【作者】 路静；

【导师】 董子明；

【作者基本信息】 郑州大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 树突状细胞(dendritic cells,DCs)是目前已知抗原呈递功能最强,唯一能激活初始T细胞的专职抗原递呈细胞,是调节免疫反应的中心环节。由DCs激活的T细胞介导的免疫反应在抗肿瘤免疫中起重要作用,DCs数量或功能缺陷是肿瘤免疫逃逸的重要原因,国内外近年来关于DCs的研究多集中于如何提高DCs的抗原递呈功能及制备有效的DCs瘤苗等方面。2004年《Nature Medicine》报道Coukos G等在高分泌血管内皮生长因子A(Vascular endothelial growth factor A,VEGF-A)的荷瘤鼠和人卵巢癌组织中发现一种新的细胞群,同时表达DCs和内皮细胞标志。VEGF-A是肿瘤细胞分泌的众多因子中一种强力的血管生成因子,是高效的内皮细胞有丝分裂原,可直接作用于内皮细胞,促进肿瘤血管生成。该研究同时发现,体外培养的小鼠骨髓来源的DCs在VEGF-A的作用下也可出现内皮样分化,提示在体内DCs可参与肿瘤血管的形成。肿瘤血管形成是肿瘤迅速生长的重要标志。传统理论认为只有通过血管新生(angiogenesis)即通过预存的内皮细胞发芽从而形成毛细血管,而血管发生(vasculogenesis)主要在胚胎期。但近来研究表明血管发生在成年生物也可实现,肿瘤的血管化作用也包括血管发生,即内皮细胞从不成熟的前体细胞发育而来。2006年Young MR报道小鼠骨髓来源的CD34~+细胞可发育为起抗原递呈功能的DCs,但加入小鼠的Lewis肺癌细胞条件培养液后,使CD34~+细胞分化发生偏移,从树突状细胞途径分化为内皮样细胞。2007年E.Gottfried研究表明肿瘤相关的DCs在VEGF和制瘤素作用下分化成为内皮样细胞,可能是肿瘤血管生成的另一条途径。DCs的内皮样分化是一新现象,具体发生机制有待深入研究。多种生长因子、细胞因子和一些血管活性物质,均可通过细胞信号转导引起细胞分化。MAPK/ERK1/2是多种细胞外刺激信号引起细胞分化、细胞内信息传递的共同通路或交汇点,那么,该通路是否介导了DCs的内皮样分化尚有待探讨。食管癌是世界上最常见的恶性肿瘤之一,有较高的发生率和死亡率。食管鳞癌是最常见的病理类型,侵袭性强,预后差。食管鳞癌组织及其代谢产物是否诱导DCs出现内皮样分化,使食管鳞癌细胞在逃避免疫监视的同时诱导血管内皮细胞的形成,目前未见报道。本研究分别采用食管鳞癌EC9706细胞上清,食管鳞癌组织匀浆上清体外模拟食管鳞癌微环境,研究其对不成熟DCs(immature DCs,iDCs)和成熟DCs(mature DCs,mDCs)内皮样分化细胞形态及功能学方面的影响;通过研究VEGF-A对DCs内皮样分化的影响,VEGF-A在EC9706细胞上清诱导iDCs内皮样分化中的作用,MAPK/ERK1/2/CREB通路在EC9706细胞上清、VEGF-A诱导iDCs内皮样分化中的作用,来探讨食管鳞癌微环境引起iDCs内皮样分化的机制,丰富食管鳞癌细胞免疫逃逸理论,提供阻断iDCs内皮样分化的策略,为DCs瘤苗在食管鳞癌患者更好地应用提供基础。第一部分食管鳞癌微环境对iDCs和mDCs内皮样分化的影响第一章EC9706上清对iDCs和mDCs内皮样分化细胞形态及相关抗原的研究方法1.实验分组对照DCs组:人外周血单核细胞经rhIL-4、rhGM-CSF、LPS联合诱导培养DCs,第9天、14天收获细胞。EC9706上清iDCs诱导组:在DCs培养的2天末(iDCs)加入40%EC9706培养上清诱导7天,第9天收获细胞。EC9706上清mDCs诱导组:在DCs培养的7天末(mDCs)加入40%EC9706培养上清诱导7天,第14天收获细胞。阳性对照人脐静脉内皮细胞组:胰蛋白酶消化配合内皮细胞培养基(endothelial cell medium,ECM)培养人脐静脉内皮细胞(human umbilicalvein endothelial cells,HUVECs)。2.光学显微镜下观察EC9706上清iDCs诱导组、mDCs诱导组细胞形态。3.流式细胞仪检测EC9706上清iDCs诱导组、mDCs诱导组细胞表达DCs特异标志CD86、CD1a及CD11c的情况。4.RT-PCR检测0d单核细胞、iDCs诱导组、mDCs诱导组细胞和对照组细胞DCs标志CD1a、内皮细胞标志CD144和血管假性血友病因子(von Willebrand Factor,vWF,又名八因子相关抗原)的表达。5.免疫荧光法检测EC9706上清iDCs诱导组、mDCs诱导组细胞和对照组细胞vWF的表达。6.Western blotting检测EC9706上清iDCs诱导组、mDCs诱导组细胞和对照组细胞vWF、CD144表达。结果1.对照DCs和HUVECs形态和相关抗原的表达电镜检测显示第7天对照DCs有明显突起,胞浆有大量线粒体、吞饮小泡和微丝;流式细胞术检测诱导前单核细胞相关抗原阳性率均较低,诱导7d后,DCs表面抗原表达明显增高(P＜0.01)。阳性对照HUVECs具有内皮细胞典型的铺路石状、长梭形、漩涡状排列生长、以及管腔样结构。高表达内皮特异性标志vWF、CD144,并具备内皮细胞强的摄取Dil荧光标记的乙酰化低密度脂蛋白(Dil-acetylated low-density lipoprotein,Dil-Ac-LDL)的能力。2.EC9706上清诱导对细胞形态学影响第8d,对照DCs逐渐增大变圆,高倍镜下细胞周围可见毛刺状突起,部分细胞悬浮,而EC9706上清iDCs诱导组大圆细胞数目较少,细胞多呈细长形,有类管腔分布趋势,少部分细胞呈条索状排列;mDCs诱导组细胞形态与其对照组无明显区别。3.EC9706上清诱导后DCs抗原CD86、CD1a及CD11c的表达流式结果显示,EC9706上清iDCs诱导组与其对照DCs相比CD86、CD1a及CD11c表达率明显下降(P＜0.05),而mDCs诱导组与其对照DCs各抗原表达率无明显差异(P＞0.05)。4.EC9706上清诱导前、后DC抗原CD1a和内皮细胞抗原CD144与vWF的表达RT-PCR结果显示,DCs诱导前单核细胞CD1a表达很弱,基本不表达CD144、vWF。iDCs诱导组细胞与其对照DCs相比CD1a表达降低,CD144与vWF表达增强;mDCs诱导组细胞与其对照DCs仅有CD1a的表达,而基本不表达CD144、vWF。免疫荧光结果显示:iDCs诱导组细胞胞浆中vWF绿色荧光颗粒表达量强于其对照DCs(P＜0.05),而mDCs诱导组与其对照DCs相比无明显差异(P＞0.05)。Western blotting结果显示:iDCs诱导组vWF、CD144蛋白表达量增加,与对照DCs相比P＜0.01;mDCs诱导组CD144、vWF蛋白均无明显表达。第二章EC9706上清对iDCs和mDCs内皮样分化功能学的研究方法1.检测EC9706上清iDCs诱导组、mDCs诱导组细胞在纤维粘连蛋白上类管腔样结构的形成能力。2.检测PBMCs、EC9706上清iDCs诱导组、mDCs诱导组细胞Dil-Ac-LDL及印度墨汁摄取情况。3.电镜检测EC9706上清iDCs诱导组细胞及其对照细胞形态及WP小体形成情况。4.活细胞计数试剂盒(Cell Counting Kit-8,CCK-8)检测EC9706上清iDCs诱导组细胞刺激T细胞增殖,及其特异性CTL对EC9706细胞杀伤效应。结果1.EC9706上清诱导后对相关内皮细胞功能的影响类管腔结构形成能力:iDCs诱导组细胞接种至纤维粘连蛋白包被的培养板上,第1个24 h细胞形态呈现梭形,漩涡状分布趋势,第2个24 h可观察到与HUVECs相似的类管腔样结构形成;而其对照DCs、mDCs诱导组和其对照DCs分布较均匀。摄取能力:单核细胞摄取Dil-Ac-LDL的能力弱,但其摄取墨汁能力强。iDCs诱导组细胞不摄取墨汁,排除诱导后细胞中混有单核细胞、巨噬细胞可能。iDCs诱导组细胞摄取Dil-Ac-LDL能力明显强于其对照DCs,而mDCs诱导组细胞摄取Dil-Ac-LDL能力与其对照DCs无明显差别。WP小体的形成:WP小体是内皮细胞特异的功能性结构单位。电镜结果示:iDCs诱导组细胞中有WP小体和大量的微丝,与HUVECs结构相似,在对照DCs中没有发现WP小体结构。2.EC9706上清诱导后对相关DC功能的影响EC9706上清iDCs诱导组细胞刺激T细胞增殖率、CTL杀伤率均明显低于对照DCs(P＜0.01),显示EC9706上清使iDCs诱导组细胞抗原递呈功能下降。第三章食管鳞癌组织匀浆上清对iDCs内皮样分化的影响方法1.采集新鲜食管鳞癌及癌旁组织标本各10例,研磨法制备组织匀浆上清。2.实验分组对照DCs组:人外周血单核细胞,rhIL-4、rhGM-CSF、LPS联合诱导培养DCs。食管鳞癌匀浆上清组:在DCs培养的2天末加入40%食管鳞癌组织匀浆上清,诱导7天,第9天收获细胞。癌旁匀浆上清组:在DCs培养的2天末加入40%食管鳞癌癌旁组织匀浆上清,诱导7天。阳性对照HUVECs。3.Western blotting检测食管鳞癌、癌旁匀浆上清组细胞vWF、CD144的表达。4.免疫荧光检测食管鳞癌、癌旁匀浆上清组细胞vWF的表达。5.Dil-Ac-LDL及印度墨汁摄取实验检测食管鳞癌、癌旁匀浆上清组细胞摄取功能。6.CCK-8检测食管鳞癌、癌旁匀浆上清组细胞刺激T细胞增殖、及其特异性CTL对EC9706细胞杀伤效应。结果1.食管鳞癌、癌旁匀浆上清诱导后对iDCs形态的影响培养第8天,正常DCs与癌旁组细胞逐渐增大变圆,细胞周围可见毛刺状突起,部分细胞悬浮;而食管癌匀浆上清组细胞呈现细长梭形,交织在一起。2.食管鳞癌、癌旁匀浆上清诱导后内皮细胞抗原CD144与vWF的表达癌匀浆上清组细胞vWF、CD144蛋白的表达量增加,与癌旁组、对照DCs相比P＜0.01;而癌旁组与对照DCs相比P＞0.05。食管癌匀浆上清组细胞胞浆中vWF绿色荧光颗粒表达量强于其癌旁组和对照DCs(P＜0.05);而癌旁组和对照DCs vWF荧光表达量均较弱(P＞0.05)。3.食管鳞癌、癌旁匀浆上清诱导后对相关内皮细胞功能的影响各诱导组细胞均不摄取墨汁,癌匀浆上清组细胞摄取Dil-Ac-LDL能力明显强于癌旁组和对照DCs。4.食管鳞癌、癌旁匀浆上清诱导后对相关DC功能的影响食管癌匀浆上清组刺激T细胞增殖率及其CTL对EC9706的杀伤率,均明显低于对照DCs组和癌旁组(P＜0.01);而癌旁组与对照DCs刺激T细胞增殖能力与CTL杀伤率差异没有统计学意义(P＞0.05)。第二部分食管鳞癌微环境引起iDCs内皮样分化机制的探讨第一章VEGF-A对DCs内皮样分化的作用方法1.实验分组对照DCs组:分离人外周血单核细胞,rhIL-4、rhGM-CSF、LPS联合诱导培养DCs。VEGF-A iDCs诱导组和mDCs诱导组,分别在DCs培养的2天末和7天末加入20 ng/ml VEGF-A进行诱导7天,收集细胞检测指标。阳性对照HUVECs。2.流式细胞仪检测20 ng/mlVEGF-A iDCs诱导组、mDCs诱导组细胞表型CD86、CD1a及CD11c。3.RT-PCR、Western blotting、免疫荧光法检测VEGF-A iDCs诱导组、mDCs诱导组CD1a、vWF、CD144表达。4.检测VEGF-A iDCs诱导组、mDCs诱导组Dil-Ac-LDL及印度墨汁摄取能力。5.电镜检测VEGF-A iDCs诱导组细胞形态及WP小体形成情况。6.CCK-8检测VEGF-A iDCs诱导组细胞刺激T细胞增殖及其CTL对EC9706细胞杀伤效应。结果1.VEGF-A对诱导组细胞形态学影响VEGF-A iDCs诱导组细胞发育迟缓,第8d,正常DCs逐渐增大变圆,而VEGF-AiDCs诱导组细胞多呈细长梭形,有部分类管腔分布趋势,少部分细胞呈条索状排列;VEGF-A mDCs诱导组细胞形态与其对照DCs无明显区别。2.VEGF-A诱导后DCs抗原CD86、CD1a及CD11c的表达VEGF-A iDCs诱导组与对照DCs相比,各抗原表达率均下降(P＜0.05),而VEGF-A mDCs诱导组与对照DCs相比无明显差异(P＞0.05)。3.VEGF-A诱导后DCs抗原CD1a和内皮细胞抗原CD144与vWF的表达VEGF-A iDCs诱导组细胞CD1a mRNA水平表达下降,同时vWF、CD144mRNA水平和蛋白水平均表达增加(P＜0.05),而VEGF-A mDCs诱导组与对照DCs相比无明显差异(P＞0.05)。4.VEGF-A诱导后对相关内皮细胞功能及相关DC功能的影响摄取能力:VEGF-A iDCs诱导组细胞不摄取墨汁,同时Dil-Ac-LDL摄取能力明显强于其对照DCs;而VEGF-A mDCs诱导组细胞摄取能力与其对照DCs无明显差别。WP小体的形成:VEGF-A iDCs诱导组细胞中发现了WP小体,并且细胞内有成束的微丝,细胞周围有细长的突起,这些结构与HUVECs很相似。VEGF-A iDCs诱导组细胞刺激T细胞增殖率及其CTL对EC9706的杀伤率,均明显低于对照DCs(P＜0.01)。第二章阻断VEGF-A对EC9706上清诱导iDCs内皮样分化的影响方法1.ELISA试剂盒检测EC9706上清及食管鳞癌、癌旁组织匀浆上清VEGF-A含量。2.实验分组对照DCs组:分离人外周血单核细胞,rhIL-4、rhGM-CSF、LPS联合诱导培养DCs。EC9706上清组:在DCs培养的2天末加入40%EC9706上清诱导7天;VEGF-A阻断组:在DCs培养的2天末加入40%EC9706上清,同时加入VEGF-A抗体阻断EC9706上清中的VEGF-A;阳性对照HUVECs。3.免疫荧光法检测VEGF-A阻断后EC9706上清组vWF表达。4.检测VEGF-A阻断后EC9706上清组细胞Dil-Ac-LDL摄取能力。5.Western blotting检测VEGF-A阻断后EC9706上清组细胞vWF、CD144表达。结果1.上清中VEGF-A的含量EC9706上清、食管癌组织匀浆上清中VEGF-A的含量大于癌旁组织匀浆上清(P＜0.05)。2.VEGF-A阻断后细胞形态EC9706上清诱导下,细胞呈现细长梭形,与HUVECs形态相似,而对照DCs呈类圆形,细胞周围有毛刺样突起;VEGF-A阻断组细胞多呈类圆形,极少有细长突起。3.VEGF-A阻断后细胞vWF和CD144的表达VEGF-A阻断组细胞胞浆中vWF绿色荧光颗粒表达量明显弱于EC9706上清组(P＜0.05),与其对照DCs相比无明显差异(P＞0.05)。VEGF-A阻断组细胞vWF、CD144蛋白表达量与EC9706上清组相比明显下降(P＜0.01)。4.VEGF-A阻断后细胞摄取能力VEGF-A阻断组细胞摄取Dil-Ac-LDL能力明显弱于EC9706上清组,而与其对照DCs相比无明显差别。第三章VEGF-A激活MAPK/ERK1/2/CREB信号通路介导食管鳞癌细胞微环境下iDC内皮样分化的研究方法1.实验分组:EC9706上清诱导组:在培养DCs的基础上于第2d末添加40%EC9706培养上清,分别诱导0 min、15 min、30 min、60 min和7d;VEGF-A诱导组:在培养DCs的基础上于第2d末添加20 ng/ml VEGF-A,分别诱导0 min、15 min、30 min、60 min和7d;PD98059 60min阻断组:PD98059 50μM先作用细胞1 h,再加入40%EC9706上清或20 ng/ml VEGF-A诱导1 h;PD98059 7d阻断组:EC9706上清或VEGF-A诱导7天过程中同时加入PD98059 10μM。2.Western blotting检测EC9706上清、VEGF-A对ERK、CREB的激活,以及PD98059阻断后EC9706上清组、VEGF-A组细胞ERK1/2、CREB磷酸化水平、及vWF、CD144表达情况。3.免疫荧光检测PD98059阻断后EC9706上清组、VEGF-A组细胞vWF的表达。4.检测PD98059阻断后EC9706上清组、VEGF-A组细胞Dil-Ac-LDL摄取能力。5.免疫荧光双染(FITC、PI)检测EC9706上清、VEGF-A激活的p-ERK1/2的核易位,以及PD98059阻断p-ERK1/2的核易位。6.Western blotting检测VEGF-A阻断后EC9706上清组细胞p-ERK1/2、p-CREB水平。7.凝胶滞留实验检测EC9706上清组、VEGF-A组细胞核里活化的CREB与CRE探针结合活性。结果1.iDCs内皮样分化过程中激活MAPK/ERK1/2、CREBEC9706上清、VEGF-A诱导MAPK/ERK1/2、CREB磷酸化均以一种时间依赖的方式。PD98059,是MEK1/2的选择性抑制剂,而MEK1/2是MAPK/ERK1/2磷酸化的上游蛋白激酶。在进行PD98059预处理后,EC9706上清60 min组、VEGF-A 60 min组磷酸化MAPK/ERK1/2、p-CREB水平明显下降(P＜0.01)。2.抑制MAPK/ERK1/2的活化就抑制了iDCs的内皮样分化PD98059处理的EC9706上清、VEGF-A诱导7天的细胞,在抑制MAPK/ERK、CREB的磷酸化的同时,伴随着诱导细胞形态和表型改变,CD144、vWF表达明显下降(P＜0.01),并且Dil-Ac-LDL摄取能力也明显下降。这表明,PD98059抑制了MAPK/ERK1/2的磷酸化,也就从形态和功能上抑制了EC9706上清、VEGF-A对iDCs的内皮样诱导分化。3.磷酸化MAPK/ERK1/2的核转位磷酸化MAPK/ERK1/2的核易位,是引起生存和分化的转录改变所必需的。EC9706上清、VEGF-A诱导iDCs 7天,均显示核(红)里出现磷酸化的MAPK/ERK1/2(绿);PD98059阻断组,EC9706上清、VEGF-A诱导的MAPK/ERK1/2的磷酸化以及核易位都被明显地抑制。4.阻断VEGF-A对EC9706上清激活MAPK/ERK1/2/CREB通路的影响阻断VEGF-A的EC9706上清组细胞中p-REK、p-CREB水平明显下降(P＜0.01)。说明在EC9706上清激活MAPK/ERK1/2/CREB通路中VEGF-A起启动作用。5活化CREB与CRE元件结合活性经EC9706上清、VEGF-A诱导后,激活的CREB具有较高的与CRE元件结合活性。CD144,vWF基因启动子上游含有CRE元件,活化的CREB与CRE元件结合后可指导CD144,vWF转录,引起表达增强。结论1.EC9706培养上清使iDCs从DCs分化途径转向内皮细胞分化途径,形成内皮样细胞,但对mDCs没有明显影响,提示mDCs可以相对安全地用于抗食管鳞癌治疗中。2.食管鳞癌组织匀浆上清也促使iDCs发生内皮样分化,并且癌旁组织匀浆上清没有产生该作用,进一步说明iDCs的内皮样分化由食管鳞癌微环境引起,这也可能是导致食管鳞癌免疫逃逸的机制之一。3.VEGF-A使iDCs表达内皮细胞标志增加,出现内皮细胞形态及功能,同时DCs标志及抗原递呈功能下降,发生内皮样分化,但不能诱导mDCs的内皮样分化。4.EC9706上清、VEGF-A时间依赖方式激活ERK1/2、CREB,并在整个诱导过程中呈持续激活状态,p-ERK1/2核易位活化CREB,可能导致内皮细胞标志CD144、vWF基因转录,表达增加。5.MAPK/ERK1/2/CREB通路介导了EC9706上清、VEGF-A诱导iDCs内皮样分化,阻断MEK,就阻断了该通路以及内皮样分化。6.阻断EC9706上清中的VEGF-A,可阻断iDCs中MAPK/ERK1/2/CREB的活化以及内皮样分化,表明VEGF-A是iDCs出现内皮样分化的主要启动信号分子。这为抑制内皮样分化提供了新策略。更多还原

【Abstract】 Dendritic cells(DCs) are the best professional antigen presenting cells,which are uniquely able to activate a naive T-cell response.DCs play the central role in regulating immune response.The T cell immune response activated by DCs plays a key role in anti-tumor immune responses.The defection on function and numbers of DCs in cancer patients is the main reason for the tumor immune escape.Therefore,many studies focus on enhancement of antigen presenting function of DCs and effective DCs vaccine preparation.On 2004,Coukos G reported a novel leukocyte subset that coexpressed EC and DC markers within murine and human ovarian carcinoma which secret a high level of. vascular endothelial growth factor A(VEGF-A).VEGF-A is one of the most potent direct-acting angiogenic molecule secreted by tumor cells,which is a highly effective and specific mitogen for endothelial cells(ECs),promoting tumor angiogenesis and metastasis.This study also proved that murine bone marrow-derived DCs could undergo endothelialization in vitro after incubation in high level of VEGF-A,and involved in tumor angiogenesis in vivo.Vessel formation is an important indicator of tumour in rapid growth.Classically, vascularization has been considered through angiogenesis,which involves sprouting from preexisting endothelial cells.Recent studies have suggested that vasculogenesis, which occurs more prominently during embryonic development,can also contribute to vessel formation in adults.Vasculogenesis is the development of vessels from precursor cells that migrate to the site of vascularization.There is a similar event in tumor.On 2006,Young MR reported that CD34~+ progenitor cells,which are capable of developing into dendritic cells,addition of conditioned medium of murine Lewis lung carcinoma cells redirected their differentiation into ECs.On 2007,E.Gottfried showed that tumor-associated DCs incubated additionally with pro-angiogenic factors,such as VEGF and oncostatin M,could trans-differentiate into endothelial-like cells,which may be an alternative pathway of tumour angiogenesis.Endothelial-like differentiation of DCs is a new phenomenon,and its mechanisms are unclear.Many growth factors/cytokines and vasoactive substances can led to cell differentiation through cellular signal transduction.MAPK/ERK1/2 is the common signaling pathway which leds to cell differentiation by various of extracellular signals. So,it is worthwhile to research further whether the MAPK/ERK1/2 signaling pathway mediate the endothelial-like differentiation of DCs.Esophageal cancer is one of the most frequently diagnosed cancers,and has high-incidence and high mortality rate.Esophageal squamous cell carcinoma(ESCC) is still the main type of esophageal cancer,which has strong invasive and bad prognosis. It is unclear wether ESCC and its metabolites can induce endothelial-like differentiation of DCs to led immune escape of ESCC ? In this study,Firstly,to simulate the ESCC microenvironment by EC9706 cell supernatant and ESCC tissue homogenate supernatant,and research the effect on the endothelial-like differentiation of immature DCs(iDCs) and mature DCs(mDCs).Secondly,to investigate the mechanisms of endothelial-like differentiation of iDCs in ESCC microenvironment through investigating the effect of VEGF-A on the endothelial-like differentiation of DCs,the effect of VEGF-A on EC9706 cell supernatant inducing the endothelial-like differentiation of iDCs,and the MAPK/ERK1/2/CREB signaling pathway mediating the endothelial-like differentiation of iDCs induced by EC9706 cell supernatant or by VEGF-A.This research will rich the theory of ESCC immune escape,and may bring forward to strategy of blocking endothelial-like differentiation of iDCs to make DCs could be used better as an anti-ESCC vaccine. PartⅠEffect of the ESCC microenvironment on the endothelial-like differentiation of iDCs and mDCsChapter 1 Study on cell morphology and associated antigen of EC9706 cell supernatant on the endothelial-like differentiation of iDCs and mDCsMethods1.Experimental grouping Control DCs:human peripheral blood monocytes were induced toward DCs by rhIL-4,rhGM-CSF and LPS.DCs were harvested at day 9.and day 14.Cells of iDCs induced group:40%EC9706 supernatant was added at the end of day 2(iDCs) for 7 days induction,and induced cells were harvested at day 9.Cells of mDCs induced group:40%EC9706 supernatant was added at the end of day 7(mDCs)for 7 days induction,and induced cells were harvested at day 14.Positive control human umbilical vein endothelial cells(HUVECs):HUVECs were acquired by digestion of trypsin and cultivation of endothelial cell medium(ECM).2.Observing cell morphology under the optical microscope.3.Flow-cytometry(FCM) analysis of DC surface antigen CD86,CD11c and CD1a on Cells of iDCs induced group and mDCs induced group.4.Detection of the expression of CD1a,vWF,CD144 on monocytes,cells of iDCs induced group and mDCs induced group,and HUVECs by RT-PCR.5.Detection of the expression of vWF on cells of iDCs induced group and mDCs induced group,and HUVECs by Immunofluorescence.6.Western blotting detection of the expression of vWF,CD144 on cells of iDCs induced group and mDCs induced group,and HUVECs.Results1.Morphology and the expression of associated antigen of control DCs and HUVECsOn day 7,control DCs displayed morphology feature of DCs under electronic microscope.There were many typical dendrites on cell surfaces,and plenty of mitochondria,microfilament in cytoplasm.Comparing with monocytes,the expression levels of related surface antigens were increased obviously(P＜0.01). Positive control HUVECs presented cobblestone,spindle,whirlpool-shaped and lumens structure,highly expressed vWF and CD144,and showed strong uptake of Dil-acetylated low-density lipoprotein(Dil-Ac-LDL).2.The effect of EC9706 supernatant on cell morphologyOn day 8,Control DCs became big and round,there were many typical dendrites on cell surfaces,some displayed suspension,iDCs induced group had slender branch, lumen-like structure,some displayed cord-arranged.There was no difference between mDCs induced group and control DCs.3.The expression of DC surface antigen CD86,CD11c and CD1a after induction of EC9706 supernatantFCM showed that the expression levels of CD86,CD1a and CD11c were decreased in iDCs induced group cells compared with the control DCs(P＜0.05),but the associated surface antigens expressed on cells of mDCs induced group were not different from the control DCs(P＞0.05).4.The expression of CD1a,CD144 and vWF pre-and post-induction of EC9706 supernatantRT-PCR showed that the expression of CD144 and vWF could not be detected and CD1a was very weak in monocytes.The CD1a expression decreased and the CD144 and vWF expression increased in iDCs induced group cells as compared with the control DCs.There was no CD144 or vWF expression in mDCs induced group cells and its control DCs,and only CD1a could be detected.Immunofluorescence analysis showed that the vWF expression level of iDCs induced group cells was greater than that in the control DCs(P＜0.05).However,there was no significant difference between mDCs induced group and its control DCs (P＞0.05).Western blot analysis demonstrated that CD144,vWF expression was higher in the iDCs induced group cells than its control DCs(P＜0.01).In both mDCs induced group cells and the control DCs,the CD144,vWF protein was not detected. Chapter 2 Study of EC9706 cell supernatant on the endothelial-like differentiation function of iDCs and mDCsMethods1.Detection of the tube-like structure formation of the cells of iDCs induced group and mDCs induced group on fibronectin-coated plates in vitro.2.Dil-Ac-LDL and India ink uptake assay of the PBMCs,cells of iDCs induced group and mDCs induced group.3.Transmission electron microscope(TEM) detection of the EC-specific structure of WP bodies in iDCs induced group cells and control DCs.4.Detection of T cell proliferation of iDCs induced group cells and killing effect of CTL on EC9706 cells by CCK-8 in vitro.Results1.The effect of EC9706 supernatant on EC associated functionsTube-like structure formation assay showed that the cells of iDCs induced group appeared in fusiform shape and had the tendency of vortex distribution in the first 24 h. They tended to form tube-like structure in the second 24 h which was very similar to the positive control HUVECs,However,the cells were distributed evenly in control DCs of iDCs induced group,mDCs induced group and its control DCs.Uptake assay showed that monocytes uptaked Indian ink and a few Dil-Ac-LDL. The cells of iDCs induced group did not uptake Indian ink,which excluded the possibility that monocytes and macrophages were mixed in with the induced cells.The increased uptake of Dil-Ac-LDL in cells of iDCs induced group.There was no difference between cells of mDCs induced group and its control DCs.WP body is the EC specific functional structure.TEM showed that WP bodies and plenty of mitochondria were presented in the cells of iDCs induced group,which was similar to HUVECs.No WP body was found in the control DCs.2.The effect of EC9706 supernatant on DC associated functionsComparing with control DCs,the T cell proliferation and CTL activity for killing EC9706 cells in iDCs induced group decreased obviously(P＜0.01),which showed that the microenviroment produced by EC9706 cell supernatant dramatically disabled the antigen presenting function of cells in iDCs induced group.Chapter 3 Effect of ESCC tissue homogenate supernatant on the endothelial-like differentiation of iDCsMethods1.Collection of fresh ESCC and peri-carcinoma tissues 10 cases to prepare homogenate supernatant by grinding method.2.Experimental grouping Control DCs:human peripheral blood monocytes were induced toward DCs by rhIL-4、rhGM-CSF and LPS.Carcinoma homogenate supernatant group:40%carcinoma homogenate supernatant was added at the end of day 2(iDCs) for 7 days induction,and induced cells were harvested at day 9. Peri-carcinoma homogenate supernatant group:40%peri-carcinoma homogenate supernatant was added at the end of day 2(iDCs) for 7 days induction.Positive control HUVECs.3.Western blotting detection of the expression of vWF,CD144 on cells of carcinoma homogenate supernatant group,peri- carcinoma homogenate supernatant group and control DCs.4.Detection of the expression of vWF on cells of carcinoma homogenate supernatant group,peri-carcinoma homogenate supernatant group and control DCs.5.Dil-Ac-LDL and India ink uptake assay of the ceils of carcinoma homogenate supernatant group,peri-carcinoma homogenate supernatant group and control DCs.6.Checking T cell proliferation of cells of carcinoma homogenate supernatant group, peri-carcinoma homogenate supernatant group and control DCs,and killing effect of CTL on EC9706 cells by CCK-8.Results1.The effect of ESSC carcinoma and peri-carcinoma homogenate supernatant on iDC morphologyOn day 8,cells of control DCs and peri-carcinoma homogenate supernatant group displayed round,some suspended,and there were many typical dendrites on cell surfaces.However,cells of carcinoma homogenate supernatant group displayed slender spindle.2.The expression of CD144 and vWF after the induction of carcinoma and peri-careinoma homogenate supernatantWestern blotting analysis demonstrated that the CD144,vWF expression was higher in the cells of carcinoma homogenate supernatant group than control DCs and peri-carcinoma group(P＜0.01).However,there was no difference between peri-carcinoma group and control DCs(P＞0.05).Immunofluorescence analysis showed that the vWF expression was stronger in the cells of carcinoma homogenate supernatant group than in control DCs and peri-carcinoma group(P＜0.05).There was no difference between peri-carcinoma group and control DCs(P＞0.05).3.The effect on uptake function of ECs after the induction of carcinoma and peri-carcinoma homogenate supernatantThe uptake of Dil-Ac-LDL in cells of carcinoma homogenate supernatan group was stronger than in peri-carcinoma group and control DCs.4.The effect of carcinoma and peri-carcinoma homogenate supernatant on antigen presentation of DCsComparing with control DCs and peri-carcinoma group,the T cell proliferation and CTL activity for killing EC9706 cells of carcinoma homogenate supernatant group decreased obviously(P＜0.01),there was no difference between peri-carcinoma group and control DCs(P＞0.05).PartⅡMechanisms of the ESCC microenvironment induction of the endothelial-like differentiation of iDCsChapter 1 Effect of VEGF-A on the endothelial-like differentiation of DCs Methods1.Experimental grouping Control DCs:human peripheral blood monocytes were induced toward DCs by rhIL-4、rhGM-CSF and LPS.VEGF-A iDCs induced group and mDCs induced group:20 ng/ml VEGF-A was added at the end of day 2(iDCs), and at the end of day 7(mDCs) respectively,after 7 days induction,induced cells were harvested at day 9 and day 14 respectively.Positive control HUVECs.2.FCM analysis of DC surface markers CD86,CD11c and CD1a on Cells of VEGF-A iDCs induced group and mDCs induced group3.Detection of the expression of CD1a,vWF and CD144 on cells of VEGF-A iDCs induced group and mDCs induced group by RT-PCR,Western blotting and Immunofluorescence.4.Dil-Ac-LDL and India ink uptake assay of the cells of VEGF-A iDCs induced group and mDCs induced group.5.TEM detection of the WP bodies in VEGF-A iDCs induced group cells and its controls.6.Detection of T cell proliferation of VEGF-A iDCs induced group cells and killing effect of CTL on EC9706 cells by CCK-8 in vitro.Results1.The effect of VEGF-A on cell morphologyThe cells of VEGF-A iDCs induced group grew slowly.On day 8,Control DCs became big and round,however,most cells of VEGF-A iDCs induced group had slender branch,spindle,lumen-like structure,some displayed cord-arranged.There was no difference between VEGF-A mDCs induced group and control DCs.2.The expression of DC surface antigen CD86,CD11c and CD1a after induction of VEGF-AFCM showed that the expression levels of CD86,CD1a and CD11c were decreased in VEGF-A iDCs induced group cells comparing with the control DCs (P＜0.05),but the surface antigens expressed in cells of VEGF-A mDCs induced group were not different from the control DCs(P＞0.05).3.The expression of CD1a,CD144 and vWF after induction of VEGF-ART-PCR,Western blotting and Immunofluorescence showed that The CD1a expression decreased and CD144 and vWF expression increased in VEGF-A iDCs induced group cells(P＜0.05).There was no difference between VEGF-A mDCs induced group and control DCs(P＞0.05).4.The effect of EC associated functions and antigen presentation of DCs after induction of VEGF-AUptake assay showed that the cells of VEGF-A iDCs induced group did not uptake Indian ink,but the uptake of Dil-Ac-LDL increased in cells of VEGF-A iDCs induced group.There was no difference between cells of mDCs induced group and its control DCs.TEM showed that WP bodies and plenty of mitochondria were present in the cells of VEGF-A iDCs induced group,which was similar to HUVECs.No WP body was found in the control DCs.Comparing with control DCs,the T cell proliferation and CTL activity for killing EC9706 cells of VEGF-A iDCs induced group decreased obviously(P＜0.01).Chapter 2 Effect of VEGF-A blocked on EC9706 cell supernatant inducing the endothelial-like differentiation of iDCsMethods1.Detection of the content of VEGF-A in EC9706 cell supernatant,esophageal carcinoma and peri-cancer tissues homogenate supernatant by ELISA.2.Experimental grouping Control DCs:human peripheral blood monocytes were induced toward DCs by rhIL-4、rhGM-CSF and LPS.EC9706 supernatant group: 40%EC9706 supernatant was added at the end of day 2(iDCs) for 7 days induction. Blocking VEGF-A group:40%EC9706 supernatant and VEGF-A antibody was added at the end of day 2(iDCs) for 7 days induction.Positive control HUVECs.3.After blocking VEGF-A,detecting the expression of vWF on cells of EC9706 supernatant group by Immunofluorescence.4.After blocking VEGF-A,detecting the expression of vWF on cells of EC9706 supematant group by Western blotting. 5.After blocking VEGF-A,detecting Dil-Ac-LDL uptake on cells of EC9706 supernatant group.Results1.The content of VEGF-A in the EC9706 cell supernatant and homogenate supematant of esophageal carcinoma was significantly higher than that of the peri-carcinoma homogenate supernatant(P＜0.05).2.Cell morphology:after blocking VEGF-A The cells of EC9706 supernatant group showed slender spindle,which was similar to HUVECs.Control DCs became big and round.There was almost no slender branch in cells of blocking VEGF-A group.3.The expression of CD144 and vWF after blocking VEGF-AImmunofluorescence showed that comparing with the cells of EC9706 supernatant group,vWF expression decreased in blocking VEGF-A group(P＜0.05).There was no difference between VEGF-A blocking Group and control DCs(P＞0.05).Western blotting showed that comparing with the cells of EC9706 supernatant group,CD144 and vWF expression decreased in blocking VEGF-A group(P＜0.01).4.The effect on uptake function of ECs after the induction of carcinoma and peri-carcinoma homogenate supernatantUptake assay showed that the uptake of Dil-Ac-LDL decreased in VEGF-A blocking group.There was no difference between blocking VEGF-A Group and control DCs.Chapter 3 MAPK/ERK1/2/CREB signal pathway activated by VEGF-A mediates endothelial-like differentiation of iDCs in the ESCC microenvironmentMethods1.Experimental grouping EC9706 supernatant group:40%EC9706 supernatant was added at the end of day 2(iDCs) for the induction of 0 min,15 min,30 min,60 min and 7 days respectively.VEGF-A group:20 ng/ml VEGF-A was added at the end of day 2(iDCs) for the induction of 0 min,15 min,30 min,60 min and 7 days respectively. PD98059 60min blocking group:after 50μM PD98059 1 h incubation,40%EC9706 supernatant or 20 ng/mlVEGF-A was added for 1 h induction.PD98059 7 d blocking group:EC9706 supernatant and 10μM PD98059 or VEGF-A and 10μM PD98059 was added for 7 days induction.2.Western blotting detection of the phospho-MAPK/ERK1/2 and phospho-CREB activated by EC9706 supernatant or VEGF-A.Detection of the level of p-ERK1/2 and p-CREB,and the expression of vWF and CD144 after PD98059 blocking.3.After PD98059 blocking,detection of the expression of vWF on cells of EC9706 supernatant group and VEGF-A group by Immunofluorescence.4.Immunofluorescence Double Staining(FITC,PI) detection of nuclear translocation of p-ERK1/2 actived by EC9706 supernatant or VEGF-A.5.After PD98059 blocking,detecting Dil-Ac-LDL uptake of cells of EC9706 supernatant group and VEGF-A group.6.After PD98059 blocking,detecting the level of p-ERK1/2 and p-CREB on cells of EC9706 supernatant group by Western blotting.7.EMSA detection of the CRE binding capacity of the activation of CREB in the nucleus of EC9706 supernatant group or VEGF-A group.Results1.The activation of MAPK/ERK1/2 and CREB during the endothelial-like differentiation of iDCsEC9706 supernatant or VEGF-A induced the phosphorylation of MAPK/ERK1 /2 and CREB in a time dependent manner.PD98059 is a selective inhibitor of MEK1/2. MEK1/2 is the upstream regulator of MAPK/ERK1/2 phosphorylation.With PD98059 pretreatment,the phosphorylation level of MAPK/ERK and CREB decreased obviously in EC9706 supernatant 60 min group or in VEGF-A 60 min group(P＜0.01).2.Inhibition of MAPK/ERK phosphorylation resulted in the inhibition of the endothelial-like differentiation of iDCsInhibition of MAPK/ERK phosphorylation by PD98059 was accompanied by morphological changes,a significantly decreased expression of CD144 and vWF (P＜0.01),and Dil-Ac-LDL uptake dramatically reduced in the cells of EC9706 supernatant group or VEGF-A group in the presence of PD98059.Thus,inhibition of MAPK/ERK1/2 phosphorylation by PD98059 resulted in the inhibition of the endothelial-like differentiation of iDCs which were induced by EC9706 supernatant or VEGF-A,both morphologically and functionally.3.Nuclear transiocation of phosphorylation of MAPK/ERK1/2Nuclear translocation following phosphorylation of MAPK/ERK1/2 is required to induce transcriptional changes leading to proliferation,survival and differentiation. iDCs incubated with EC9706 supernatant or VEGF-A for 7 days showed that the presence of phosphorylation of MAPK/ERK1/2(green) in the nuclei(red) of iDCs.In the presence of PD98059,however,EC9706 supernatant or VEGF-induced MAPK/ ERK1/2 phosphorylation as well as nuclear translocation were significantly inhibited on day 9.4.Effect of VEGF-A blocked on EC9706 cell supernatant inducing the endothelial-like differentiation of iDCsWestern blotting showed that comparing with EC9706 supernatant,the level of p-ERK1/2 and p-CREB decreased in VEGF-A blocking group(P＜0.01),which showed VEGF-A played a starter role during the process of MAPK/ERK1/2/CREB activated by EC9706 supernatant.5.Activation of CREB binding activity to CREEMSA showed that CREB activated by EC9706 supernatant or VEGF-A had high binding capacity of CRE DNA sequence.Promoter sequences upstream of CD144 and vWF gene contain the CRE element.Activation of CREB may bind CRE element to mediate CD144 and vWF transcription to up-regulate their expression levels.Conclusions1.EC9706 cell supernatant induces iDCs to differentiate from the DC pathway toward endothelial cells,and form endothelial-like cells.But it has no obvious influence on mDCs,which may bring forward evidence that the mDCs can be used relatively safely in an anti-ESCC vaccine.2.The ESCC tissue homogenate supernatant also induce the endothelial-like differentiation of iDCs,however,peri-carcinoma homogenate supernatant has no this effect,which shows that the endothelial-like differentiation of iDCs is induced by the ESCC microenvironment,which may be one of the mechanisms of ESCC immune escape.3.VEGF-A induces iDCs to up-regulate some EC markers and down-regulate some DC markers,simultaneously,EC morphology features and functions observed,and form endothelial-like cells.But VEGF-A doesn’t induce the endothelial-like differentiation of mDCs.4.EC9706 supernatant or VEGF-A induces the phosphorylation of MAPK/ERK1/2 and CREB in a time dependent manner,and the activation persists in whole induction process.The nuclear translocation of p-ERK1/2 activates CREB,may led to CD144, vWF gene transcription.5.MAPK/ERK1/2/CREB signal pathway mediates the endothelial-like differentiation of iDCs induced by EC9706 supernatant or VEGF-A.Blocking MEK by PD98059 results in the inhibition of MAPK/ERK1/2 phosphorylation,and inhibition of the endothelial-like differentiation of imDCs induced by EC9706 supernatant or VEGF-A.6.Blocking VEGF-A in EC9706 supernatant results in the inhibition of MAPK/ERK 1/2 with CREB phosphorylation,and inhibition of the endothelial-like differentiation of iDCs,which shows that VEGF-A in EC9706 supernatant is a starter to activate MAPK/ERK 1/2/CREB signal pathway,which mediates the endothelial-like differentiation of iDCs in the ESCC microenvironment.This study provids the new strategy to inhibite the endothelial-like differentiation of iDCs.更多还原