效率的检测:
慢病毒载体转染一周后颈动脉AS斑块内可见绿色荧光蛋白GFP;转染二周后斑块内GFP的表达量增加;当实验结束,转染四周后斑块内仍可见GFP的表达。说明慢病毒载体有效的转入斑块内。
TLR1mRNA水平分别下降57%和48%在TLR1干扰组和TLR1+2干扰组与对照组相比(P＜0.05);TLR2干扰组的TLR1mRNA与对照组相比没有统计学差异;TLR1蛋白水平在TLR1干扰组和TLR1+2干扰组与其他组相比明显降低。
TLR2mRNA水平分别下降50%和59%在TLR2干扰组和TLR1+2干扰组与对照组相比(P＜0.05);TLR1干扰组的TLR2mRNA与对照组相比没有统计学差异;TLR2蛋白水平在TLR2干扰组和TLR1+2干扰组与其他组相比明显降低。
3.血液生化指标检测:
RNAi组和对照组血清TC、TG、HDL-C、LDL-C未见统计学差异。
4.病理学检测:
对照组,空载体组,TLR1干扰组,TLR1干扰组和TLR1+2联合干扰组的巨噬细胞含量百分比分别为31.8%,30.8%,27.0%,26.9%和22.2%;平滑肌细胞含量百分比分别为8.7%,8.7%,8.5%,9.2%和10.2%;斑块内脂质含量百分比分别为30.0%,29.2%,28.4%,24.7%和21.9%;斑块内胶原含量百分比分别为21.9%,23.2%,23.4%,29.2%和29.4%;纤维帽的厚度分别为9.96±0.97μm,10.03±1.38μm,12.62±1.99μm,16.97±4.2μm和17.26±2.00μm;易损指数分别为2.09±0.17,2.06±0.14,1.94±0.20,1.40±0.27和1.22±0.15。
基因干扰各组与非干扰组相比,其巨噬细胞含量明显减少(P＜0.05);在TLR1+2联合干扰组上述指标较TLR1,TLR2单独作用变化更为明显(P＜0.05)。
联合干扰组与非干扰组及TLR1单独干扰组相比,平滑肌细胞含量明显增加(P＜0.05)。
TLR1干扰和TLR2干扰可以增加斑块纤维帽的厚度(P＜0.05)。
TLR2干扰组易损指数与非干扰组相比明显降低(P＜0.05),且联合干扰组与TLR1单独干扰组相比,降低更为明显(P＜0.05)。
5.斑块内炎症因子的检测:
TLR1干扰组的IL-6的mRNA水平与非干扰组相比显著降低(P＜0.05):TLR2干扰组的和TLR1+2联合干扰组的IL-6和MCP-1的mRNA水平与其余三组相比显著降低(P＜0.05)。
结论
1.本研究成功地应用RNA干扰技术构建了针对小鼠TLR1和TLR2基因的慢病毒干扰载体。
2.体内实验结果表明,TLR1和TLR2基因沉默能有效的抑制斑块局部TLR1和TLR2基因的表达,显著减少斑块内巨噬细胞的含量,降低斑块内炎症因子的表达,从而抑制斑块内炎症反应,增强斑块的稳定性。
3.联合应用TLR1干扰和TLR2干扰,能够更有效的增加斑块的稳定性。
Background
Atherosclerosis is a long-term chronic disease characterized by the accumulation of lipids and fibrous connective tissue in the large arteries,accompanied by a local inflammatory response.Inflammatory cells,like T lymphocytes and macrophages, play an important role in all stages of atherosclerotic lesion development.Moreover, cells from the vessel wall,like endothelial cells,smooth muscle cells,adventitial fibroblasts and mast cells,are able to act as immunological cells that produce proinflammatory cytokines.The triggers and pathways of initiation and regulation of the immune responses in atherosclerotic disease are largely unknown.In general,the human immune system has two closely related pathways to respond to potentially harmful agents:the innate and the adaptive immune recognition systems.The adaptive immune system involves dynamic adaptation to unique antigenic epitopes that are present in the environment.The innate immune response is the first line of defense in which highly conserved pathogen motifs,entitled pathogen-associated molecular patterns(PAMPs),are recognized.
That atherosclerosis is a chronic inflammatory disease,highlighting the pivotal role played by the immune system,leads inevitably to the question of which molecule and downstream signal pathway might dominate inflammatory responses within arteries.In many current discussions of inflammatory states,the subject of toll-like receptors(TLRs) is bound to arise.The response of TLRs in atherosclerosis and finding a novel potential target for intervention in these processes is important.
Toll-like receptors(TLRs) are a group of receptors that play a key role in innate immune signaling and in initiating inflammatory responses,possibly in atherosclerosis.To date,11 human and 13 mouse TLRs have been cloned.TLRs have a short intracellular TolI/IL-1R(TIR) domain,which,upon ligand binding,results in recruitment of adapter molecules and induction of downstream activation.Activation of one or more TLRs(either as homomeric or heteromeric assemblies) causes the recruitment and activation of adaptor proteins such as myeloid differentiation factor 88(MyD88),then recruitment of members of the IL-1R-associated kinase(IRAK) family and activation of tumor necrosis factor(TNF) receptor-associated factor 6 (TRAF-6) and ultimately nuclear factor-kappa B(NF-kB).
Of the 11 human TLR-subtypes,TLR4 is strongly expressed in the healthy human artery,although each of the four main TLRs(TLR 1,2,4 and 6) involved in the recognition of lipids are considerably upregulated in diseased arteries.TLR2 and TLR4 are the best studied and are generally believed to be involved in the pathogenesis of cardiovascular disease.They are expressed in most cardiovascular cells,such as endothelial cells,smooth muscle cells,macrophages and adventitial fibroblasts.Activation of these cells as a response to self- and non-self ligands appears to be mediated by TLR2 and TLR4.In atherosclerosis,apolipoprotein E (apoE)-/- mice show accelerated atherosclerosis after TLR2 or TLR4 ligand stimulation.Several lines of evidence suggest that TLRs are proatherogenic.In mice lacking TLR4,small neointimal lesions developed after vascular injury,and TLR2 has similar effects.Recent study showed both TLR2 and TLR4 expression significantly increased over time in the arterial lesions of apoE-/- mice.Recent animal studies showed that MyD88 and TLR4 play an important role in atherosclerotic plaque accumulation.As well,local TLR2 stimulation in the femoral artery in apoE-/- mice accelerated atherosclerosis.These results support those of a study demonstrating impaired atherosclerotic lesion development in TLR2~(-/-)/LDLR~(-/-) mice.
Recently,small interfering RNAs(siRNAs) have been efficacious in silencing target genes by means of RNA interference(RNAi),a natural mechanism conserved in nature from yeast to humans,and lentiviral vectors can efficiently deliver siRNA because they are capable of stably transducing both dividing and nondividing cells.In this study,we constructed two lentiviral vectors aimed at knocking down TLR2 and TLR4 to elucidate their roles in atherosclerosis in apoE-/- mice by stabilizing plaque.
Objectives
1.To construct lentiviruses targeting mouse TLR2 or TLR4 small interfering RNA (siRNA).
2.Constricting collars were placed around the carotid arteries of apolipoprotein E (apoE)-/- mice to induce formation of atherosclerosis plaque.The two lentiviruses were transfected into carotid lesions separately or together.Plaque characteristics were analyzed.
3.Combined interfering TLR2 and TLR4 gene on atherosclerosis plaque to study the synthetic effect between TLR2 and TLR4.
Methods
1.Cell culture
RAW264.7 mouse macrophage cell line purchased from the American Type Culture Collection(Rockville,MD) was routinely maintained in RPMI-1640 medium (Sigma,St.Louis,MO) supplemented with 10%fetal bovine serum,at 37℃in humidified air containing 5%CO_2.The 293FT cell line(invitrogen,Carlsbad,CA), which stably expresses SV40 large T antigen and facilitates the optimal production of viruses,was cultured in Dulbecco's modified Eagle's medium(GIBCO,Grand Island, NY).
2.Lentiviral vector construction
First,we selected three different sequences(siteA,siteB,siteC) for targeting the TLR2 and TLR4 genes using the BLOCK-R RNAi Designer(Invitrogen) (http://maidesigner.invitrogen.com).targeted sequence of TLR2-siteA was 5'-TGCTGAATAGAGGTGAAAGACCTGGAGTTTTGGCCACTGACTGACTCCA GGTCTCACCTCTATT-3';targeted sequence of TLR2-siteB was 5'-TGCTGTGTACTGGACAAATTCAGGAAGTTTTGGCCACTGACTGACTTCCT GAATGTCCAGTACA-3';targeted sequence of TLR2-siteC was 5'-TGCTGAAAGGCATCATAGCAAACGTCGTTTTGGCCACTGACTGACGACG TTTGATGATGCCTTT-3';targeted sequence of TLR4-siteA was 5'-TGCTGTATGCAGGTAACTTACAGGAAGTTTTGGCCACTGACTGACTTCCT GTATTACCTGCATA-3';targeted sequence of TLR4-siteB was 5'-TGCTGTTCACGTAGAAACTGTAAGTCGTTTTGGCCACTGACTGACGACTT ACATTCTACGTGAA-3';targeted sequence of TLR4-siteC was 5'-TGCTGTGCACATGTCATTTGTTCAACGTTTTGGCCACTGACTGACGTTGA ACATGACATGTGCA-3'.A scrambled siRNA sequence(named mock-siRNA) with no known homology to mammal genes served as a control.The preparation of lentiviral vectors expressing human TLR2 and TLR4 involved use of the BLOCK-It Lentiviral PolⅡmiR RNAi Expression System(catalog no.K4948-00;Invitrogen) following the manufacturer's instructions.In brief,three TLR2(siteA,B,C) and three TLR4(siteA,B,C) PolⅡsiRNA expression vectors containing the respective mouse TLR2 and TLR4 siRNA-expressing cassettes were constructed.A replication-incompetent lentivirus was produced by cotransfeetion of the siRNA-expression vector and ViraPower packaging mix(Invitrogen) containing an optimized mixture of three packaging plasmids--pLP1,pLP2,and pLP/VSVG--into 293FT cells.Viral supernatant was harvested 48 h after transfection,filtered through a 0.45-μm cellulose acetate filter and frozen at -70℃.The three TLR2i lentiviruses (siteA,B,C) and three TLR4i lentiviruses(siteA,B,C) were transfected into RAW264.7 cells.Five days after transfection,cells were collected for real-time PCR and western blot analysis to select the most effective targeting site for the TLR2 and TLR4 genes to amplify.The siRNA mock vector containing lentivirus only was used as a control.Finally,three lentivirus vectors were produced:TLR2i-,TLR4i-,and mock,and the terminal virus titer was 2×10~9 TU/mL.
3.Animal protocol
Male apoE-/- mice(n=140),aged 10 to 12 weeks,were purchased from Beijing Laboratory Animal Research Center.Mice were kept on a 12-h light/12-h dark cycle with food and water freely available.All animals received a high-fat diet(0.25% cholesterol and 15%cocoa butter) for 12 weeks.All animal care and procedures were approved by Shandong University Institutional Animal Care and Use Committee and complied with the Guide for the Use and Care of Laboratory Animals published by the US National Institutes of Health(NIH publication 80-23,revised 1996)
4.Carotid collar placement and lentivirus injection
All mice were anesthetized with an intraperitoneal injection of pentobarbital sodium(40 mg/kg).As described by vonder Th(u|¨)sen et al.,a constricting silastic tube (0.30-mm inner diameter,0.50-mm outer diameter,and 2.5-mm long;Shandong Key Laboratory of Medical Polymer Materials,Jinan,China) was placed on the right common carotid artery near its bifurcation.Eight weeks after surgery,the collars were removed and lentiviral suspension was instilled into the right common carotid artery via the external carotid.The suspension was left in situ for 15 min after temporary ligation of the proximal common carotid artery and the internal carotid artery,and subsequently drawn off before ligation of the external carotid and closure of the skin wound with silk sutures as described.The mice were randomly divided into five groups according to lentivirus injection:control,no lentivirus;mock,10μL mock lentiviral suspension;TLR2 interfering(TLR2i),10μL TLR2i lentiviral suspension; TLR4 interfering(TLR4i),10μL TLR4i lentiviral suspension;and TLR2+4i,5μL each of TLR2i and TLR4i lentiviral suspension.Control and mock groups were non-knockdown groups,and TLR2i,TLR4i and TLR2+4i groups were gene-knockdown groups.
5.Serum lipid measurement
Before perfusion-fixation,blood was collected from the inferior vena cava. Serum total cholesterol,triglycerides,low-density lipoprotein(LDL) cholesterol,and high-density lipoprotein(HDL) cholesterol concentrations were measured by enzymatic assay(Zhejiang Dongou Biotechnology,Wenzhou,China)
6.Tissue preparation and histological analysis
Mice were perfused with phosphate-buffered saline through the left ventricle and then underwent peffusion fixation at 100 mm Hg with 4%formaldehyde(pH 7.2) for 2 and 15 min.The right common carotid artery with bifurcation was carefully excised and immersed in 4%formaldehyde overnight(4℃),embedded in OCT compound, and stored at -20℃until use.Each vessel throughout the entire length of the carotid artery underwent histological analysis.
In the 6 mice of the mock group sacrificed at week 9 and week 10,Sky Blue 6B (Sigma C8679) was used to eliminate background autofluorescence and cryosections were viewed under fluorescence microscopy to identify GFP expression.In all of the mice,OCT-embedded carotid artery was cross-sectioned into pieces 6μm thick at 50-μm intervals.Five cross sections in each mouse were used for a particular type of staining and the whole cross section viewed in one field was used for quantitative measurements.Sections were stained with hematoxylin and eosin for histological analysis.The plaque area was calculated by subtracting the lumen area from the area circumscribed by the internal elastic lamina.The plaque was subdivided into a fibrous cap and a necrotic core on the basis of extracellular matrix staining by HE,which was further confirmed by staining for collagen and lipids using picrosirius red and Oil-red O,respectively.Corresponding sections on separate slides were immunostained with monoclonal antibodies.Smooth muscle cells(SMCs) and macrophages were immunostained with anti-α-actin antibodies(1:1000;Sigma) and MOMA-2(1:25; Serotec,Oxford,UK),respectively.After incubation with the appropriate horseradish peroxidase(HRP)-conjugated secondary antibodies(1:100;Zhongshan Biological Technology,Wenzhou,China),the sections were incubated with 3',3'-diaminobenzidine and counterstained with hematoxylin.Sections reacted with non-immune IgG,secondary antibody only and no primary and secondary antibodies were used as negative controls.An automated image analysis system(Image-Pro Plus 5.0,Media Cybernetics,USA) was used for quantitative measurements.The positive staining area of macrophages,SMCs,lipids and collagen was quantified by computer-assisted color-gated measurement,and the ratio of positive staining area to the intimal area was calculated.The vulnerability index was calculated by the following formula:positive staining area of(macrophages +lipid)/positive staining area of(α-SMCs +collagen).
7.Transmission electron microscopy
Tissues for TEM were prepared as previously described.Tissue samples were trimmed into small pieces and fixed in chilled 2.5%glutaraldehyde in 0.1 M cacodylate buffer(pH 7.4) for 2 h and then postfixed in osmium tetroxide dissolved in the same buffer for 1-2 h.They were dehydrated through a graded series of ethanol and propylene oxide and embedded in epoxy resin.The embedded blocks were cut into ultrathin sections,then stained with lead citrate and uranyl acetate and observed under a 12A electron microscope(Hitachi,Japan).
8.Quantitative real-time RT-PCR analysis
RNA was extracted with TriZol Reagent(Invitrogen) following the manufacturer's instruction.The mRNA levels of TLR2,TLR4,interleukin-1β(IL-1β), IL-6,tumor necrosis factor-α(TNF-α),and monocyte chemoattractant protein-1 (MCP-1) were quantified in in vitro RAW264.7 cells and in vivo fresh carotid plaque tissue by use of SYBR Green technology(Applied Biosystems).Quantitative values were obtained from the threshold cycle value(Ct).The housekeeping geneβ-actin was quantified as an internal RNA control.The "2~(-△△CT) method" for comparing relative expression results was applied as described.
9.Western blot analysis
Equal amounts of protein from in vitro RAW264.7 cells and in vivo fresh carotid plaque tissue were separated on 14%SDS-PAGE and transferred to nitrocellulose membrane.Following blocking with 5%non-fat milk,the blots were washed with PBS containing 0.1%Tween 20 and incubated with an appropriate primary antibody at 4℃overnight.The blots were probed with antibodies againstβ-actin(1:100, Zhongshan,China),as well as TLR2(1:500),TLR4(1:200).After overnight incubation,the blots were washed with TBST and incubated with secondary antibody conjugated to HRP(1:1000,Zhongshan),then washed again.The blots were then visualized by enhanced chemiluminescence.
10.Data Analysis
Data were expressed as mean±SD.Multiple comparisons were analyzed by LSD post hoc tests.Factorial ANOVA was used to analyze the individual and interactive effects of two independent variables,TLR2i and TLR4i.With the statistical design of 2 x 2 factorial,the main effects of TLR2i,main effects of TLR4i and the interaction between TLR2i and TLR4i were compared.In such an analysis,if a significant interaction is present,the effects between TLR2i and TLR4i should be interpreted as synergistic rather than additive.All analyses involved use of SPSS 11.5 (SPSS Inc.,Chicago,IL).P<0.05 was considered statistically significant.
Results
1.Gene silencing in vitro:screening the most effective targeting sites for the TLR2 and TLR4 genes by real-time PCR and western blot analysis.
RAW264.7 cell line was transfected with lentivirus-based vectors expressing three different TLR2-siRNAs and gene silencing analysis showed that the TLR2i-site B lentivirus was the most effective vector in blocking TLR2 expression. TLR2-knockdown clone A,clone B,and clone C exhibited 56%,81%,and 62% reduction,respectively,in the level of TLR2 mRNA expression and 50%,62%,and 35%reduction,respectively,in the level of TLR2 protein expression.
The TLR4i-site C lentivirus was found to be the most effective vector in blocking TLR4 expression.TLR4 knockdown clone A,clone B,and clone C exhibited 61%,54%,and 72%reduction,respectively,in the level of TLR4 mRNA expression and 45%,32%,and 61%reduction,respectively,in the level of TLR4 protein expression.As a result,TLR2-site B and TLR4-site C lentiviruses were selected for further in vivo studies.
2.Efficient transfection of lentivirus in atherosclerosis plaque
Previous local virus delivery to pre-collar-induced carotid artery lesions of apoE-/- mice had resulted in efficient transfection to atherosclerosis plaque.Since GFP expression provides an efficient,convenient monitor to check transfection efficiency of lentivirus,we detected GFP fluorescence in carotid artery plaques 1 week after transfection,which indicated transfection of the siRNA,and greater fluorescence at 2 weeks after transfection.When the study was terminated,four weeks after transfection,GFP was still visible,although weak.These results demonstrated the efficient in vivo transfection of lentivirus by siRNA in atheroselerotie plaque.The virus local transfection did not affect normal functioning of the animals and did not result in weight change(30.6±3.1g in transfected mice vs.29.9±3.5g in controls) or serum cholesterol level(29.6±2.6 vs.30.1±2.0 mmol/L).
3.Gene silencing in vivo:effect of knockdown of siRNA lentivirus on TLR2 and TLR4 expression in atheroselerosis plaque by real-time PCR and western blot analysis.
To evaluate the efficacy of lentivirus-mediated gene silencing in vivo,we tested atherosclerosis plaque for TLR2 and TLR4 mRNA and protein expression levels.The TLR2 mRNA levels were lower,by 50%and 59%,in the TLR2i group and TLR2+4i group,respectively,than in the control group(P<0.05 for each).The TLR4 mRNA levels were lower,by 48%and 53%,in the TLR4i and TLR2+4i groups,respectively, than in the control group(P<0.05 for each).The mock,TLR2i and TLR4i groups showed no significant change in TLR4 or TLR2 mRNA level,respectively.
TLR2 protein was lower in the TLR2i and TLR2+4i groups than in the other groups,as was TLR4 protein lower in the TLR4i group and TLR2+4i group than in the other groups.So the local application of siRNA-lentivirus can efficiently silence gene expression in vivo.
4.Effect of TLR2i and TLR4i on plaque features
The atherosclerotie plaque composition of collagen,lipid,macrophages,and SMCs was demonstrated by histological and immunohistochemieal staining.The gene-knockdown groups showed higher content of collagen and lower content of lipid and macrophages than the non-knockdown groups(P<0.05 for all).Moreover,plaques in the TLR2+4i group showed a significant change as compared with plaques in the TLR2i- and TLR4i-alone groups(P<0.05 each).ANOVA revealed TLR2i and TLR4i significantly related in terms of macrophage and collagen content(P<0.05 for each) and TLR4i was shown to increaseα-SMC content in TLR4i and TLR2+4i groups (P<0.05 for each),with no differences in SMCs among other groups.
Both TLR2i and TLR4i induced markedly increased cap thickness(P<0.05 for all).Furthermore,the combination of TLR2i and TLR4i resulted in significantly increased mean cap thickness(P<0.05).
The vulnerable index differed significantly between gene-knockdown groups and non-knockdown groups,with 1.00±0.25 for the TLR2+4i group,signifieantly smaller than that of the other groups(2.09±0.17 for control,2.06±0.14 for mock,1.40±0.27 for TLR2i,and 1.38±0.19 for TLR4i groups;P<0.05 for all).
The gene-knockdown groups showed a lower accumulation of lipids and macrophages,a higher content of collagen,thicker fibrous caps and reduced vulnerable index than non-knockdown groups,with a synergistic combination effect of TLR2i and TLR4i on maerophage and collagen content and the mean cap thickness.
5.Gene knockdown effect on the ultrastructure of atherosclerosis plaque
The non-knockdown group ultrastucture consisted of foam cells and severely discontinuous innermost lining of endothelial cells,with wide gaps between adjacent cells and more synthetic(increased rough endoplasmic reticulum) SMCs than a contractile phenotype(cytoplasmic filaments prominent).Lipid-filled macrophages, characterized by numerous variable-sized granules and lipid droplets,were also present in the intima.The extracellular matrix contained fragmented elastic tissue, myelin figures,necrotic debris,and cholesterol clefts.
In the gene-knockdown group ultrastructure,the endothelial cells were smooth with integrity.Both synthetic and contractile types of SMCs were present,and the cytoplasm had more fine filaments,with dense bodies,decreased rough endoplasmic reticulum,mitoehondrial number and cytoplasmic lipid level.Foam cells were reduced in number as were extracellular matrix lipid droplets.
6.Effect of TLR2i and TLR4i on inflammation within lesions
The mRNA expression level of IL-6 and MCP-1 in the TLR2i subgroup and that of IL-1β,IL-6,TNF-αand MCP-1 in the TLR4i subgroup were lower than the corresponding measurements in the control or mock subgroups(all P<0.05).The TLR2+4i subgroup showed a significantly lower mRNA expression level of IL-1β, IL-6,TNF-α,MCP-1 than the other four subgroups(P<0.05).Furthermore,the synergistic effect of TLR2i and TLR4i in the reduction of the mRNA expression level of these cytokines was confirmed by Factorial ANOVA analysis(P<0.05).
Conclusions
1.In the present study,we applied lentivirus-mediated RNAi methods to efficiently knock down mice TLR2 and TLR4 genes in vitro and in vivo.
2.In vivo study demonstrated that transfected lentiviruses could silence TLR2 and TLR4 expression in apoE-/- mice with collar-induced carotid plaques,then decrease the level of inflammatory cytokines,and finally postpone the progression of atherosclerosis by stabilizing plaques.
3.Moreover,we found a synergistic effect between TLR2i and TLR4i in preventing atherosclerosis progression.These results point to a potential role for knockdown of TLR2 and TLR4 in local therapy for atherosclerosis.
Background
Atherosclerosis is a complicated inflammatory process with immune reactions during the initiation and progression of the disease.Toll-like receptors(TLRs) are known to play a key role in innate immune signaling and initiating inflammatory responses and their potential roles in the pathogenesis of atherosclerosis have received great attention.To date,11 human and 13 mouse TLRs have been cloned.TLRs have a short intracellular Toll/IL-1R domain,which,upon ligands binding,results in recruitment of adapter molecules and induction of downstream activation.Activation of one or more TLRs(either as homomeric or heteromeric assemblies) causes the recruitment and activation of adaptor proteins,and ultimately,nuclear factor-kappa B (NF-κB),which leads to stimulated release of a large number of inflammatory cytokines such as interleukin-6(IL-6),and chemoattractant cytokines such as monocyte chemoattractant protein-1(MCP-1).
Of the 11 human TLR-subtypes,TLR2 and TLR4 are the best studied and are generally believed to be involved in the pathogenesis of atherosclerosis.They are highly expressed in most vascular cells,such as endothelial cells,smooth muscle cells (SMCs),macrophages and adventitial fibroblasts.Studies in mice found that TLR2 played a critical role in the progression of atherosclerosis that was independent of dietary lipids,and TLR2 has to form dimers with other receptors such as TLR1 or TLR6 for signaling.TLR1 was also detected in atherosclerotic plaques,especially in areas infiltrated with inflammatory cells although the expression of TLR1 was found to be lower than that of TLR2 and TLR4.However,whether TLR1 and TLR2 are involved in the pathogenesis of vulnerable plaques is unknown.Since both TLR1 and TLR2 are expressed in atherosclerotic plaques and TLR2 may integrate with TLR1 or other TLRs for signaling,we hypothesized that TLR1 and TLR2 may play different roles in the formation of vulnerable plaques and combinatorial knockdown of TLR1 and TLR2 genes may enhance the effects of isolated knockdown of TLRI or TLR2 gene on plaque stabilization.
Recently,small interfering RNAs(siRNAs) have proven efficacious in silencing target genes and lentivirus can efficiently deliver siRNA because they are able to transduce both dividing and nondividing cells stably.In this study,we constructed two lentiviral vectors to knockdown TLR1 and TLR2 to elucidate their individual and synergistic roles in stabilizing atherosclerotic plaques in apolipoprotein E(apoE)-/-mice.
Objectives
1.To construct lentiviruses targeting mouse TLRI or TLR2 small interfering RNA(siRNA).
2.Constricting collars were placed around the carotid arteries of apolipoprotein E(apoE)-/- mice to induce formation of atherosclerosis plaque.The two lentiviruses were transfected into carotid lesions separately or together.Plaque characteristics were analyzed.
3.Combined interfering TLR1 and TLR2 gene on atherosclerosis plaque to study the synthetic effect between TLR1 and TLR2.
Methods
1.Cell culture
RAW264.7 mouse macrophage cell line purchased from the American Type Culture Collection(Rockville,MD) was routinely maintained in RPMI-1640 medium (Sigma,St.Louis,MO) supplemented with 10%fetal bovine serum,at 37℃in humidified air containing 5%CO_2.The 293FT cell line(Invitrogen,Carlsbad,CA), which stably expresses SV40 large T antigen and facilitates the optimal production of viruses,was cultured in Dulbecco's modified Eagle's medium(GIBCO,Grand Island, NY).
2.Lentiviral vector construction
First,three different sequences(site A,B and C) of TLRI and TLR2 genes in mice were selected as the target for RNA interference(RNAi),using the BLOCK-It RNAi Designer(invitrogen)(http://maidesigner.invitrogen.com).Targeted sequence of TLR1-siteA was 5'-TGCTGATTAAAGGAGAGGTCCAAATGGTTTTGGCCACTGACTGACCATTT GGATCTCCTTTAAT-3';targeted sequence of TLR1-siteB was 5'-TGCTGTAAAGTAGTTGTTTGCAAGGGGTTTTGGCCACTGACTGACCCCTT GCACAACTACTTTA-3';targeted sequence of TLRl-siteC was 5'-TGCTGTAATGAAGGAATTCCACGTTGGTTTTGGCCACTGACTGACCAAC GTGGTTCCTTCATTA-3';targeted sequence of TLR2-siteA was 5'-TGCTGAATAGAGGTGAAAGACCTGGAGTTTTGGCCACTGACTGACTCCA GGTCTCACCTCTATT-3';targeted sequence of TLR2-siteB was 5'-TGCTGTGTACTGGACAAATTCAGGAAGTTTTGGCCACTGACTGACTTCCT GAATGTCCAGTACA-3';targeted sequence of TLR2-siteC was 5'-TGCTGAAAGGCATCATAGCAAACGTCGTTTTGGCCACTGACTGACGACG TTTGATGATGCCTTT-3'.A scrambled siRNA sequence(named moek-siRNA) with no known homology to mammal genes served as a control.The preparation of lentiviral vectors expressing human TLR1 and TLR2 involved use of the BLOCK-It Lentiviral PolⅡmiR RNAi Expression System(catalog no.K4948-00;Invitrogen) following the manufacturer's instructions.In brief,three TLR1(siteA,B,C) and three TLR2(siteA,B,C) PolⅡsiRNA expression vectors containing the respective mouse TLR1 and TLR2 siRNA-expressing cassettes were constructed.A replication-incompetent lentivirus was produced by cotransfection of the siRNA-expression vector and ViraPower packaging mix(invitrogen) containing an optimized mixture of three packaging plasmids--pLP1,pLP2,and pLP/VSVG--into 293FT ceils.Viral supernatant was harvested 48 h after transfection,filtered through a 0.45-μm cellulose acetate filter and frozen at -70℃.The three TLRIi lentiviruses (siteA,B,C) and three TLR2i lentiviruses(siteA,B,C) were transfected into RAW264.7 cells.Five days after transfection,cells were collected for real-time PCR and western blot analysis to select the most effective targeting site for the TLR1 and TLR2 genes to amplify.The siRNA mock vector containing lentivirus only was used as a control.Finally,three lentivirus vectors were produced:TLR1i-,TLR2i-,and mock,and the terminal virus titer was 2×10~9 TU/mL.
3.Animal protocol
Male apoE-/- mice(n=140),aged 10 to 12 weeks,were purchased from Beijing Laboratory Animal Research Center.Mice were kept on a 12-h light/12-h dark cycle with food and water freely available.All animals received a high-fat diet(0.25% cholesterol and 15%cocoa butter) for 12 weeks.All animal care and procedures were approved by Shandong University Institutional Animal Care and Use Committee and complied with the Guide for the Use and Care of Laboratory Animals published by the US National Institutes of Health(NIH publication 80-23,revised 1996)
4.Carotid collar placement and lentivirus injection
All mice were anesthetized with an intraperitoneal injection of pentobarbital sodium(40 mg/kg).As described by vonder Th(u|¨)sen et al.,a constricting silastic tube (0.30-mm inner diameter,0.50-mm outer diameter,and 2.5-mm long;Shandong Key Laboratory of Medical Polymer Materials,Jinan,China) was placed on the right common carotid artery near its bifurcation.Eight weeks after surgery,the collars were removed and lentiviral suspension was instilled into the right common carotid artery via the external carotid.The suspension was left in situ for 15 min after temporary ligation of the proximal common carotid artery and the internal carotid artery,and subsequently drawn off before ligation of the external carotid and closure of the skin wound with silk sutures as described.The mice were randomly divided into five groups according to lentivirus injection:control,no lentivirus;mock,10μL mock lentiviral suspension;TLR1 interfering(TLRli),10μL TLR1i lentiviral suspension; TLR2 interfering(TLR2i),10μL TLR2i lentiviral suspension;and TLR1+2i,5μL each of TLR1i and TLR2i lentiviral suspension.Control and mock groups were non-knockdown groups,and TLR1i,TLR2i and TLR1+2i groups were gene-knockdown groups.
5.Serum lipid measurement
Before perfusion-fixation,blood was collected from the inferior vena cava. Serum total cholesterol,triglycerides,low-density lipoprotein(LDL) cholesterol,and high-density lipoprotein(HDL) cholesterol concentrations were measured by enzymatic assay(Zhejiang Dongou Biotechnology,Wenzhou,China)
6.Tissue preparation and histological analysis
Mice were perfused with phosphate-buffered saline through the left ventricle and then underwent perfusion fixation at 100 mm Hg with 4%formaldehyde(pH 7.2) for 2 and 15 min.The right common carotid artery with bifurcation was carefully excised and immersed in 4%formaldehyde overnight(4℃),embedded in OCT compound, and stored at -20℃until use.Each vessel throughout the entire length of the carotid artery underwent histological analysis.
In the 6 mice of the mock group sacrificed at week 9 and week 10,Sky Blue 6B (Sigma C8679) was used to eliminate background autofluorescence and cryosections were viewed under fluorescence microscopy to identify GFP expression.In all of the mice,OCT-embedded carotid artery was cross-sectrned into pieces 6 lira thick at 50-μm intervals.Five cross sections in each mouse were used for a particular type of staining and the whole cross section viewed in one field was used for quantitative measurements.Sections were stained with hematoxylin and eosin for histological analysis.The plaque area was calculated by subtracting the lumen area from the area circumscribed by the internal elastic lamina.The plaque was subdivided into a fibrous cap and a necrotic core on the basis of extracellular matrix staining by HE,which was further confirmed by staining for collagen and lipids using picrosirius red and Oil-red O,respectively.Corresponding sections on separate slides were immunostained with monoclonal antibodies.Smooth muscle cells(SMCs) and macrophages were immunostained with anti-α-actin antibodies(1:1000;Sigma) and MOMA-2(1:25; Serotec,Oxford,UK),respectively.After incubation with the appropriate horseradish peroxidase(HRP)-conjugated secondary antibodies(1:100;Zhongshan Biological Technology,Wenzhou,China),the sections were incubated with 3',3'-diaminobenzidine and counterstained with hematoxylin.Sections reacted with non-immune IgG,secondary antibody only and no primary and secondary antibodies were used as negative controls.An automated image analysis system(Image-Pro Plus 5.0,Media Cybernetics,USA) was used for quantitative measurements.The positive staining area of macrophages,SMCs,lipids and collagen was quantified by computer-assisted color-gated measurement,and the ratio of positive staining area to the intimal area was calculated.The vulnerability index was calculated by the following formula:positive staining area of(macrophages +lipid)/positive staining area of(α-SMCs +collagen).
7.Quantitative real-time RT-PCR analysis
RNA was extracted with TriZol Reagent(Invitrogen) following the manufacturer's instruction.The mRNA levels of TLR1,TLR2,interleukin-6(IL-6), and monocyte chemoattractant protein-1(MCP-1) were quantified in in vitro RAW264.7 cells and in vivo fresh carotid plaque tissue by use of SYBR Green technology(Applied Biosystems).Quantitative values were obtained from the threshold cycle value(Ct).The housekeeping geneβ-actin was quantified as an internal RNA control.The "2~(-△△CT) method" for comparing relative expression results was applied as described.
8.Western blot analysis
Equal amounts of protein from in vitro RAW264.7 cells and in vivo fresh carotid plaque tissue were separated on 14%SDS-PAGE and t
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