【作者】 王颖；

【导师】 张运；

【作者基本信息】 山东大学 ， 内科学， 2007， 博士

【摘要】 背景动脉粥样硬化（athcrosclcrosis，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促炎细胞因子。但在动脉粥样硬化疾病的发生和发展中，对其发病分子机制至今尚未阐明。近年来认为免疫应答，在AS的发生发展中起重要作用，特别是晚近对天然免疫在AS中的作用的研究成为新的研究热点。已知人类具有天然和获得性两个免疫识别系统来抵御潜在的有害物质。获得性免疫系统特异性识别环境中存在的独特抗原决定簇。天然免疫是机体抵御微生物的第一道屏障，识别被称为病原相关分子模式（pathogen-associated molecular patterns，PAMP）的高度保守抗原域。Toll样受体（Toll-like receptor，TLR）识别种类繁多的PAMP，属于模式识别受体。目前为止，人类TLR家族至少有10名成员已被发现。不同的PAMP活化人类TLR家族的不同成员。TLR通过与内、外源性配体结合，激发天然免疫和获得性免疫应答，启动促炎因子的释放，导致炎症反应的发生，在炎症反应的信号转导系统中发挥极其重要的作用。实验发现：TLR调控胆固醇代谢、炎症和免疫反应、细胞凋亡、斑块稳定性以及血管重塑等病理过程，而这些贯穿于AS病程始终。因此，本研究认为TLR将为动脉粥样硬化疾病的治疗提供一个新的靶点。虽然近年来国内外学者在TLR4与AS的相互关系研究中已取得很大的进展但仍存在许多重大问题亟待解决：①既然TLR在AS形成和斑块破裂中扮演重要角色，而动脉粥样硬化斑块病变处的巨噬细胞和内皮细胞以表达TLR4为主，能否通过封闭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目前造成基因失活的方式主要有反义寡核苷酸技术、核酶或核酸技术、三链DNA技术、单克隆抗体等。反义技术虽能抑制“有害”基因的表达而造成基因失活，但由于反义RNA的选择设计难度较大、反义RNA与mRNA的亲和力及其结合的特异性受到结合位点两侧序列的二级或三级结构的影响、反义寡核苷酸的多聚阴离子性质会引起一系列副作用等问题，故反义RNA目前只应用于恶性肿瘤、病毒感染等疾病。三链DNA技术是将脱氧寡核苷酸与双螺旋双链DNA专一性序列结合形成三链DNA，达到阻止基因转录或DNA复制的目的，此脱氧寡核苷酸称为三链DNA形成脱氧寡核苷酸（TFO），这一技术存在稳定性差、半衰期短的缺点，而且TFO必须与同聚嘌呤同聚嘧啶片段结合，而这样的DNA片段在真核细胞中很少。同样，利用单克隆抗体封闭治疗亦存在半衰期短等缺点。能否找寻一种有效的方法对基因进行干预。③针对TLR4的基因干预与药物治疗谁能在炎性信号转导通路和斑块易损性中起“闸门”作用，它们各自的机制如何?因此，本课题率先开展了封闭TLR4基因阻止AS进程，稳定易损斑块的机制研究，并取得了一定研究成果。目的1．建立TLR4基因RNA干扰载体并转染细胞筛选阳性克隆细胞；2．体外对比研究TLR4-RNAi与阿托伐他汀药物干预抑制TLR4基因继而引起细胞生物学行为和相关基因的表达变化，并探讨其机制；3．体外对比观察TLR4-RNAi与阿托伐他汀药物对TLR4信号通路诱导炎症反应的作用，为在体研究提供分子生物学依据；4．动物实验对比研究RNAi与药物封闭TLR4基因对阻止AS进程，降低动脉粥样硬化斑块易损性的作用机制并评估其治疗效果。方法1．RNA小分子干扰表达载体的构建筛选1．1有效封闭TLR4表达的小分子干扰RNA载体的构建设计、合成两对特异性针对TLR4基因的小分子干扰RNA序列，同时设计与人类所有已知基因序列均无同源性的片段作为阴性对照，以pRNAT-U6.1neo为母本，分别构建小分子干扰RNA载体pRNAT-TLR4-1、pRNAT-TLR4-2及对照载pRNAT-Neg，DNA序列测定鉴定重组质粒的正确性。1．2阳性细胞克隆筛选质粒经阳离子脂质体介导分别转染处于对数生长期的ECV304细胞，利用含G418的DMEM培养液筛选阳性克隆。根据转染质粒的不同，分别命名为ECV304-pTLR4-1，ECV304-pTLR4-2，ECV304-pNeg细胞。1．3有效封闭TLR4表达的小分子干扰RNA载体的筛选收集稳定筛选获得的ECV304-pTLR4-1，ECV304-pTLR4-2，ECV304-pNeg及ECV304对照细胞。荧光定量RT-PCR、Western blot分别检测各转染细胞TLR4 mRNA水平、蛋白质水平的表达，筛选有效阻断TLR4表达的小分子干扰RNA载体。2．体外对比研究RNAi与阿托伐他汀抑制内毒素诱导的TLR4表达的机制及逆转炎症反应的效应2．1将细胞随机分成下列4组进行干预：对照组、LPS组、LPS+RNAi组及LPS+阿托伐他汀组；观察各组TLR4基因和蛋白的表达变化，以及引起NF-κB信号通路蛋白的变化，初步探讨基因干预与阿托伐他汀引起其变化机制的异同。2．2将LPS+RNAi组、LPS+阿托伐他汀组选择性加入NF-κB抑制剂（SN50）、PI3K抑制剂（Ly294002）、ERK抑制剂（PD98059）和P38抑制剂（SB203580），深入探讨基因治疗与药物干预调节NF-κB的内在分子机制的的异同。2．3将细胞随机分成下列4组进行干预：对照组、LPS组、LPS+RNAi组、及LPS+阿托伐他汀组；观察不同方法（RNAi与阿托伐他汀）抑制TLR4基因与下游效应因子IL-6（Th-2细胞因子）及IL-10（促使T细胞向T调节细胞发育）产生的关系。同时观察两种不同干预与炎性因子TNFα产生的关系。以次探讨比较基因治疗与药物干预对TLR4诱导免疫反应与炎症反应的逆转效应。3．动物实验对比研究RNAi与药物封闭TLR4基因对阻止AS进程，稳定AS斑块易损性的作用机制并评估其治疗效果3．1小分子干扰腺病毒载体的体外扩增、滴度测定及体外活性检测pSuppressorAd-TLR4，pSuppressorAd-Neg腺病毒感染HEK293细胞，72h后出现明显细胞病变，分别收集培养上清和细胞。反复冻融裂解细胞，收集病毒颗粒。低熔点琼脂糖空斑形成试验测定病毒滴度，并将其作用于小鼠巨噬细胞RAW264.7，荧光定量检测TLR4mRNA表达。3．2 RNAi基因治疗与阿托伐他汀药物治疗ApoE-／-小鼠的实验研究：8周龄ApoE-／-小鼠，予西方饮食（含21％脂肪和0.15％胆固醇）和SPF级饲养10周，并随机分为下列4组（n=27）对照载体组、RNAi组、安慰剂组和阿托伐他汀组。对照载体组：先单纯高脂喂养5周。于五周末将小分子干扰腺病毒Ad-siRNA-Neg经尾静脉注射至小鼠体内，继续高脂喂养5周。RNAi组：先单纯高脂喂养5周。于五周末将小分子干扰腺病毒Ad-siRNA-TLR4经尾静脉注射至小鼠体内，继续高脂喂养5周。安慰剂组：将安慰剂溶于超纯水灌胃10周。阿托伐他汀组：将阿托伐他汀（10mg／kg／d）溶于超纯水灌胃10周。干预10周后处死动物，颈静脉取血检测血胆固醇、甘油三酯、LDL和HDL（禁食8h）。3．3测定血脂、HDL、LDL水平，比较4组小鼠头臂干斑块面积均值；油红O染色分析斑块脂质含量；斑块破裂评价斑块破裂率；易损指数评价斑块易损性；检测斑块内TLR4、SM-actin、MAMO-2等抗原的免疫活性；荧光定量PCR定量分析各组小鼠头臂干TLR4、IL-6、IL-10、IL-12、TNF-α、VCAM-1及MMP-9基因表达变化。结果1．构建质粒pRNAT-TLR4-1，pRNAT-TLR4-2，pRNAT-Neg，经阳离子脂质体介导分别转染ECV304细胞，利用含500ug／mL G418的DMEM培养液筛选获得阳性克隆。ECV304-pTLR4-1细胞TLR4 mRNA表达水平显著低于未转染组ECV304细胞沉默率达61％。TLR4蛋白相对表达强度显著低于未转染ECV304细，沉默率达52％。2．阿托伐他汀作用于ECV304细胞对LPS上调TLR4的影响Western blot结果显示，LPS+阿托伐他汀（1μmol／L，12h）组TLR4蛋白表达较LPS组显著降低（P＜0.05）。LPS+阿托伐他汀（1μmol／L，24h）组TLR4蛋白表达较LPS组显著降低（P＜0.05）。LPS+阿托伐他汀（1μmol／L，12h）组与LPS+阿托伐他汀（1μmol／L，24h）组TLR4蛋白表达差异无统计学意义（P＞0.05）。Western blot结果显示，阿托伐他汀预处理细胞，TLR4蛋白表达呈量效依赖性降低。LPS+阿托伐他汀（1μmol／L）组TLR4蛋白表达较LPS组显著降低（5.56±0.26 vs 111.20±1.93），LPS+阿托伐他汀（10μmol／L）组TLR4蛋白表达较LPS组显著降低（4.72±0.22 vs 111.20±1.93），LPS+阿托伐他汀（1μmol／L）组TLR4蛋白表达较LPS+阿托伐他汀（10μmol／L）组差异无统计学意义（P＞0.05）。3．TLR4小分子干扰RNA与阿托伐他汀干预细胞对LPS-TLR4诱导NF-κB活性的影响ELISA结果显示，与对照组相比（0.67±0.002），LPS作用30min NF-κB活性显著增加到1.89±0.04（P＜0.05）。LPS（30min）+阿托伐他汀（1μmol／L，12h）组NF-κB活性较LPS（30min）组显著降低（0.96±0.12 vs 1.89±0.04）（P＜0.05）。LPS（30min）+RNAi组NF-κB活性较LPS（30min）组显著降低（0.72±0.02 vs 1.89±0.04），且较LPS（30min）+阿托伐他汀（1μmol／L，12h）组显著降低（0.72±0.02 vs 0.96±0.12）（P＜0.05）。Western blot结果显示，与对照组相比，LPS作用30min胞浆IκB-α蛋白表达显著减少（P＜0.01）。IκB-α蛋白表达于LPS（30min）+阿托伐他汀（1μmol／L，12h）组较LPS（30min）组显著增加（P＜0.01）。然而LPS（30min）+RNAi组IκB-α蛋白表达较LPS（30min）组差异无统计学意义（P＞0.05）。4．TLR4小分子干扰RNA对LPS-TLR4通路诱导PI3K-Akt的作用Western blot结果显示，与LPS（30min）组相比，RNAi+LPS（30min）组p-Akt／Akt蛋白表达（0.73±0.07 vs 0.2±0.02），P65蛋白表达（43.4±1.29 vs 23.78±0.26）显著减少（P均＜0.05）与LPS（30min）组相比，Ly294002+LPS（30min）组胞浆P-Akt／Akt蛋白表达显著减少（0.73±0.07 vs 0.13±0.008），核蛋白p65蛋白表达显著减少（43.4±1.29 vs 12.16±0.33）（P均＜0.05），胞浆IκB-α蛋白表达差异无统计学意义（10.00±0.06 vs 9.89±0.19）（P＞0.05）。5．阿托伐他汀作用于ECV304细胞对LPS-TLR4通路诱导磷酸化MAPKs表达的影响Western blot结果显示，与对照组相比，LPS（30min）组P-ERK／ERK蛋白表达显著增加（0.018±0.0001 vs 0.786±0.0276）（P＜0.01）。LPS（30min）+阿托伐他汀（1μmol／L，12h）组较LPS（30min）组P-ERK／ERK蛋白表达显著减少（0.215±0.0060 vs 0.786±0.0276）（P＜0.05）。Western blot结果显示，与对照组相比，LPS（30min）组P-P38／P38蛋白表达显著增加（0.027±0.002 vs 0.782±0.015）（P＜0.05）。LPS（30min）+阿托伐他汀（1μmol／L，12h）组较LPS（30min）组P-P38／P38蛋白表达显著减少（0.690±0.008 vs 0.782±0.015）（P＜0.05）。Western blot结果显示，与对照组相比，LPS（30min）组p-JNK／JNK蛋白表达显著增加（0.053±0.004 vs 0.584±0.011）（P＜0.05）。LPS（30min）+阿托伐他汀（1μmol／L，12h）组较LPS（30min）组P-JNK／JNK蛋白表达差异无统计学意义（P＜0.05）。6．阿托伐他汀诱导LPS-TLR4-MAPKs信号对NF-κB转录活化的作用（1） ERK活性的抑制对NF-κB转录活化作用ELISA结果显示，LPS（30min）+PD98059+阿托伐他汀（1μmol／L，12h）组较LPS（30min）+阿托伐他汀（1μmol／L，12h）组NF-κB活性显著降低（0.75±0.049 vs 0.96±0.12）（P＜0.05）。Western blot结果显示，LPS（30min）+SN50+阿托伐他汀（1μmol／L，12h）组较阿托伐他汀（1μmol／L，12h）+LPS（30min）组P-ERK／ERK蛋白表达差异无统计学意义（0.221±0.0049 vs 0.215±0.0060）（P＜0.05）。（2） P38活性的抑制对NF-κB转录活化作用ELISA结果显示，LPS（30min）+SB203580+阿托伐他汀（1μmol／L，12h）组较LPS（30min）+阿托伐他汀（1μmol／L，12h）组NF-κB活性增加差异无统计学意义（1.08±0.21 vs 0.96±0.12）（P＞0.05）。Western blot结果显示，LPS（30min）+SN50+阿托伐他汀（1μmol／L，12h）组较LPS（30min）+阿托伐他汀（1μmol／L，12h）组P-P38／P38蛋白表达差异无统计学意义（0.695±0.0011 vs 0.690±0.0079）（P＞0.05）。（3）ERK与P38的相互作用Western blot结果显示，LPS（30min）+SB203580+阿托伐他汀（1μmol／L，12h）组较LPS（30min）+阿托伐他汀（1μmol／L，12h）组P-ERK／ERK蛋白表达显著增加（0.240±0.0051 vs 0.215±0.0060）（P＜0.05）。LPS（30min）+PD98059+阿托伐他汀（1μmol／L，12h）组较LPS（30min）+阿托伐他汀（1μmol／L，12h）组P-P38／P38蛋白表达差异无统计学意义（0.695±0.0023 vs 0.690±0.0079）（P＞0.05）。7．RNAi与阿托伐他汀对LPS-TLR4通路诱导效应蛋白表达的逆转作用ELISA结果显示，IL-6：LPS作用12h：LPS+阿托伐他汀组较LPS组IL-6蛋白表达差异无统计学意义。LPS+RNAi组较LPS组IL-6蛋白表达显著降低。LPS作用24h：LPS+阿托伐他汀组及LPS+RNAi组较LPS组IL-6蛋白表达显著降低。LPS+RNAi组较LPS+阿托伐他汀组IL-6蛋白表达显著降低。LPS作用48h：LPS+阿托伐他汀组较LPS组IL-6蛋白表达差异无统计学意义，LPS+RNAi组较LPS组IL-6蛋白表达显著降低。IL-10：LPS作用24h：LPS+阿托伐他汀组及LPS+RNAi组较LPS组IL-10蛋白表达显著增加。LPS+RNAi组较LPS+阿托伐他汀组IL-10蛋白表达显著增加。LPS作用48h：LPS+阿托伐他汀组及LPS+RNAi组较LPS组IL-10蛋白表达显著增加。LPS+RNAi组较LPS+阿托伐他汀组IL-10蛋白表达显著增加。TNF-α：LPS作用12h：LPS+阿托伐他汀组及LPS+RNAi组较LPS组TNF-α蛋白表达显著降低。LPS+RNAi组较LPS+阿托伐他汀组TNF-α蛋白表达显著降低。LPS作用24h：LPS+阿托伐他汀组及LPS+RNAi组较LPS组TNF-α蛋白表达显著降低。LPS+RNAi组较LPS+阿托伐他汀组TNF-α蛋白表达显著降低。LPS作用48h：LPS+阿托伐他汀组较LPS组TNF-α蛋白表达差异无统计学意义，LPS+RNAi组较LPS组TNF-α蛋白表达显著降低。8．腺病毒干扰载体的体外扩增、滴度测定及体外活性检测体外大量扩增pSuppressorAd-TLR4，pSuppressorAd-Neg腺病毒，空斑形成实验测定病毒滴度为6×10¹¹pfu／mL。荧光定量-PCR检测Ad-siRNA-TLR4组较对照组细胞RAW264.7抑制TLR4 mRNA表达达81％。Ad-siRNA-Neg组细胞无抑制作用。9．TLR4腺病毒干扰载体基因治疗与阿托伐他汀药物治疗ApoE-／-小鼠的实验研究：结果发现：（1）各组血TC、TG、HDL及LDL无明显变化，统计学无显著性差异（P＞0.05）（2）与安慰剂组比较，阿托伐他汀组ApoE-／-小鼠头臂干动脉斑块面积，斑块面积／血管面积减少但差异均无统计学意义（P＞0.05）。与安慰剂组比较，阿托伐他汀组ApoE-／-小鼠纤维帽增加，差异无统计学意义（P＞0.05）。与对照载体组比较，RNAi组ApoE-／-小鼠斑块面积，斑块面积／血管面积减少但差异均无统计学意义（P＞0.05）。与对照载体组比较，RNAi组ApoE-／-小鼠纤维帽显著增加（4.53±0.32 vs 8.62±0.39μm）（P＜0.05）。与阿托伐他汀组比较，RNAi组ApoE-／-小鼠纤维帽显著增加（P＜0.05）。（3）油红O染色分析各组小鼠头臂干切片阳性染色区域面积均值：与安慰剂组比较，阿托伐他汀组ApoE-／-小鼠斑块脂质含量显著减少（P＜0.05）。与对照载体组比较，RNAi组ApoE-／-小鼠斑块面积脂质含量减少，差异无显著性（P＞0.05）。与阿托伐他汀组比较，RNAi组ApoE-／-小鼠斑块面积脂质含量显著增加（P＜0.05）。（4）斑块破裂率比较：各组斑块破裂率差异均无统计学意义。（5）易损指数比较：与安慰剂组比较，阿托伐他汀组ApoE-／-小鼠斑块易损指数显著减少，（1.72±0.09 vs 1.44±0.04）（P＜0.05）。与对照载体组比较，RNAi组ApoE-／-小鼠斑块易损指数显著减少，（1.75±0.06 vs 1.10±0.08）（P＜0.05）。与阿托伐他汀组比较，RNAi组ApoE-／-小鼠斑块面积易损指数显著减少（P＜0.05）。（6）免疫组织化学染色：与安慰剂组比较，阿托伐他汀组ApoE-／-小鼠斑块TLR4、MAMO-2抗原含量显著减少（P均＜0.05）。与对照载体组比较，RNAi组ApoE-／-小鼠斑块TLR4、MAMO-2抗原含量显著减少，SM-actin抗原含量显著增加（P均＜0.05）。与阿托伐他汀组比较，RNAi组ApoE-／-小鼠斑块面积TLR4、MAMO-2抗原含量显著减少，SM-actin抗原含量显著增加（P均＜0.05）。（7）荧光定量PCR检测：与安慰剂组比较，阿托伐他汀组ApoE-／-小鼠头臂干动脉TLR4、IL-6、IL-12和VCAM mRNA表达显著降低，IL-10 mRNA表达显著增加。与对照载体组比较，RNAi组ApoE-／-小鼠头臂干动脉TLR4、IL-6、IL-12、TNF-α、VCAM和MMP-9 mRNA表达显著降低，IL-10 mRNA表达显著增加。与阿托伐他汀组比较，RNAi组ApoE-／-小鼠头臂干动脉TLR4、IL-6、IL-12和TNF-αmRNA表达显著降低，IL-10 mRNA表达显著增加。结论1．实验室构建了TLR4基因RNA干扰载体pRNAT-TLR4-1，其可视的转染效率可确保试验的可靠性，且操作简单，适合长期研究基因功能。2．实验室建立了稳定抑制TLR4蛋白的细胞模型（ECV304-pTLR4-1细胞），可长期研究TLR4功能及其介导的信号通路。3．NF-κB是炎症免疫反应的枢纽，我们对比研究TLR4的小分子干扰RNA干预与阿托伐他汀干预对NF-κB的作用，发现RNAi较阿托伐他汀干预能更强的抑制NF-κB的活性，其机制可能为RNAi较阿托伐他汀干预能更有效的抑制LPS-TLR4通路且P38在阿托伐他汀干预抑制NF-κB活性中起负反馈作用。4．TLR4的小分子干扰RNA抑制NF-κB活性的作用不依赖IκB-α。其可能通过抑制PI3K／AKT而抑制p65活化，进而抑制NF-κB活性。5．阿托伐他汀通过稳定IκB-α抑制NF-κB活性。其机制可能为（1）通过稳定TAK1-IKKα依赖的IκBα活性而抑制NF-κB活性；（2）抑制ERK进而稳定IκBα，导致NF-κB转录活性降低。抑制P38活性可能对于ERK和NF-κB活性有负调控作用。6．阿托伐他汀能显著抑制IL-6、IL-10、TNF-α等炎性因子的释放，与之相比RNAi抑制炎性因子的释放时间更持久，效果更显著。7．ApoE-／-小鼠体内试验表明，TLR4小分子干扰腺病毒的基因治疗与阿托伐他汀的药物治疗均可降低斑块的易损性，但是基因治疗效果更优于药物治疗。8．ApoE-／-小鼠体内试验表明，TLR4小分子干扰腺病毒的基因治疗与阿托伐他汀的药物治疗抗AS作用机制可能与促炎／抗炎的平衡和胶原合成／降解的平衡、Th1／Th2免疫调控的平衡有关。更多还原

【Abstract】 BackgroundAtherosclerosis is a long-term chronic disease characterized by the accumulation oflipids 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. The receptors capable of recognizing these PAMPs are toll-like receptors （TLRs）. Until now, 10 TLRs have been identified in humans. Different members of this receptor family are activated by different PAMPs. Ligand binding of the extracellular domain induces dimerization of the TLRs and activating immune system, which leads to the activation of signals. Subsequently, TLR activation induces the expression of a wide variety of genes encoding proinflammatory proteins. Moreover, TLR4 has been shown to be involved in modulating the recruitment and adhesion of leukocytes and monocytes to atherosclerotic lesions, even in plaque stabilizing and embolism. Thus, TLR4 is an interesting gene therapeutic target for the treatment of diseases related to atherosclerosis. Although in recent yeas, many scholars had made great progress in the realationg of TLR4 and AS,there are many question waiting for answer. Can we block the gene expression of TLR4 then prevent the progression of atherosclerosis and stable the plaque of AS. Now there are many ways to inactive genes, for example antisense oligonucleotides, nucleic acid technique, monoclonal antibody,et al.Although antisense technology can inactive the expression of harmful gene ,the design of antisense RNA is difficult. The chemical force of antisense RNA and mRNA and the specificity of their binding of are affected by second order or tertiary structure of ambi- sequential binding sites. The polyanion character of antisense oligonucleotides also cause a series of side effects. Triplex DNA technique applies through binding deoxy-oligonucleotide and double helix double strands DNA to TFO.This technique is short of stability and half life. Morever,monoclonal antibody treatment also exist the short half life. Whether can find an effect method block the gene? Which treatment gene interference or drug treatment play the key point in inflammation signal transduction and the rupture of vulnerable plaque?So we develop the research about block TLR4 to prevent AS progress and stable vulnerable plaque.Aims1. To construct an expression vector of TLR4 using pRNAT-U6.1/Neo and screen the positive clones successfully;2. To compare TLR4-RNAi with atorvastatin treatments on influence of cell biology in Ecv304 cells and explores their mechanism;3. To compare TLR4-RNAi with atorvastatin treatments on influence of TLR4 induced the effects of mmune and inflammation in order to provide experience in vivo; 4. To compare TLR4-RNAi with atorvastatin treatments on influence of TLR4blocking effects in orde to reveal the mechanism that prevent AS progress and stablevulnerable plaque.Methods1. selection of effective siRNA vector for TLR41.1 Construction and identification of siRNA vector for TLR4According to the design principles, two pairs of siRNA sequences were design At the same time design the negatve control fragment.All the siRNA oligos were ligated with pRNAT-U6.lneo. The positive recombinants were sequenced and called pRNAT-TLR4-1、 pRNAT-TLR4-2 and pRNAT-Neg.1.2 screening of positive clonesTransfected with plasmid,most of ECV304 cells was lyzed and died in G418-containing medium.The positive clones stably grew in G418-containing medium and subcultrued for every 4 days.The G418-resistant clones ,named as ECV304-pTLR4-1 , ECV304-pTLR4-2 and ECV304- pNeg respectively,were obtained according to the transfected vectors.1.3 selection of efficient siRNA vector for TLR4Total RNA from cells were extracted.Real time PCR, western blot showed the expression of TLR4 and select the efficient siRNA.2. Comparation TLR4-RNAi with atorvastatin treatments on mechanism of TLR4 induced by LPS and retroconversion of inflammatory action induced by TLR4 in vivo2.1 Cells were divided into the following groups: control group, LPS group, LPS+RNAi group and LPS+atorvastatin group. We observe the change of TLR4 and the downstream signal protein NF-κB and explore the difference of mechanism between gene treatment and atorvastatin treatment.2.2 SN50,Ly294002 ,PD98059 or SB203580 was added into LPS+RNAi group or LPS+atorvastatin group respectively in order to explore the inner mechanism between gene treatment and atorvastatin treatment.2.3 Cells were divided into the following groups: control group, LPS group, LPS+RNAi group and LPS+atorvastatin group. We observe the relationship between downstream effector including IL-6 , IL-10 and TNFα.In this way ,we can explore and compare the reverse effect of immune and inflammation between gene treatment and atorvastatin.3. To compare TLR4-RNAi with atorvastatin treatments on influence of TLR4 blocking effects in orde to reveal the mechanism that prevent AS progress and stable vulnerable plaque.3.1 Proliferation ,titration and activity detection of TLR4 RNAi adenovirus HEK293 cells were infected with pSuppressorAd-TLR4 ,pSuppressorAd-Neg.72h later,the cytopathic effects were observed and the culture supernant and cells were collected respectively.The collected cells were fully frozen and thawed for 3 times in order to obtain the viral paticles.Titer of virus was detected by plaque-forming assay.Then RAW247.5 cells were treated with Real-time PCR to detect TLR4 mRNA.3.2 The research of TLR4 RNAi adenovirus treatment and atorvastatin treatment on ApoE-knock out mice.ApoE-knock out mice with 8 week old were fed on western-diet. At the same time either pSAd-TLR4 or pSAd-Neg diluted to a total volume of of 100μL was injected into the tail vein of each group of mice （n=27）. Mice （n=27） injected with pSAd-Neg （100μL） served as control. Mice（n=22） in atorvastatin group were intervened with atorvastatin （dissolved in ultrapure water, 10mg/kg/d,ig） . Mice （n=22） were intervened with placebo served as control. All the animals sacrificed 10 weeks later. The lipid core and composition of plaque were characterized with oil red O staining. The vulnerable plaque was characterized with vulnerability index.The expression of antigens in brachiocephalic trunk and their plaques among 5 groups such as TLR4、 SM-actin and MAMO-2 were compared semi-quantitatively using immunohistochemical technology. The differential displays of mRNAs for TLR4、 IL-6、 IL-10、 IL-12、 TNF-α VCAM-1 MMP-9 and ICAM-1 were determined using relative quantitative real-time PCR analysis.Results 1. We construct an expression vector of TLR4 using pRNAT-U6.1/Neo and screen the positive clones successfully; Real time PCR and Westemblot displayed that the expression of TLR4 mRNA and protein were significantly downregulated in ECV304-pTLR4-1 cells when compared with those of other groups.2. The effection of atorvastatin on LPS upregulated TLR4 in ECV304 cells Western blot showed that LPS+atorvastatin group reversed the LPS-inducedincreases in TLR4 dose dependently. Maximal effects were obtained after 12h of exposure to atorvastatin （1 μmol/L）3. The effection of TLR4-RNAi and atorvastatin on the NF-κB activation induced by LPS-TLR4ELISA showed that relative to the basal activity detected in control cells（0.67±0.002） , NF-κB binding activity was distinctly enhanced in cells treated for30 min with LPS（1.89±0.04） （P<0.05） , and this augmented NF-κB binding activitywas significantly inhibited in RNAi+ LPS group （0.72±0.02） and atorvastatin +LPS group （0.96±0.12 ） .Western blot showed that comared with control group ,IkBa levels obtained after 30 min of LPS stimulation have a rapid disappearance（P<0.05） .Comared with LPS group, IkBa levels were significantly increase inatorvastatin（ 1 μmol/L , 12h）+ LPS（30min）group.While compared with LPS group,IkBa levels have no significant difference in RNAi+LPS group. （P>0.05）4. The effection of TLR4-RNAi on the PI3K-Akt induced by LPS -TLR4 Western blot showed that comared with LPS （30min） group, p-Akt/Akt（0.73±0.07 vs 0.2±0.02） and p65 （43.4±1.29 vs 23.78 ±0.26） levels were significantly decrease in RNAi+ LPS （30min） group. Comared with LPS （ 30min） group, p-Akt/Akt （0.73±0.07 vs 0.13±0.008） and p65 levels （43.4±1.29 vs 12.16 ±0.33） were significantly decrease in RNAi+ LPS （30min） group.While IxB-α level had no significant difference among these groups （10.00±0.06 vs 9.89±0.19） （P >0.05） .5. The effection of atorvastatin on the MAPKs activation induced by LPS -TLR4 in ECV304 cell Western blot showed that comared with control group, p-ERK/ERK levels were significantly increase in LPS （100 ng/mL, 30min） group （0.018±0.0001 vs 0.786±0.0276） （P<0.01） .Comared with LPS group, p-ERK/ERK levels were significantly decrease in （1μmol/L , 12h）+LPS （100 ng/mL, 30min） group（0.215±0.0060 vs 0.786±0.0276） （P<0.05） . Comared with control group, p-P38/P38 levels were significantly increase in LPS （100 ng/mL, 15min） group（P<0.01） .Comared with LPS group, p-P38/P38 levels were significantly decrease in atorvastatin （1 μmol/L , 12h）+LPS （100ng/mL, 15min） group （0.690±0.008 vs 0.782±0.015 ） （P<0.05） . Comared with control group, p-JNK/JNK levels were significantly increase in LPS （100 ng/mL, 30min） group （0.053±0.004 vs 0.584±0.011 ） .There was no significant difference in the expression of p-JNK/JNKlevels between LPS group and atorvastatin（1 μmol/L , 12h）+LPS （100 ng/mL, 15min） group.6. The effection of LPS-TLR4- MAPKs induced by atorvastatin on the activity of NF-κB（1） ELISA showed that comared with atorvastatin （1 μmol/L , 12h）+LPS （100 ng/mL, 30min） group group, NF-κB activitions were significantly decrease in atorvastatin （ 1μmol/L , 12h） +PD98059+LPS （ 100 ng/mL , 30min ） group（0.75±0.049 vs 0.96±0.12） （P<0.05） .Westernblot showed that there was no significant difference in the expression of p-ERK/ERK between atorvastatin（ 1μmol/L , 12h） + SN50+LPS （ 100 ng/mL , 30min ） group and atorvastatin（1μmol/L , 12h）+LPS （100 ng/mL, 30min） group（ 0.221 ±0.0049 vs 0.215±0.0060） （P<0.05） .（2） ELISA showed that there was no significant difference in the expression of NF-κB activitions between atorvastatin （1μmol/L , 12h） + SB203580+LPS （100 ng/mL, 30min） group and atorvastatin（1 μmol/L , 12h）+LPS（ 100 ng/mL, 30min） group（1.08±0.21 vs 0.96±0.12） （P>0.05） . Westernblot showed that there was no significant difference in the expression of p-P38/P38 between atorvastatin（ 1 μmol/L , 12h） + SN50+LPS （ 100 ng/mL , 30min ） group and atorvastatin（1 μmol/L , 12h）+LPS （100 ng/mL, 30min） group（ 0.695±0.0011 vs 0.690±0.0079） （P>0.05） .（3） Westernblot showed that compared with atorvastatin （1μmol/L , 12h）+LPS （100 ng/mL, 30min） group group, p-ERK/ERK levels were significantly increase in atorvastatin （1 μmol/L , 12h） + SB203580+LPS （100 ng/mL, 30min） group （0.240±0.0051 vs 0.215±0.0060） （P<0.05）,but there was no significant difference between the two groups.There was no significant difference in the expression of p-P38/P38 between atorvastatin （1μmol/L , 12h） + PD98059+LPS （100ng/mL, 30min） group and atorvastatin（1 μmol/L , 12h）+LPS （100 ng/mL, 30min） group（ 0.695±0.0023 vs 0.690±0.0079） （P>0.05） .7. The effection of TLR4-RNAi and atorvastatin on the downstream factors induced by LPS -TLR4IL-6: 12h:ELISA showed that there was no significant difference between atorvastatin group and LPSgroup. Compared with LPS group , IL-6 levels were significantly decrease in RNAi group. 24h: Compared with LPS group , IL-6 levels were significantly decrease in atorvastatin group. Compared with atorvastatin group, IL-6 levels were significantly decrease in RNAi group. 48h: there was no significant difference between atorvastatin group and LPS group. Compared with LPS group , IL-6 levels were significantly decrease in RNAi group.IL-10: 24h: Compared with LPS group , IL-10 levels were significantly increase in RNAi group. Compared with atorvastatin group , EL-10 levels were significantly increase in RNAi group.TNF-α: 6h: there was no significant difference between atorvastatin group and LPS group. Compared with LPS group , TNF-α levels were significantly decrease in RNAi group. 12h: Compared with LPS group , TNF-α levels were significantly decrease in atorvastatin group. Compared with atorvastatin group , TNF-α levels were significantly decrease in RNAi group. 24h: Compared with LPS group , TNF-α levels were significantly decrease in atorvastatin group. Compared with atorvastatin group , TNF-α levels were significantly decrease in RNAi group. 48h: there was no significant difference between atorvastatin group and LPS group. Compared with LPS group , TNF-α levels were significantly decrease in RNAi group.8. Proliferation ,titration and activity detection of TLR4 RNAi adenovirus pSuppressorAd-TLR4 and pSuppressorAd-Neg were amplified in scale.Plaque-forming assay showed that viral titer was 6×10¹¹ pfu/mL . Real-time PCR showed TLR4 mRNA in pSAd-TLR4 group was suppressed.9. The research of TLR4 RNAi adenovirus treatment and atorvastatin treatment on ApoE-knock out miceAfter 10 week treatment, there was no significant difference between RNAi and control group （p>0.05）. Moreover, no significant differences in either body weight or total cholesterol and triglyceride concentrations in plasma among four groups were observed. Compared with control group, the area of plaque lipid contents have no significant difference in RNAi group,but there is a thinker fiber cap in RNAi group. Compared with placebo group, the area of plaque has no significant difference but lipid contents were decreased in atorvastatin group. Compared with control or placebo group, the vulnerability index was decreased in RNAi or atorvastatin group. Compared with atorvastatin group, the vulnerability index was decreased in RNAi group. Upregulation of SM-actin antigen activity and downregulation of TLR4 and MAMO-2 antigen activity in RNAi group was observed （P<0.05）. Downregulation of TLR4 and MAMO-2 antigen activity in atorvastatin group was observed （P<0.05）. Real-time detection of PCR showed increasing of mRNAs for IL-10, while TLR4,IL-6,IL-12,TNF-α,VCAM-1,MMP-9 and ICAM-1 gene expressions decreasing in RNAi group.There is an increasing of mRNAs for IL-10, while TLR4,IL-6,IL-12, VCAM-1 and ICAM-1 gene expressions decreasing in RNAi group.Conclusions1. Construct an expression vector of TLR4 using pRNAT-U6.1/Neo and screen the positive clones （ECV304-pTLR4-1, ECV304-pTLR4-2 and ECV304- pNeg cell） successfully.2. ECV304-pTLR4-1 can effective inhibit the expression of TLR4 mRNA and TLR4 protein3. NF-κB is the key point in immunity reaction. So we study the effection of TLR4-RNAi or atorvastatin on NF-κB and found RNAi may be inhibit the activity of NF-κB better.The mechanism maybe the negative feedback of P38 in atorvastatin treatment.4. TLR4-RNAi prevents NF-κB transactivation independently of the IκB-α pathway.The mechanism is inhibiting PI3K/AKT then p-p65 and then NF- κB.5. Atorvastatin may block NF-κB activation by causing abolition the TLR4 pathway then stabilization of the IkBa in cellular cytoplasm.The mechanism maybe as follows: First, atorvastatin regulate the NF-κB translocation dependent pathway through TAK1- IKKα dependent IκBα stabilization. Second, the inaction of phosphorylation of ERK leads to the IκBα stabilization and then the inhibition of NF-kB translocation. While the suppression of p38MAPK may exert slightly up regulation on ERK and NF-κB.6. Atorvastatin may supprss the expression of IL-6 and TNF-α, and increase the expression of IL-10.Howerver, RNAi had better effect.7. In vivo, TLR4-RNAi treatment and atorvastatin treatment can decrease the vulnerability of plaque. However RNAi had better effect.8. The anti-atheroscleris of TLR4-RNAi and atorvastatin treatment may be attributed to balance of proinflammation/antiinflammation, synthesis and degradation of collagen and immunoregulation更多还原