TNFR在下咽鳞状细胞癌中的表达及利用基因开关调控TNFR表达对下咽癌化疗、TNF治疗的增敏作用
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摘要
下咽癌（hypopharyngeal carcinoma）恶性度较高，邻近扩散、淋巴结转移都较早。下咽癌的病理分型以下咽鳞状细胞癌（hypopharyngealsquamous cell carcinoma，HPSCC）最多见。因为发病部位隐蔽，下咽癌很难早期发现，且易沿粘膜下扩散，临床发现的时候很多已经达到中晚期，能够接受手术的病例非常有限，大约只在60%左右，五年生存率大约为20%-40%。尽管现代医学飞速发展，诊疗设备、外科手术和放化疗等综合诊疗手段在不断改进，但在最近的二十余年里HPSCC的五年生存率并没有明显的改善和提高。开发和应用更加有效的肿瘤治疗手段，寻求更加有效的治疗策略对提高HPSCC的治疗水平至关重要。
已经证实在肿瘤发生、发展过程中，分子生物学机制发挥着至关重要的作用，因而越来越多的学者关注于基因治疗，试图从肿瘤的发生根源寻找遏制肿瘤的方法。人们理想的基因治疗是可以根据疾病的严重程度来调节治疗基因在特定器官内的表达水平。但是在基因治疗领域如何对特定基因定时、定量地调控并研究其不同表达水平下的作用是一个难题。依赖于热休克蛋白、激素或重金属等的传统基因表达诱导系统，存在着高本底、多嗜效应和环境污染等缺陷，限制了其应用和发展。Gossen等创建的基因开关系统，又称为四环素诱导表达开关系统（Tet-on/off系统）克服了传统诱导系统的不足。这种系统是通过原核大肠杆菌Tnol转座子调控元件，在真核细胞中定量并特异地控制外源基因表达的体系。该系统被认为是目前最理想的真核生物基因表达系统，具有高效、特异、严密等特点，在基因功能和治疗等领域已被国内、外学者广泛地应用。但是，尚未有HPSCC方面的相关研究报道。
TNF-α(tumor necrosis factor-α，以下简称TNF)于30年前被发现，是到目前为止发现的抗肿瘤活性最强的细胞因子，对多种肿瘤细胞具有选择性的细胞毒作用。TNF既能直接杀死肿瘤细胞，又能通过激活免疫系统发挥抗肿瘤作用。它的最显著的活性特征是可以在体内外特异的杀伤肿瘤细胞，而对正常细胞无明显的毒性作用。但是，由于TNF在体内的半衰期较短，低剂量的应用抗肿瘤效果不太显著，甚至在某些肿瘤，还有促进肿瘤细胞增殖的作用，要达到疗效水平，需持续大剂量注射，这通常会引起发热、寒战、疲劳、头痛、休克等全身症状，临床应用的效果不太理想，大大限制了TNF的使用范围。
TNF主要通过与两个受体：TNF受体1（tumor necrosis factor receptor
1，TNFR1）和TNF受体2（tumor necrosis factor receptor2，TNFR2）结合来发挥生物学作用。这两类受体均由信号肽、胞浆结构区、跨膜结构区和胞外结构区组成。两类受体的胞外区同源性为28%，胞内区没有同源性，提示两类受体介导的是不同的信号传递通路。TNFR1可产生两种相反的作用效应，它既可诱导产生TNF的毒性效应，促进细胞凋亡、坏死，又可以通过激活NF-κB促进增殖。TNFR2也具有两种作用途径，它既可以增强TNFR1引起的凋亡，又可独立诱导凋亡，还可以促进增殖。两种受体在什么情况下选择哪种通路，仍不完全清楚。有研究表明在细胞表面TNFR的表达水平变化可以改变细胞对TNF的敏感性，细胞表面的TNFR数目越高，对TNF的细胞毒性作用就越敏感，而对TNF作用不敏感的肿瘤细胞，细胞表面的TNF受体缺如或几乎观察不到，受体数目与肿瘤细胞敏感性呈正相关。可见，TNFR对机体的TNF作用具有重要的调节作用。
TNF与多种化疗药物具有协同作用。经研究表明TNF与化疗药物联合应用可明显增强软组织肉瘤、黑色素瘤、胃癌、肺癌等多种肿瘤的化疗效果。但是尚未有相关的研究在头颈部肿瘤中开展。并且，TNF与化疗药物协同作用的机理及调控机制未完全清楚。不管其确切的机制如何，在下咽癌的治疗过程中，如何提高肿瘤的杀伤作用并同时减少TNF和化疗药物的用量，是肿瘤治疗的核心问题。肿瘤细胞的TNFR表达水平对这一协同作用可能具有重要影响，与其相关的研究可能具有重要意义。
本研究用免疫组化和Western blot的方法检测TNFR1和TNFR2在HPSCC组织中的表达，分析TNFR表达与HPSCC临床病理因素的关系。通过慢病毒构建利用基因开关系统稳定高表达人TNFR2基因的下咽癌FaDu细胞系。研究TNF、顺铂（cisplatin，DDP）及二者联合作用对TNFR正常表达的下咽癌FaDu细胞增殖、凋亡的影响。观察当用中和抗体阻断TNFR1的作用、利用基因开关系统高表达TNFR2后、以及同时阻断TNFR1作用、高表达TNFR2后，TNF、DDP作用后，细胞系增殖、凋亡情况的变化，分析TNFR表达变化与TNF、DDP敏感性的关系，探讨下咽癌的TNF、DDP治疗抵抗与TNFR表达水平之间的关系，为临床减少化疗药物的用量，提高下咽癌的治疗水平提供理论基础和依据。研究分为三部分：
第一部分TNFR在下咽鳞状细胞癌中的表达及与临床病理因素的关系
目的：检测TNFR1和TNFR2在HPSCC组织中的表达，并且与临床诸病理因素进行相关分析，探讨TNFR的表达与HPSCC发生、发展的关系。
方法：
1用免疫组织化学SP染色的方法检测45例HPSCC组织及配对癌旁下咽粘膜组织中TNFR1和TNFR2的表达，并与临床病理因素进行分析。
2Western blot检测3例HPSCC患者癌组织和癌旁下咽粘膜组织中TNFR1及TNFR2的表达。
结果：
1免疫组织化学染色结果
1.1TNFR在HPSCC组织及癌旁组织中的表达：45例HPSCC癌组织中，TNFR1和TNFR2的阳性表达率均为100%。TNFR1在癌旁粘膜组织中的表达率为50%，TNFR2在癌旁下咽粘膜组织中的表达率为10%。与癌旁下咽粘膜组织比较，TNFR1与TNFR2在HPSCC癌组织中的阳性表达率均明显升高（P＜0.01），表达水平也显著高于癌旁下咽粘膜组织（P＜0.01）
1.2TNFR与HPSCC临床病理因素的关系：
在45例HPSCC病人中，TNFR1和TNFR2的AOD在60岁以下组和60岁以上组的差异经统计学分析无明显差异（TNFR1：t=0.125，P=0.901；TNFR2：t=0.149，P=0.883）。在37例男性病人和8例女性病人中，TNFR1和TNFR2的表达与性别无差异（TNFR1：t=0.060，P=0.952;TNFR2：t=0.959，P=0.343）。45例HPSCC中，30例为梨状窝型，10例环后区型，5例为下咽后壁区，TNFR1和TNFR2的表达在三种分型中的表达无统计学差异（TNFR1：F=0.169，P=0.845；TNFR2：F=1.888，P=0.164）。
TNFR1在13例高分化鳞癌中的AOD为0.192±0.051，在32例低分化鳞癌中的表达为0.239±0.029，两者之间有显著差异（t=3.822，P＜0.01）；TNFR1在T3-T4表达的AOD为0.239±0.030，明显高于其在T1-T2期的表达（AOD为0.178±0.044）两者之间有显著性差异（t=5.087，P＜0.01）；TNFR1在临床分期Ⅲ-Ⅳ级的AOD为0.241±0.029，表达高于II期的AOD为0.187±0.045，两者之间有显著统计学差异（t=4.751，P＜0.01）；TNFR1在淋巴结转移的病例中的表达0.241±0.030明显高于无淋巴结转移组0.194±0.046，差异具有统计学意义（t=4.189，P＜0.01）。TNFR1在HPSCC癌组织中的表达与肿瘤的分化程度、肿瘤的T分期、临床分期、淋巴结转移密切相关，而与性别、年龄、分型无关（P＞0.05）。
TNFR2在高分化鳞癌13例中AOD为0.226±0.055，在32例低分化鳞癌中的表达为0.205±0.040，在10例T1-2分期组织中表达的AOD为0.227±0.061，在T3-4期AOD为0.206±0.040，在临床分期为Ⅱ期的AOD为0.221±0.057，Ⅲ-Ⅳ0.207±0.040，在30例淋巴结转移的组织中表达的AOD为0.202±0.036，在15例无淋巴结转移的AOD为0.229±0.056。经统计学分析，TNFR2的表达与HPSCC患者的年龄、性别、肿瘤的生长部位、分化等级、临床分期、T分期均无相关性（P均＞0.05）。
2TNFR1与TNFR2的相关性分析
TNFR1和TNFR2的表达经统计学分析呈负相关（rs=-0.305），TNFR1与TNFR2的AOD比值与HPSCC的分化程度、T分期、临床分期、淋巴结转移密切相关，而与性别、年龄、分型无关（P均＞0.05）。TNFR1与TNFR2在HPSCC的发生、发展中存在一定的相互调节作用，其确切机制有待我们进一步研究。
3Western blot结果：
TNFR1在3例HPSCC癌组织中的表达为0.822±0.144，在3例癌旁下咽粘膜组织表达为0.080±0.104，TNFR1在癌组织中的表达明显高于在癌旁下咽粘膜组织的表达，两者之间有统计学差异（t=5.079，P＜0.05）。TNFR2在3例HPSCC癌组织中的表达为0.775±0.072，在3例癌旁粘膜组织中的表达为0.010±0.005，TNFR2在HPSCC癌组织中的表达显著高于癌旁粘膜组织中的表达，经统计学分析，两者之间有显著差异（t=17.333，P＜0.01）。
结论：TNFR1和TNFR2在HPSCC癌组织中的表达较癌旁下咽粘膜组织均明显升高。TNFR1的表达水平与肿瘤的病理分化程度、肿瘤T分期、临床分期和淋巴结转移密切相关；TNFR2的表达与肿瘤的临床病理因素无显著关系；TNFR1与TNFR2的表达呈负相关，两者的比值与HPSCC的临床病理因素呈正相关，说明TNFR1和TNFR2之间存在着一定的负调节关系。TNFR在HPSCC的发生、发展中发挥着重要的作用。
第二部分慢病毒介导的利用基因开关稳定高表达人TNFR2基因的下咽癌FaDu细胞系的建立
目的：构建慢病毒介导的利用基因开关系统稳定高表达人TNFR2基因的下咽癌FaDu细胞系，为后续研究调控TNFR表达对TNF、DDP作用的影响提供细胞模型。
方法：
1Tet-on-Puro-TNFR2慢病毒载体的构建
TNFR2基因序列调取引物由上海桑尼生物合成，TNFR2原始ORF（open reading frame, ORF）购自汉恒生物科技（上海）有限公司，PCR扩增TNFR2ORF序列，并将PCR产物与Tet-on系列慢病毒载体pHBTet-on-Puro同步XhoI和EcoRI双酶切，回收。将酶切的PCR产物与载体连接过夜后转化感受态细胞，转化后的TNFR2平板挑菌，菌液进行菌落PCR鉴定，将阳性克隆送上海桑尼生物技术有限公司测序。
2构建质粒的包装前准备
2.1慢病毒系统构成：三质粒Tet-on慢病毒系统包括慢病毒质粒pHBTet-on-Puro-TNFR2（对照为pHBTet-on-Puro空质粒,该Tet-on慢病毒系统购自上海汉恒生物科技有限公司），包装辅助质粒：pspax2、pMD2G，其中pHBTet-on-Puro-TNFR2具有Puromycin药物抗性，可以进行药物筛选；同时带有药物诱导下启动子UBC，可以通过强力霉素（Doxorubicin，Dox）诱导目的基因TNFR2的表达。
2.2慢病毒载体高纯度制备：将制备的慢病毒质粒pHBTet-on-Puro-TNFR2（对照为pHBTet-on-Puro空质粒）及其辅助包装原件载体质粒pspax2、pMD2G，分别进行高纯度无内毒素抽提，制备出高纯度的慢病毒质粒及包装辅助质粒。
3慢病毒包装
用LipoFiterTM脂质体介导慢病毒载体pHBTet-on-Puro-TNFR2（对照为pHBTet-on-Puro空质粒）与pspax2，pMD2G共转染293T细胞，进行慢病毒包装。72小时后收取病毒上清并进行超速离心浓缩，用500ulDMEM重悬慢病毒颗粒。
4稳定可调控表达TNFR2基因FaDu细胞系的构建
将浓缩的慢病毒颗粒感染人下咽癌FaDu细胞，嘌呤霉素筛选一周，得到稳定可调控表达TNFR2基因的下咽癌FaDu细胞。进一步扩增细胞并进行传代，保种，扩增及下一步检测实验。
5Tet-on调控TNFR2基因的稳定转染FaDu细胞系的验证
扩增后一部分细胞用于保种，一部分细胞加入2ug/ml的Doxorudicin诱导24小时之后，收集细胞蛋白，实时定量PCR和Western Blot检测转染前后FaDu细胞中TNFR2的mRNA及蛋白的表达。
结果：
1将质粒转化感受态细胞，挑取阳性克隆扩增后提取质粒，经测序分析，结果与实验设计一致。
2用最优筛选浓度为2.0μg/ml的嘌呤霉素筛选出稳定表达Tet-on系统的FaDu细胞克隆。
3经实时定量PCR检测稳定表达Tet-on-Puro-TNFR2下咽癌FaDu细胞与对照组在加入Dox诱导24小时后比较，TNFR2的mRNA水平明显增高，差异有显著统计学意义（P＜0.01）。
4Western blot法检测稳定表达Tet-on-Puro-TNFR2下咽癌FaDu细胞与对照组在加入Dox诱导24小时后比较，TNFR2的蛋白水平明显增高，差异有统计学意义（P＜0.01）。
结论：利用慢病毒载体成功构建利用Tet-on系统稳定高表达人TNFR2基因的下咽癌FaDu细胞系，为后续研究该基因的功能提供细胞模型。
第三部分利用基因开关调控TNFR表达对下咽癌化疗、TNF治疗的增敏作用
目的：观察在TNFR1和TNFR2正常表达的Fadu细胞，TNF和DDP对细胞增殖、凋亡的影响；在TNFR1正常作用，TNFR2高表达的Fadu细胞，TNF和顺铂对细胞增殖、凋亡的影响变化；以及在TNFR1作用被阻断后，TNF和DDP对细胞增殖、凋亡的影响变化；最后观察阻断TNFR1作用联合TNFR2高表达情况下，TNF和DDP对Fadu细胞增殖、凋亡的影响变化，分析TNFR1和TNFR2表达的变化对TNF和DDP作用敏感性的影响。
方法：
本部分主要研究人为调控TNFR1和TNFR2表达变化时，TNF、DDP以及二者联合作用情况下，Fadu细胞增殖、凋亡情况的变化。利用MTT法检测Fadu细胞的增殖抑制作用；流式细胞Annexin V/PI双染色法检测细胞凋亡情况。
1研究TNF、DDP以及两者联合作用对TNFR正常作用的Fadu细胞增殖、凋亡的影响。
2研究利用中和抗体阻断TNFR1作用后，TNF、DDP以及两者联合作用对Fadu细胞增殖、凋亡情况的影响变化。
3研究在TNFR1正常作用，TNFR2高表达后，TNF、DDP以及两者联合作用对Fadu细胞系增殖、凋亡情况的影响变化。
4研究高表达TNFR2联合阻断TNFR1作用后，TNF、DDP以及两者联合作用对Fadu细胞系增殖、凋亡情况的影响变化。
结果：
1MTT检测结果
1.1稳定表达Tet-on-Puro的Fadu细胞系（A组），TNF在低浓度（0.1，1，10ng/ml）时对Fadu细胞有促增殖作用，而高浓度（50-100ng/ml）具有抑制肿瘤生长的作用。Fadu细胞系对DDP作用抵抗，6μg/ml的DDP作用24h后，肿瘤细胞存活率为81.8%。TNF在低浓度与DDP联合应用即可明显增强DDP的抗肿瘤效果。
1.2稳定表达Tet-on-Puro的Fadu细胞系，利用中和抗体阻断TNFR1作用后（B组），可逆转小剂量TNF（0.1-10ng/ml）的促增殖作用（t=4.390，t=5.886，t=5.258，P均＜0.05），并且低浓度与DDP联合作用对肿瘤的抑制作用较A组增强，高剂量（50-100ng/ml）TNF单独或联合DDP应用，均与A组无显著差异（P均＞0.05）。
1.3稳定表达Tet-on-Puro-TNFR2的Fadu细胞系，Dox诱导TNFR2高表达（C组）后与A组相比，小剂量（0.1，1，10ng/ml）即对细胞具有增殖抑制作用，具有显著差异（P均＜0.01），高浓度（50-100ng/ml）的TNF作用无差异（P均＞0.05）。Dox诱导后，可提高各浓度TNF与DDP联合应用的抗肿瘤效果，与A组相比，有统计学差异（P均＜0.05）。
1.4稳定表达Tet-on-Puro-TNFR2的Fadu细胞系，同时高表达TNFR2和中和TNFR1作用（D组），与A组相比，TNF各浓度对Fadu细胞均有明显的增殖抑制作用（P均＜0.05）；与B、C组相比，除TNF浓度为0.1ng/ml无差异，其他浓度均有更明显的增殖抑制作用，差异有统计学意义（P均＜0.05）；TNF和DDP联合作用于D组细胞后，与A、B组相比，各浓度对Fadu细胞的增殖抑制作用均明显加强（P均＜0.05）；与C组相比，在TNF低浓度（0.1，1，10ng/ml）时联合DDP具有明显抑制作用，高浓度（50-100ng/ml）无显著差异。
2流式细胞术Annexin V/PI双染色法检测TNF、TNF联合DDP对各实验组FaDu细胞凋亡率的影响
2.1在10ng/ml的TNF作用下，B组与A组的凋亡率无显著差异（t=0.918，P=0.456），C组比A组凋亡率明显增高（t=13.504，P＜0.01）。D组与A、B组相比凋亡率明显升高，具有统计学差异（t=12.424，t=10.058，P＜0.05）；与C组相比，凋亡率无明显变化（t=0.624，P＞0.05）
2.210ng/ml的TNF和6μg/ml的DDP共同作用后，B组与A组凋亡率无显著差异；C组比A组凋亡率明显增高（t=8.498，P＜0.05）；D组与A组、B组相比，凋亡率明显升高（t=4.606，t=4.737，P＜0.05）；与C组相比，凋亡率无明显变化（t=0.795，P＞0.05）
结论：
1在TNFR正常表达的下咽癌Fadu细胞系， TNF在低浓度（0.1-10ng/ml）时对Fadu细胞有促增殖作用，而高浓度（50-100ng/ml）具有抑制肿瘤生长的作用。TNF在低浓度与DDP联合应用即可明显增强DDP的抗肿瘤效果。
2在下咽癌Fadu细胞系中和TNFR1作用后，可逆转低浓度TNF的促肿瘤增殖作用，并且可增强低浓度TNF与DDP联合应用的抗肿瘤效果。
3在下咽癌Fadu细胞系中，TNFR2的表达增高可增强低浓度TNF和各浓度TNF联合DDP对肿瘤的增殖抑制作用，并可促进肿瘤细胞凋亡。
4在下咽癌Fadu细胞系中，同时中和TNFR1作用和高表达TNFR2，可显著促进TNF和DDP对肿瘤细胞的增殖抑制作用，并促进肿瘤细胞凋亡。
5TNFR1在下咽癌中很可能介导增殖通路，TNFR2可能介导凋亡通路，两者的作用途径有待我们进一步证实。同时调控TNFR1和TNFR2的表达有望明显提高下咽癌的TNF和DDP的治疗效果，克服治疗抵抗。
Hypopharyngeal carcinoma is a kind of tumor with highly malignantproperties including easy diffusion to adjacent structures and early lymphnode metastasis. Squamous cell carcinoma (SCC）is the most commonpathological type. Because of its special biological characters, the prognosis ofhypopharyngeal SCC (HPSCC) is poor, with an overall5-year survival rate ofapproximately20%to40%. Surgical operation is still restricted to a subset ofabout60%of all cases because early-stage cases of HPSCC are difficult toidentify. Despite improvements in diagnostic tools, surgical techniques, andconcurrent chemo-radiation therapy, the5-year survival rate of HPSCCremains nearly unimproved in the past two decades. Therefore, it is urgent toseek more effective treatment strategies to improve the therapeutic outcomesof HPSCC.
It has been confirmed that the molecular biological aspects playimportant parts in the process of the tumorigenesis and progression of cancers.Nowadays, more and more researchers focus on gene therapy, trying toestablish methodologies to prevent cancers from occuring. The ideal genetherapy should reside on regulating target genes in specific organs accordingto the severity of the diseases. However, it is a diffcult problem on how toregulate the expression of a certain gene in accordance with the amount inneed at punctual time. The traditional gene inducible systems relying on thethermal shock protein systems, hormone systems and the heavy metalssystems have many drawbakcs. For example, they possess commonshortcomings like high background, polytrophic effect and environmentalpollution, etc. These disadvantages limit their applications and development.The gene inducible system created by Gossen, also known as Tetracyclin inducible gene expression system (Tet-on/off system), overcomes thedisadvantages of the traditional inducible systems. This system canspecifically regulate exogenous gene expression through E.coli Tnolregulatory elements. Tet system is considered to be the most ideal induciblegene expression system. It has many advantages such as high efficiency,specificity and restriction. It has been applied to many areas of research, suchas the study of gene function and the treatment of disease. However, therehave been are no relevant reports on study of HPSCC at present.
Tumor Necrosis Factor-α (TNF-α) was discovered30years ago. It isregarded as the strongest antitumor cytokine identified till present. TNF-αcankill the tumor cells directly or exert its antitumor effects by activating theimmune system, without influencing on normal cell functions simultaneously.However, the half-life of TNF-αis short. The low-dose TNF-αhas nosignificant antitumor effect but rather promote the cell proliferation in sometumors. Continuous injection of large doses of TNF-α for therapeuticpurposes would eventually cause fever, chills, fatigue, headache, shock andother side effects. Therefore, the clinical application of TNF-αin cancerpatients and its practical effects remains fairly questionable and very limited.
TNF-α exerts its biological effects via two known transmembranereceptors, TNFR1(p55) and TNFR2(p75), both of which are composed ofsignal peptide, cytoplasmic domains, transmembrane region and extracellulardomains. The cytoplasmic domains of TNFR1and TNFR2are obviouslydifferent, indicating that TNFR1and TNFR2mediate different intacellularsignaling pathways. Functionally, TNFR1can mediate both apoptosis andnecrosis, and promote the proliferation of cells as well by activating NF-κ B.TNFR2also mediate two pathways. It can either promote the apoptosisthrough or independent of TNFR1, or promote the proliferation of cells. Theproliferation or apoptosis pathways mediated by TNFR are still not fullyunderstood. Previous studies have shown that TNFR levels of cell surface canalter the sensitivity of cells to TNF-α. The higher the TNFR expression, themore cytotoxic effect of TNF-α is noticed. Therefore, TNFRs play important roles in the regulation of TNF-αeffects.
It has been reported that TNF-α has synergistic effects withchemotherapy drugs in induing cell death in many tumors, such as soft tissuesarcoma, melanoma, gastric cancer, lung cancer and some other malignancies.However, there have been no similar reports on head and neck cancers.Besides, the mechanism by which TNF-α and chemotherapy drug interactwith each other is not completely clear. In management of hypopharyngealcancer, how to reduce application dosage of TNF-α and chemotherapy drugsto avoid sides effects with simultaneous improvement of their treatmenteffects remains a core problem to solve. The expression levels of TNFRs oncertain tumor cells may determine the outcomes of above treatments.
In this study, we used immunohistochemistry and Western blot to detectthe expression of TNFR1and TNFR2in HPSCC tissues and analyzed therelationship between TNFR expression and associated clinicopathologicfactors. Stable Tet-on-TNFR2Fadu cell lines were established by lentiviraltransfection. The proliferation-inhibiting and apoptosis-inducing effects ofTNF-α and cisplatin (DDP) in cells with different TNFR-expression levelswere investigated, for a deep insight into relationship between TNF-αandDDP resistance of hypopharyngeal cancer and the expression of TNFRs. Thepresent investigation is divided into three parts as follows.
Part one: Expression of TNFR1and TNFR2and its clinocopathologicsignificance in HPSCC
Objective: To investigate TNFR1and TNFR2expression and thus therelationship between TNFR expression and clinicopathologic features inHPSCC, for preliminary definition of their roles in tumorigenesis anddevelopment.
Methods:
1The expression levels of TNFR1and TNFR2were detected byimmunohistochemical staining in45HPSCC specimens and correspondingtumor-adjacent hypopharyngeal tissues, and their relationships withclinicopathologic characteristics were evaluated.
2The expression of TNFR1and TNFR2was also detected by WesternBlot in3HPSCC specimens and tumor-adjacent hypopharyngeal tissues.
Results:
1Immunohistochemistry analysis.
1.1The expression of TNFR in HPSCC and paracancerous hypopharyngealmucosal tissues.
Positive staining of both TNFR1and TNFR2was seen in all45HPSCCspecimens, and there was no significant difference of the expression levelsbetween TNFR1and TNFR2among the45HPSCC samples. However,expression of TNFR1and TNFR2was observed in50%and10%of the pairedtumor-adjacent hypopharyngeal tissues, respectively. Further statisticalanalyses showed that the average optical density (AOD) values of bothTNFR1and TNFR2in HPSCC were significantly higher than those in normalhypopharyngeal tissues (P<0.01). These data indicate that the expression ofboth TNFR1and TNFR2is upregulated in HPSCC.
1.2Relationship between TNFR expression and clinicopathologic factors inHPSCC.
The data revealed that both TNFR1and TNFR2expression had nothingto do with age (TNFR1, t=0.125，P=0.901. TNFR2, t=0.149, P=0.883), sex(TNFR1, t=0.060, P=0.952. TNFR2, t=0.959，P=0.343) and tumor location(TNFR1, F=0.169, P=0.845. TNFR2, F=1.888, P=0.164).
However, TNFR1expression was significantly correlated withhistological grade (t=3.822，P＜0.01), tumor clinical stage (t=4.751，P＜0.01),T stage (t=5.087，P＜0.01), and lymph node metastasis (t=4.189，P＜0.01).We next analyzed the correlation of the expression status of TNFR2withclinicopathologic factors. Unlike TNFR1, TNFR2expression status was notsignificantly associated with any of the above mentioned clinicopathologicfactors in HPSCC(P＞0.05).
2Correlation of TNFR1expression with TNFR2expression in HPSCC.
The distinct correlation of the expression levels of TNFR1and TNFR2with the clinicopathologic factors suggests that there exist a correlation between the expression levels of TNFR1and TNFR2in HPSCC. Indeed, ourstatistical analyses demonstrated a reverse correlation between the expressionof TNFR1and TNFR2in HPSCC (rs=-0.305, P＜0.05). The ratio of TNFR1and TNFR2AOD was significantly correlated with histological grade, tumorclinical stage, T stage, and lymph node metastasis (all P＜0.01), and hadnothing to do with age, sex and tumor location(P＞0.05).
3Western blotting showed that compared with tumor-adjacenthypopharyngeal tissues, the protein expression of TNFR1(t=5.079，P＜0.05)and TNFR2(t=17.333，P＜0.01) were both significantly increased in HPSCC.
Conclusions: The expression of both TNFR1and TNFR2wassignificantly higher in HPSCC than in tumor-adjacent hypopharyngeal tissues.TNFR1expression and the ratio of TNFR1and TNFR2was significantlycorrelated with histological grade, tumor clinical stage, T stage, and lymphnode metastasis. But TNFR2expression status was not significantly associatedwith any of the clinicopathologic factors in HPSCC. There is a reversecorrelation between the expression of TNFR1and TNFR2in HPSCC. TNFRplay an important role in the occurrence and development of HPSCC.
Part two: Establishment of a stable Tet-on-TNFR2system cell linemediated by lentivirus in FaDu cell
Objective: To establish a stable Tet-on-TNFR2cell line transfected withlentivirus in FaDu cell, and to provide a cell line for further study on therelationship between TNFR expression and the sensitivity of TNF and DDP.
Methods:
1Construction of a Tet-on-TNFR2recombinant lentiviral vector
The open reading frame (ORF) sequences of TNFR2gene was purchasedfrom Shanghai Hanbio Biological Company. Amplification of TNFR2ORFsequence was carried out through PCR. Then, the products and Tet-onlentiviral vector carriers were subjected to XhoI and EcoRI double enzymedigestion. The plasmid acquired from enzyme-linked products was trasformedin competent cells overnight, after which time the formed bacteria clones werepicked and identified by PCR. The PCR products were sent for sequencing tests for final confirmation.
2Preparation before packaging plasmids
2.1The lentivirus system contains lentiviral plasmid pHBTet-on-Puro-TNFR2(empty vector control pHBTet-on-Puro), and auxiliary packaging plasmidspspax2and pMD2G. Among them, pHBTet-on-Puro-TNFR2with Puromycinresistance and drug-induced UBC promoter can induce the TNFR2geneexpression by Doxorubicin(Dox).2.2The lentiviral plasmid (pHBTet-on-Puro-TNFR2) preparation plasmidpspax2and pMD2G were extracted without endotoxin to obtain the highpurity lentiviral vector and auxiliary packaging plasmids.3The lentivirus vectors pHBTet-on-Puro-TNFR2and lentivirus auxiliarypackaging plasmids were co-transfected into293T cells for72h. Then thesupernatant was collected, concentrated and resuspended with500ul DMEMfor acquisition of infection viral paticles.4FaDu cells were infected with lentivirus vectors. Puromycin was used toselect the stable Tet-on-Puro-TNFR2viral system infected cells for a week.5The stable Tet-on-Puro-TNFR2cells were detected by quantitativereal-time PCR (QPCR) and Western blotting.
Results:
1Though identification the recombinant plasmid sequence wasconsistent with the experimental design.
2Stable Tet-on-Puro-TNFR2FaDu cell clones were selected by2.0μg/ml of puromycin.
3QPCR showed that the mRNA level of TNFR2in Tet-on-Puro FaDucell without induction by Dox was0.126±0.046. The mRNA level of TNFR2in Tet-on-Puro FaDu cells induced by Dox for24h was0.215±0.124. Therewas no significant difference between the two groups (t=1.606, P＞0.05). ThemRNA level of TNFR2in stable Tet-on-Puro-TNFR2FaDu cells withoutinduction by Dox was0.149±0.056. Wheras, the mRNA level of TNFR2was3.518±0.733in the same cells induced by Dox for24h. The mRNA level ofTNFR2was significantly increased in stable Tet-on-Puro-TNFR2FaDu cell (t=8.125, P＜0.05) after Dox induction.
4Western blot showed that TNFR2protein was significantly increasedin stable Tet-on-Puro-TNFR2FaDu cells stimulated with Dox for24h than innomal Fadu cells with or without Dox induction and Tet-on-Puro FaDu cellswith or without Dox stimulation.
Conclusions:
The stable Tet-on-Puro-TNFR2Fadu cell line was successfullyestablished by lentivirus transfection, which provides a new cell line forfurther investigation on the function of TNFRs.Part Three: Regulation of TNFR expression by Tet-on system to increase
the sensitization of FaDu cells to TNF and DDP
Objective: To observe the proliferation and apoptosis rates induced bycisplatin (DDP) and TNF-ɑ in Fadu cells and changes in the proliferation andapoptosis rates after neutralizing the effect of TNFR1or up-regulating TNFR2expression by Tet-on system for definition of the relationship between theTNFR expression and sensitivity to TNF-ɑ and DDP.
Methods:
This part of investigation mainly foucused on evaluation of proliferationand apoptosis changes in Fadu cells when TNF-ɑ and DDP were applicatedunder different TNFR expression conditions. Inhibition of proliferation wasdetected by MTT method. The apoptosis rate was detected by flow cytometrythrough Annexin V/PI double staining.
1The study of proliferation-inhibiting and apoptosis-inducing effects ofTNF-ɑ, DDP and their combinations in nomal TNFR-expressing Fadu cells.
2The study of difference in proliferation inhibition and apoptosisinduced by TNF-ɑ, DDP or both combined in Fadu cells after neutralizingTNFR1.
3The study of changes in proliferation and apoptosis rates induced byTNF-ɑ, DDP and both combined upon Fadu cells after upregulating theexpression of TNFR2by Tet-on systerm.
4The study of changes in proliferation and apoptosis induced by TNF-ɑ, DDP and both combined after upregulating the expression of TNFR2by Tet-on systerm and neutralizing TNFR1with the blocking antibody.
Results:
1The results of MTT
1.1TNF-ɑ can promote cell proliferation at low concentrations (0.1-10ng/ml)and can inhibit the tumor growth at high concentrations (50-100ng/ml) instable Tet-on-Puro Fadu cell (Group A). TNF-ɑ combined with DDP cansignificantly enhance the antitumor effect of DDP.
1.2In stable Tet-on-Puro Fadu cells, neutralizing TNFR1with its blockingantibody (Group B) can reverse the proliferation-promoting effects oflow-dose (0.1-10ng/ml) TNF-ɑ prominently (t=4.390，t=5.886，t=5.258, all P<0.05); high-dose TNF-ɑ (50-100ng/ml) induced no significant difference inproliferation inhibition compared to Group A (t=2.657, t=1.083, t=1.113, allP>0.05). TNF-ɑ in low concentration combined with DDP can significantlyenhance the antitumor effect of DDP, but high concentration was nosignificant difference compared with Group A (P>0.05).
1.3In stable Tet-on-Puro-TNFR2Fadu cells induced by Dox (Group C), lowdoses of TNF-ɑ can inhibit the proliferation of cells significantly compared toGroup A (P <0.01). The effects of high concentration TNF-ɑ (50-100ng/ml)were of no significant difference with Group A (P>0.05). In this group, theeffect of each concentration TNF-ɑ combined with DDP increased. The effectwas statistically difference (P <0.05).
1.4In stable Tet-on-Puro-TNFR2expressing Fadu cells induced by Dox,with simultaneous neutralization of TNFR1(Group D), TNF-ɑ cansignificantly inhibit the proliferation of cells compared with Group A, GroupB (except TNF-ɑ0.1ng/ml) and C (except TNF-ɑ0.1ng/ml). The antitumoreffects of TNF-ɑ combined with DDP were significantly greater compared tothe three groups (all P＜0.05), except for that of TNF-ɑ at50-100ng/ml inGroup C.
2The apoptosis results by Annexin V/PI double staining in flow cytometry
2.1The apoptosis rate induced by10ng/ml TNF-ɑ revealed no significant difference (t=0.918，P＞0.05) between Group B and Group A. The apoptosisrate was obviously increased in Group C compared to Group A (t=13.504，P＜0.01). The apoptosis rate was significantly increased in Group D comparedto Group A and Group B (t=12.424，t=10.058，P＜0.05). But there was nosignificant change in apoptosis rates compared to Group C (t=0.624，P＞0.05).
2.2The apoptosis rate induced by10ng/ml TNF-ɑ and6μg/ml DDP bearedno significant difference between Group B and Group A(all P＞0.05).Apoptosis rate in Group C increased significantly compared to Group A(t=8.498，P＜0.05). The apoptosis rate of Group D was significantly higherthan Group A and Group B (all P<0.05). There was no significant differencein apoptosis rate in comparison with Group C (t=0.795，P＞0.05).
Conclusions:
1In Fadu cells without changes in TNFR expression, TNF-ɑ inducescell proliferation at low concentrations (0.1-10ng/ml), while highconcentration TNF-ɑ (50-100ng/ml) exhibits effects of proliferation inhibitionon tumor cells. TNF-ɑ and DDP have a synergistic effect in inducingproliferation inhibition.
2Neutralizatiion of TNFR1with its blocking antibody reversesproliferation-promoting effects of TNF-ɑ and can increase the antitumoreffects of TNF-ɑ combined with DDP at low concentrations, but does notincrease the apoptosis rate induced by TNF-ɑ and/or DDP in Fadu cells.
3Upregulation of TNFR2in Fadu cells obviously promotes theproliferation inhibition induced by TNF-ɑ at low concentrations and TNF-ɑ ateach concentrations plus DDP, and can obviously promotes apoptosis inducedby TNF-ɑ and TNF-ɑ plus DDP.
4Upregulation of TNFR2with simultaneous neutralization of TNFR1in Fadu cells can significantly increase the therapeutic effect of TNF-ɑ andDDP.
5In hypopharyngeal carcinoma Fadu cells, it is highly likely thatTNFR1mediates proliferation pathway whereas TNFR2mediates apoptosispathway. Regulating the expression levels of TNFR1and TNFR2may shed light on improving the therapeutic effects of TNF-ɑ and DDP throughinterfering with the crosstalk between the two receptors.
已经证实在肿瘤发生、发展过程中，分子生物学机制发挥着至关重要的作用，因而越来越多的学者关注于基因治疗，试图从肿瘤的发生根源寻找遏制肿瘤的方法。人们理想的基因治疗是可以根据疾病的严重程度来调节治疗基因在特定器官内的表达水平。但是在基因治疗领域如何对特定基因定时、定量地调控并研究其不同表达水平下的作用是一个难题。依赖于热休克蛋白、激素或重金属等的传统基因表达诱导系统，存在着高本底、多嗜效应和环境污染等缺陷，限制了其应用和发展。Gossen等创建的基因开关系统，又称为四环素诱导表达开关系统（Tet-on/off系统）克服了传统诱导系统的不足。这种系统是通过原核大肠杆菌Tnol转座子调控元件，在真核细胞中定量并特异地控制外源基因表达的体系。该系统被认为是目前最理想的真核生物基因表达系统，具有高效、特异、严密等特点，在基因功能和治疗等领域已被国内、外学者广泛地应用。但是，尚未有HPSCC方面的相关研究报道。
TNF-α(tumor necrosis factor-α，以下简称TNF)于30年前被发现，是到目前为止发现的抗肿瘤活性最强的细胞因子，对多种肿瘤细胞具有选择性的细胞毒作用。TNF既能直接杀死肿瘤细胞，又能通过激活免疫系统发挥抗肿瘤作用。它的最显著的活性特征是可以在体内外特异的杀伤肿瘤细胞，而对正常细胞无明显的毒性作用。但是，由于TNF在体内的半衰期较短，低剂量的应用抗肿瘤效果不太显著，甚至在某些肿瘤，还有促进肿瘤细胞增殖的作用，要达到疗效水平，需持续大剂量注射，这通常会引起发热、寒战、疲劳、头痛、休克等全身症状，临床应用的效果不太理想，大大限制了TNF的使用范围。
TNF主要通过与两个受体：TNF受体1（tumor necrosis factor receptor
1，TNFR1）和TNF受体2（tumor necrosis factor receptor2，TNFR2）结合来发挥生物学作用。这两类受体均由信号肽、胞浆结构区、跨膜结构区和胞外结构区组成。两类受体的胞外区同源性为28%，胞内区没有同源性，提示两类受体介导的是不同的信号传递通路。TNFR1可产生两种相反的作用效应，它既可诱导产生TNF的毒性效应，促进细胞凋亡、坏死，又可以通过激活NF-κB促进增殖。TNFR2也具有两种作用途径，它既可以增强TNFR1引起的凋亡，又可独立诱导凋亡，还可以促进增殖。两种受体在什么情况下选择哪种通路，仍不完全清楚。有研究表明在细胞表面TNFR的表达水平变化可以改变细胞对TNF的敏感性，细胞表面的TNFR数目越高，对TNF的细胞毒性作用就越敏感，而对TNF作用不敏感的肿瘤细胞，细胞表面的TNF受体缺如或几乎观察不到，受体数目与肿瘤细胞敏感性呈正相关。可见，TNFR对机体的TNF作用具有重要的调节作用。
TNF与多种化疗药物具有协同作用。经研究表明TNF与化疗药物联合应用可明显增强软组织肉瘤、黑色素瘤、胃癌、肺癌等多种肿瘤的化疗效果。但是尚未有相关的研究在头颈部肿瘤中开展。并且，TNF与化疗药物协同作用的机理及调控机制未完全清楚。不管其确切的机制如何，在下咽癌的治疗过程中，如何提高肿瘤的杀伤作用并同时减少TNF和化疗药物的用量，是肿瘤治疗的核心问题。肿瘤细胞的TNFR表达水平对这一协同作用可能具有重要影响，与其相关的研究可能具有重要意义。
本研究用免疫组化和Western blot的方法检测TNFR1和TNFR2在HPSCC组织中的表达，分析TNFR表达与HPSCC临床病理因素的关系。通过慢病毒构建利用基因开关系统稳定高表达人TNFR2基因的下咽癌FaDu细胞系。研究TNF、顺铂（cisplatin，DDP）及二者联合作用对TNFR正常表达的下咽癌FaDu细胞增殖、凋亡的影响。观察当用中和抗体阻断TNFR1的作用、利用基因开关系统高表达TNFR2后、以及同时阻断TNFR1作用、高表达TNFR2后，TNF、DDP作用后，细胞系增殖、凋亡情况的变化，分析TNFR表达变化与TNF、DDP敏感性的关系，探讨下咽癌的TNF、DDP治疗抵抗与TNFR表达水平之间的关系，为临床减少化疗药物的用量，提高下咽癌的治疗水平提供理论基础和依据。研究分为三部分：
第一部分TNFR在下咽鳞状细胞癌中的表达及与临床病理因素的关系
目的：检测TNFR1和TNFR2在HPSCC组织中的表达，并且与临床诸病理因素进行相关分析，探讨TNFR的表达与HPSCC发生、发展的关系。
方法：
1用免疫组织化学SP染色的方法检测45例HPSCC组织及配对癌旁下咽粘膜组织中TNFR1和TNFR2的表达，并与临床病理因素进行分析。
2Western blot检测3例HPSCC患者癌组织和癌旁下咽粘膜组织中TNFR1及TNFR2的表达。
结果：
1免疫组织化学染色结果
1.1TNFR在HPSCC组织及癌旁组织中的表达：45例HPSCC癌组织中，TNFR1和TNFR2的阳性表达率均为100%。TNFR1在癌旁粘膜组织中的表达率为50%，TNFR2在癌旁下咽粘膜组织中的表达率为10%。与癌旁下咽粘膜组织比较，TNFR1与TNFR2在HPSCC癌组织中的阳性表达率均明显升高（P＜0.01），表达水平也显著高于癌旁下咽粘膜组织（P＜0.01）
1.2TNFR与HPSCC临床病理因素的关系：
在45例HPSCC病人中，TNFR1和TNFR2的AOD在60岁以下组和60岁以上组的差异经统计学分析无明显差异（TNFR1：t=0.125，P=0.901；TNFR2：t=0.149，P=0.883）。在37例男性病人和8例女性病人中，TNFR1和TNFR2的表达与性别无差异（TNFR1：t=0.060，P=0.952;TNFR2：t=0.959，P=0.343）。45例HPSCC中，30例为梨状窝型，10例环后区型，5例为下咽后壁区，TNFR1和TNFR2的表达在三种分型中的表达无统计学差异（TNFR1：F=0.169，P=0.845；TNFR2：F=1.888，P=0.164）。
TNFR1在13例高分化鳞癌中的AOD为0.192±0.051，在32例低分化鳞癌中的表达为0.239±0.029，两者之间有显著差异（t=3.822，P＜0.01）；TNFR1在T3-T4表达的AOD为0.239±0.030，明显高于其在T1-T2期的表达（AOD为0.178±0.044）两者之间有显著性差异（t=5.087，P＜0.01）；TNFR1在临床分期Ⅲ-Ⅳ级的AOD为0.241±0.029，表达高于II期的AOD为0.187±0.045，两者之间有显著统计学差异（t=4.751，P＜0.01）；TNFR1在淋巴结转移的病例中的表达0.241±0.030明显高于无淋巴结转移组0.194±0.046，差异具有统计学意义（t=4.189，P＜0.01）。TNFR1在HPSCC癌组织中的表达与肿瘤的分化程度、肿瘤的T分期、临床分期、淋巴结转移密切相关，而与性别、年龄、分型无关（P＞0.05）。
TNFR2在高分化鳞癌13例中AOD为0.226±0.055，在32例低分化鳞癌中的表达为0.205±0.040，在10例T1-2分期组织中表达的AOD为0.227±0.061，在T3-4期AOD为0.206±0.040，在临床分期为Ⅱ期的AOD为0.221±0.057，Ⅲ-Ⅳ0.207±0.040，在30例淋巴结转移的组织中表达的AOD为0.202±0.036，在15例无淋巴结转移的AOD为0.229±0.056。经统计学分析，TNFR2的表达与HPSCC患者的年龄、性别、肿瘤的生长部位、分化等级、临床分期、T分期均无相关性（P均＞0.05）。
2TNFR1与TNFR2的相关性分析
TNFR1和TNFR2的表达经统计学分析呈负相关（rs=-0.305），TNFR1与TNFR2的AOD比值与HPSCC的分化程度、T分期、临床分期、淋巴结转移密切相关，而与性别、年龄、分型无关（P均＞0.05）。TNFR1与TNFR2在HPSCC的发生、发展中存在一定的相互调节作用，其确切机制有待我们进一步研究。
3Western blot结果：
TNFR1在3例HPSCC癌组织中的表达为0.822±0.144，在3例癌旁下咽粘膜组织表达为0.080±0.104，TNFR1在癌组织中的表达明显高于在癌旁下咽粘膜组织的表达，两者之间有统计学差异（t=5.079，P＜0.05）。TNFR2在3例HPSCC癌组织中的表达为0.775±0.072，在3例癌旁粘膜组织中的表达为0.010±0.005，TNFR2在HPSCC癌组织中的表达显著高于癌旁粘膜组织中的表达，经统计学分析，两者之间有显著差异（t=17.333，P＜0.01）。
结论：TNFR1和TNFR2在HPSCC癌组织中的表达较癌旁下咽粘膜组织均明显升高。TNFR1的表达水平与肿瘤的病理分化程度、肿瘤T分期、临床分期和淋巴结转移密切相关；TNFR2的表达与肿瘤的临床病理因素无显著关系；TNFR1与TNFR2的表达呈负相关，两者的比值与HPSCC的临床病理因素呈正相关，说明TNFR1和TNFR2之间存在着一定的负调节关系。TNFR在HPSCC的发生、发展中发挥着重要的作用。
第二部分慢病毒介导的利用基因开关稳定高表达人TNFR2基因的下咽癌FaDu细胞系的建立
目的：构建慢病毒介导的利用基因开关系统稳定高表达人TNFR2基因的下咽癌FaDu细胞系，为后续研究调控TNFR表达对TNF、DDP作用的影响提供细胞模型。
方法：
1Tet-on-Puro-TNFR2慢病毒载体的构建
TNFR2基因序列调取引物由上海桑尼生物合成，TNFR2原始ORF（open reading frame, ORF）购自汉恒生物科技（上海）有限公司，PCR扩增TNFR2ORF序列，并将PCR产物与Tet-on系列慢病毒载体pHBTet-on-Puro同步XhoI和EcoRI双酶切，回收。将酶切的PCR产物与载体连接过夜后转化感受态细胞，转化后的TNFR2平板挑菌，菌液进行菌落PCR鉴定，将阳性克隆送上海桑尼生物技术有限公司测序。
2构建质粒的包装前准备
2.1慢病毒系统构成：三质粒Tet-on慢病毒系统包括慢病毒质粒pHBTet-on-Puro-TNFR2（对照为pHBTet-on-Puro空质粒,该Tet-on慢病毒系统购自上海汉恒生物科技有限公司），包装辅助质粒：pspax2、pMD2G，其中pHBTet-on-Puro-TNFR2具有Puromycin药物抗性，可以进行药物筛选；同时带有药物诱导下启动子UBC，可以通过强力霉素（Doxorubicin，Dox）诱导目的基因TNFR2的表达。
2.2慢病毒载体高纯度制备：将制备的慢病毒质粒pHBTet-on-Puro-TNFR2（对照为pHBTet-on-Puro空质粒）及其辅助包装原件载体质粒pspax2、pMD2G，分别进行高纯度无内毒素抽提，制备出高纯度的慢病毒质粒及包装辅助质粒。
3慢病毒包装
用LipoFiterTM脂质体介导慢病毒载体pHBTet-on-Puro-TNFR2（对照为pHBTet-on-Puro空质粒）与pspax2，pMD2G共转染293T细胞，进行慢病毒包装。72小时后收取病毒上清并进行超速离心浓缩，用500ulDMEM重悬慢病毒颗粒。
4稳定可调控表达TNFR2基因FaDu细胞系的构建
将浓缩的慢病毒颗粒感染人下咽癌FaDu细胞，嘌呤霉素筛选一周，得到稳定可调控表达TNFR2基因的下咽癌FaDu细胞。进一步扩增细胞并进行传代，保种，扩增及下一步检测实验。
5Tet-on调控TNFR2基因的稳定转染FaDu细胞系的验证
扩增后一部分细胞用于保种，一部分细胞加入2ug/ml的Doxorudicin诱导24小时之后，收集细胞蛋白，实时定量PCR和Western Blot检测转染前后FaDu细胞中TNFR2的mRNA及蛋白的表达。
结果：
1将质粒转化感受态细胞，挑取阳性克隆扩增后提取质粒，经测序分析，结果与实验设计一致。
2用最优筛选浓度为2.0μg/ml的嘌呤霉素筛选出稳定表达Tet-on系统的FaDu细胞克隆。
3经实时定量PCR检测稳定表达Tet-on-Puro-TNFR2下咽癌FaDu细胞与对照组在加入Dox诱导24小时后比较，TNFR2的mRNA水平明显增高，差异有显著统计学意义（P＜0.01）。
4Western blot法检测稳定表达Tet-on-Puro-TNFR2下咽癌FaDu细胞与对照组在加入Dox诱导24小时后比较，TNFR2的蛋白水平明显增高，差异有统计学意义（P＜0.01）。
结论：利用慢病毒载体成功构建利用Tet-on系统稳定高表达人TNFR2基因的下咽癌FaDu细胞系，为后续研究该基因的功能提供细胞模型。
第三部分利用基因开关调控TNFR表达对下咽癌化疗、TNF治疗的增敏作用
目的：观察在TNFR1和TNFR2正常表达的Fadu细胞，TNF和DDP对细胞增殖、凋亡的影响；在TNFR1正常作用，TNFR2高表达的Fadu细胞，TNF和顺铂对细胞增殖、凋亡的影响变化；以及在TNFR1作用被阻断后，TNF和DDP对细胞增殖、凋亡的影响变化；最后观察阻断TNFR1作用联合TNFR2高表达情况下，TNF和DDP对Fadu细胞增殖、凋亡的影响变化，分析TNFR1和TNFR2表达的变化对TNF和DDP作用敏感性的影响。
方法：
本部分主要研究人为调控TNFR1和TNFR2表达变化时，TNF、DDP以及二者联合作用情况下，Fadu细胞增殖、凋亡情况的变化。利用MTT法检测Fadu细胞的增殖抑制作用；流式细胞Annexin V/PI双染色法检测细胞凋亡情况。
1研究TNF、DDP以及两者联合作用对TNFR正常作用的Fadu细胞增殖、凋亡的影响。
2研究利用中和抗体阻断TNFR1作用后，TNF、DDP以及两者联合作用对Fadu细胞增殖、凋亡情况的影响变化。
3研究在TNFR1正常作用，TNFR2高表达后，TNF、DDP以及两者联合作用对Fadu细胞系增殖、凋亡情况的影响变化。
4研究高表达TNFR2联合阻断TNFR1作用后，TNF、DDP以及两者联合作用对Fadu细胞系增殖、凋亡情况的影响变化。
结果：
1MTT检测结果
1.1稳定表达Tet-on-Puro的Fadu细胞系（A组），TNF在低浓度（0.1，1，10ng/ml）时对Fadu细胞有促增殖作用，而高浓度（50-100ng/ml）具有抑制肿瘤生长的作用。Fadu细胞系对DDP作用抵抗，6μg/ml的DDP作用24h后，肿瘤细胞存活率为81.8%。TNF在低浓度与DDP联合应用即可明显增强DDP的抗肿瘤效果。
1.2稳定表达Tet-on-Puro的Fadu细胞系，利用中和抗体阻断TNFR1作用后（B组），可逆转小剂量TNF（0.1-10ng/ml）的促增殖作用（t=4.390，t=5.886，t=5.258，P均＜0.05），并且低浓度与DDP联合作用对肿瘤的抑制作用较A组增强，高剂量（50-100ng/ml）TNF单独或联合DDP应用，均与A组无显著差异（P均＞0.05）。
1.3稳定表达Tet-on-Puro-TNFR2的Fadu细胞系，Dox诱导TNFR2高表达（C组）后与A组相比，小剂量（0.1，1，10ng/ml）即对细胞具有增殖抑制作用，具有显著差异（P均＜0.01），高浓度（50-100ng/ml）的TNF作用无差异（P均＞0.05）。Dox诱导后，可提高各浓度TNF与DDP联合应用的抗肿瘤效果，与A组相比，有统计学差异（P均＜0.05）。
1.4稳定表达Tet-on-Puro-TNFR2的Fadu细胞系，同时高表达TNFR2和中和TNFR1作用（D组），与A组相比，TNF各浓度对Fadu细胞均有明显的增殖抑制作用（P均＜0.05）；与B、C组相比，除TNF浓度为0.1ng/ml无差异，其他浓度均有更明显的增殖抑制作用，差异有统计学意义（P均＜0.05）；TNF和DDP联合作用于D组细胞后，与A、B组相比，各浓度对Fadu细胞的增殖抑制作用均明显加强（P均＜0.05）；与C组相比，在TNF低浓度（0.1，1，10ng/ml）时联合DDP具有明显抑制作用，高浓度（50-100ng/ml）无显著差异。
2流式细胞术Annexin V/PI双染色法检测TNF、TNF联合DDP对各实验组FaDu细胞凋亡率的影响
2.1在10ng/ml的TNF作用下，B组与A组的凋亡率无显著差异（t=0.918，P=0.456），C组比A组凋亡率明显增高（t=13.504，P＜0.01）。D组与A、B组相比凋亡率明显升高，具有统计学差异（t=12.424，t=10.058，P＜0.05）；与C组相比，凋亡率无明显变化（t=0.624，P＞0.05）
2.210ng/ml的TNF和6μg/ml的DDP共同作用后，B组与A组凋亡率无显著差异；C组比A组凋亡率明显增高（t=8.498，P＜0.05）；D组与A组、B组相比，凋亡率明显升高（t=4.606，t=4.737，P＜0.05）；与C组相比，凋亡率无明显变化（t=0.795，P＞0.05）
结论：
1在TNFR正常表达的下咽癌Fadu细胞系， TNF在低浓度（0.1-10ng/ml）时对Fadu细胞有促增殖作用，而高浓度（50-100ng/ml）具有抑制肿瘤生长的作用。TNF在低浓度与DDP联合应用即可明显增强DDP的抗肿瘤效果。
2在下咽癌Fadu细胞系中和TNFR1作用后，可逆转低浓度TNF的促肿瘤增殖作用，并且可增强低浓度TNF与DDP联合应用的抗肿瘤效果。
3在下咽癌Fadu细胞系中，TNFR2的表达增高可增强低浓度TNF和各浓度TNF联合DDP对肿瘤的增殖抑制作用，并可促进肿瘤细胞凋亡。
4在下咽癌Fadu细胞系中，同时中和TNFR1作用和高表达TNFR2，可显著促进TNF和DDP对肿瘤细胞的增殖抑制作用，并促进肿瘤细胞凋亡。
5TNFR1在下咽癌中很可能介导增殖通路，TNFR2可能介导凋亡通路，两者的作用途径有待我们进一步证实。同时调控TNFR1和TNFR2的表达有望明显提高下咽癌的TNF和DDP的治疗效果，克服治疗抵抗。
Hypopharyngeal carcinoma is a kind of tumor with highly malignantproperties including easy diffusion to adjacent structures and early lymphnode metastasis. Squamous cell carcinoma (SCC）is the most commonpathological type. Because of its special biological characters, the prognosis ofhypopharyngeal SCC (HPSCC) is poor, with an overall5-year survival rate ofapproximately20%to40%. Surgical operation is still restricted to a subset ofabout60%of all cases because early-stage cases of HPSCC are difficult toidentify. Despite improvements in diagnostic tools, surgical techniques, andconcurrent chemo-radiation therapy, the5-year survival rate of HPSCCremains nearly unimproved in the past two decades. Therefore, it is urgent toseek more effective treatment strategies to improve the therapeutic outcomesof HPSCC.
It has been confirmed that the molecular biological aspects playimportant parts in the process of the tumorigenesis and progression of cancers.Nowadays, more and more researchers focus on gene therapy, trying toestablish methodologies to prevent cancers from occuring. The ideal genetherapy should reside on regulating target genes in specific organs accordingto the severity of the diseases. However, it is a diffcult problem on how toregulate the expression of a certain gene in accordance with the amount inneed at punctual time. The traditional gene inducible systems relying on thethermal shock protein systems, hormone systems and the heavy metalssystems have many drawbakcs. For example, they possess commonshortcomings like high background, polytrophic effect and environmentalpollution, etc. These disadvantages limit their applications and development.The gene inducible system created by Gossen, also known as Tetracyclin inducible gene expression system (Tet-on/off system), overcomes thedisadvantages of the traditional inducible systems. This system canspecifically regulate exogenous gene expression through E.coli Tnolregulatory elements. Tet system is considered to be the most ideal induciblegene expression system. It has many advantages such as high efficiency,specificity and restriction. It has been applied to many areas of research, suchas the study of gene function and the treatment of disease. However, therehave been are no relevant reports on study of HPSCC at present.
Tumor Necrosis Factor-α (TNF-α) was discovered30years ago. It isregarded as the strongest antitumor cytokine identified till present. TNF-αcankill the tumor cells directly or exert its antitumor effects by activating theimmune system, without influencing on normal cell functions simultaneously.However, the half-life of TNF-αis short. The low-dose TNF-αhas nosignificant antitumor effect but rather promote the cell proliferation in sometumors. Continuous injection of large doses of TNF-α for therapeuticpurposes would eventually cause fever, chills, fatigue, headache, shock andother side effects. Therefore, the clinical application of TNF-αin cancerpatients and its practical effects remains fairly questionable and very limited.
TNF-α exerts its biological effects via two known transmembranereceptors, TNFR1(p55) and TNFR2(p75), both of which are composed ofsignal peptide, cytoplasmic domains, transmembrane region and extracellulardomains. The cytoplasmic domains of TNFR1and TNFR2are obviouslydifferent, indicating that TNFR1and TNFR2mediate different intacellularsignaling pathways. Functionally, TNFR1can mediate both apoptosis andnecrosis, and promote the proliferation of cells as well by activating NF-κ B.TNFR2also mediate two pathways. It can either promote the apoptosisthrough or independent of TNFR1, or promote the proliferation of cells. Theproliferation or apoptosis pathways mediated by TNFR are still not fullyunderstood. Previous studies have shown that TNFR levels of cell surface canalter the sensitivity of cells to TNF-α. The higher the TNFR expression, themore cytotoxic effect of TNF-α is noticed. Therefore, TNFRs play important roles in the regulation of TNF-αeffects.
It has been reported that TNF-α has synergistic effects withchemotherapy drugs in induing cell death in many tumors, such as soft tissuesarcoma, melanoma, gastric cancer, lung cancer and some other malignancies.However, there have been no similar reports on head and neck cancers.Besides, the mechanism by which TNF-α and chemotherapy drug interactwith each other is not completely clear. In management of hypopharyngealcancer, how to reduce application dosage of TNF-α and chemotherapy drugsto avoid sides effects with simultaneous improvement of their treatmenteffects remains a core problem to solve. The expression levels of TNFRs oncertain tumor cells may determine the outcomes of above treatments.
In this study, we used immunohistochemistry and Western blot to detectthe expression of TNFR1and TNFR2in HPSCC tissues and analyzed therelationship between TNFR expression and associated clinicopathologicfactors. Stable Tet-on-TNFR2Fadu cell lines were established by lentiviraltransfection. The proliferation-inhibiting and apoptosis-inducing effects ofTNF-α and cisplatin (DDP) in cells with different TNFR-expression levelswere investigated, for a deep insight into relationship between TNF-αandDDP resistance of hypopharyngeal cancer and the expression of TNFRs. Thepresent investigation is divided into three parts as follows.
Part one: Expression of TNFR1and TNFR2and its clinocopathologicsignificance in HPSCC
Objective: To investigate TNFR1and TNFR2expression and thus therelationship between TNFR expression and clinicopathologic features inHPSCC, for preliminary definition of their roles in tumorigenesis anddevelopment.
Methods:
1The expression levels of TNFR1and TNFR2were detected byimmunohistochemical staining in45HPSCC specimens and correspondingtumor-adjacent hypopharyngeal tissues, and their relationships withclinicopathologic characteristics were evaluated.
2The expression of TNFR1and TNFR2was also detected by WesternBlot in3HPSCC specimens and tumor-adjacent hypopharyngeal tissues.
Results:
1Immunohistochemistry analysis.
1.1The expression of TNFR in HPSCC and paracancerous hypopharyngealmucosal tissues.
Positive staining of both TNFR1and TNFR2was seen in all45HPSCCspecimens, and there was no significant difference of the expression levelsbetween TNFR1and TNFR2among the45HPSCC samples. However,expression of TNFR1and TNFR2was observed in50%and10%of the pairedtumor-adjacent hypopharyngeal tissues, respectively. Further statisticalanalyses showed that the average optical density (AOD) values of bothTNFR1and TNFR2in HPSCC were significantly higher than those in normalhypopharyngeal tissues (P<0.01). These data indicate that the expression ofboth TNFR1and TNFR2is upregulated in HPSCC.
1.2Relationship between TNFR expression and clinicopathologic factors inHPSCC.
The data revealed that both TNFR1and TNFR2expression had nothingto do with age (TNFR1, t=0.125，P=0.901. TNFR2, t=0.149, P=0.883), sex(TNFR1, t=0.060, P=0.952. TNFR2, t=0.959，P=0.343) and tumor location(TNFR1, F=0.169, P=0.845. TNFR2, F=1.888, P=0.164).
However, TNFR1expression was significantly correlated withhistological grade (t=3.822，P＜0.01), tumor clinical stage (t=4.751，P＜0.01),T stage (t=5.087，P＜0.01), and lymph node metastasis (t=4.189，P＜0.01).We next analyzed the correlation of the expression status of TNFR2withclinicopathologic factors. Unlike TNFR1, TNFR2expression status was notsignificantly associated with any of the above mentioned clinicopathologicfactors in HPSCC(P＞0.05).
2Correlation of TNFR1expression with TNFR2expression in HPSCC.
The distinct correlation of the expression levels of TNFR1and TNFR2with the clinicopathologic factors suggests that there exist a correlation between the expression levels of TNFR1and TNFR2in HPSCC. Indeed, ourstatistical analyses demonstrated a reverse correlation between the expressionof TNFR1and TNFR2in HPSCC (rs=-0.305, P＜0.05). The ratio of TNFR1and TNFR2AOD was significantly correlated with histological grade, tumorclinical stage, T stage, and lymph node metastasis (all P＜0.01), and hadnothing to do with age, sex and tumor location(P＞0.05).
3Western blotting showed that compared with tumor-adjacenthypopharyngeal tissues, the protein expression of TNFR1(t=5.079，P＜0.05)and TNFR2(t=17.333，P＜0.01) were both significantly increased in HPSCC.
Conclusions: The expression of both TNFR1and TNFR2wassignificantly higher in HPSCC than in tumor-adjacent hypopharyngeal tissues.TNFR1expression and the ratio of TNFR1and TNFR2was significantlycorrelated with histological grade, tumor clinical stage, T stage, and lymphnode metastasis. But TNFR2expression status was not significantly associatedwith any of the clinicopathologic factors in HPSCC. There is a reversecorrelation between the expression of TNFR1and TNFR2in HPSCC. TNFRplay an important role in the occurrence and development of HPSCC.
Part two: Establishment of a stable Tet-on-TNFR2system cell linemediated by lentivirus in FaDu cell
Objective: To establish a stable Tet-on-TNFR2cell line transfected withlentivirus in FaDu cell, and to provide a cell line for further study on therelationship between TNFR expression and the sensitivity of TNF and DDP.
Methods:
1Construction of a Tet-on-TNFR2recombinant lentiviral vector
The open reading frame (ORF) sequences of TNFR2gene was purchasedfrom Shanghai Hanbio Biological Company. Amplification of TNFR2ORFsequence was carried out through PCR. Then, the products and Tet-onlentiviral vector carriers were subjected to XhoI and EcoRI double enzymedigestion. The plasmid acquired from enzyme-linked products was trasformedin competent cells overnight, after which time the formed bacteria clones werepicked and identified by PCR. The PCR products were sent for sequencing tests for final confirmation.
2Preparation before packaging plasmids
2.1The lentivirus system contains lentiviral plasmid pHBTet-on-Puro-TNFR2(empty vector control pHBTet-on-Puro), and auxiliary packaging plasmidspspax2and pMD2G. Among them, pHBTet-on-Puro-TNFR2with Puromycinresistance and drug-induced UBC promoter can induce the TNFR2geneexpression by Doxorubicin(Dox).2.2The lentiviral plasmid (pHBTet-on-Puro-TNFR2) preparation plasmidpspax2and pMD2G were extracted without endotoxin to obtain the highpurity lentiviral vector and auxiliary packaging plasmids.3The lentivirus vectors pHBTet-on-Puro-TNFR2and lentivirus auxiliarypackaging plasmids were co-transfected into293T cells for72h. Then thesupernatant was collected, concentrated and resuspended with500ul DMEMfor acquisition of infection viral paticles.4FaDu cells were infected with lentivirus vectors. Puromycin was used toselect the stable Tet-on-Puro-TNFR2viral system infected cells for a week.5The stable Tet-on-Puro-TNFR2cells were detected by quantitativereal-time PCR (QPCR) and Western blotting.
Results:
1Though identification the recombinant plasmid sequence wasconsistent with the experimental design.
2Stable Tet-on-Puro-TNFR2FaDu cell clones were selected by2.0μg/ml of puromycin.
3QPCR showed that the mRNA level of TNFR2in Tet-on-Puro FaDucell without induction by Dox was0.126±0.046. The mRNA level of TNFR2in Tet-on-Puro FaDu cells induced by Dox for24h was0.215±0.124. Therewas no significant difference between the two groups (t=1.606, P＞0.05). ThemRNA level of TNFR2in stable Tet-on-Puro-TNFR2FaDu cells withoutinduction by Dox was0.149±0.056. Wheras, the mRNA level of TNFR2was3.518±0.733in the same cells induced by Dox for24h. The mRNA level ofTNFR2was significantly increased in stable Tet-on-Puro-TNFR2FaDu cell (t=8.125, P＜0.05) after Dox induction.
4Western blot showed that TNFR2protein was significantly increasedin stable Tet-on-Puro-TNFR2FaDu cells stimulated with Dox for24h than innomal Fadu cells with or without Dox induction and Tet-on-Puro FaDu cellswith or without Dox stimulation.
Conclusions:
The stable Tet-on-Puro-TNFR2Fadu cell line was successfullyestablished by lentivirus transfection, which provides a new cell line forfurther investigation on the function of TNFRs.Part Three: Regulation of TNFR expression by Tet-on system to increase
the sensitization of FaDu cells to TNF and DDP
Objective: To observe the proliferation and apoptosis rates induced bycisplatin (DDP) and TNF-ɑ in Fadu cells and changes in the proliferation andapoptosis rates after neutralizing the effect of TNFR1or up-regulating TNFR2expression by Tet-on system for definition of the relationship between theTNFR expression and sensitivity to TNF-ɑ and DDP.
Methods:
This part of investigation mainly foucused on evaluation of proliferationand apoptosis changes in Fadu cells when TNF-ɑ and DDP were applicatedunder different TNFR expression conditions. Inhibition of proliferation wasdetected by MTT method. The apoptosis rate was detected by flow cytometrythrough Annexin V/PI double staining.
1The study of proliferation-inhibiting and apoptosis-inducing effects ofTNF-ɑ, DDP and their combinations in nomal TNFR-expressing Fadu cells.
2The study of difference in proliferation inhibition and apoptosisinduced by TNF-ɑ, DDP or both combined in Fadu cells after neutralizingTNFR1.
3The study of changes in proliferation and apoptosis rates induced byTNF-ɑ, DDP and both combined upon Fadu cells after upregulating theexpression of TNFR2by Tet-on systerm.
4The study of changes in proliferation and apoptosis induced by TNF-ɑ, DDP and both combined after upregulating the expression of TNFR2by Tet-on systerm and neutralizing TNFR1with the blocking antibody.
Results:
1The results of MTT
1.1TNF-ɑ can promote cell proliferation at low concentrations (0.1-10ng/ml)and can inhibit the tumor growth at high concentrations (50-100ng/ml) instable Tet-on-Puro Fadu cell (Group A). TNF-ɑ combined with DDP cansignificantly enhance the antitumor effect of DDP.
1.2In stable Tet-on-Puro Fadu cells, neutralizing TNFR1with its blockingantibody (Group B) can reverse the proliferation-promoting effects oflow-dose (0.1-10ng/ml) TNF-ɑ prominently (t=4.390，t=5.886，t=5.258, all P<0.05); high-dose TNF-ɑ (50-100ng/ml) induced no significant difference inproliferation inhibition compared to Group A (t=2.657, t=1.083, t=1.113, allP>0.05). TNF-ɑ in low concentration combined with DDP can significantlyenhance the antitumor effect of DDP, but high concentration was nosignificant difference compared with Group A (P>0.05).
1.3In stable Tet-on-Puro-TNFR2Fadu cells induced by Dox (Group C), lowdoses of TNF-ɑ can inhibit the proliferation of cells significantly compared toGroup A (P <0.01). The effects of high concentration TNF-ɑ (50-100ng/ml)were of no significant difference with Group A (P>0.05). In this group, theeffect of each concentration TNF-ɑ combined with DDP increased. The effectwas statistically difference (P <0.05).
1.4In stable Tet-on-Puro-TNFR2expressing Fadu cells induced by Dox,with simultaneous neutralization of TNFR1(Group D), TNF-ɑ cansignificantly inhibit the proliferation of cells compared with Group A, GroupB (except TNF-ɑ0.1ng/ml) and C (except TNF-ɑ0.1ng/ml). The antitumoreffects of TNF-ɑ combined with DDP were significantly greater compared tothe three groups (all P＜0.05), except for that of TNF-ɑ at50-100ng/ml inGroup C.
2The apoptosis results by Annexin V/PI double staining in flow cytometry
2.1The apoptosis rate induced by10ng/ml TNF-ɑ revealed no significant difference (t=0.918，P＞0.05) between Group B and Group A. The apoptosisrate was obviously increased in Group C compared to Group A (t=13.504，P＜0.01). The apoptosis rate was significantly increased in Group D comparedto Group A and Group B (t=12.424，t=10.058，P＜0.05). But there was nosignificant change in apoptosis rates compared to Group C (t=0.624，P＞0.05).
2.2The apoptosis rate induced by10ng/ml TNF-ɑ and6μg/ml DDP bearedno significant difference between Group B and Group A(all P＞0.05).Apoptosis rate in Group C increased significantly compared to Group A(t=8.498，P＜0.05). The apoptosis rate of Group D was significantly higherthan Group A and Group B (all P<0.05). There was no significant differencein apoptosis rate in comparison with Group C (t=0.795，P＞0.05).
Conclusions:
1In Fadu cells without changes in TNFR expression, TNF-ɑ inducescell proliferation at low concentrations (0.1-10ng/ml), while highconcentration TNF-ɑ (50-100ng/ml) exhibits effects of proliferation inhibitionon tumor cells. TNF-ɑ and DDP have a synergistic effect in inducingproliferation inhibition.
2Neutralizatiion of TNFR1with its blocking antibody reversesproliferation-promoting effects of TNF-ɑ and can increase the antitumoreffects of TNF-ɑ combined with DDP at low concentrations, but does notincrease the apoptosis rate induced by TNF-ɑ and/or DDP in Fadu cells.
3Upregulation of TNFR2in Fadu cells obviously promotes theproliferation inhibition induced by TNF-ɑ at low concentrations and TNF-ɑ ateach concentrations plus DDP, and can obviously promotes apoptosis inducedby TNF-ɑ and TNF-ɑ plus DDP.
4Upregulation of TNFR2with simultaneous neutralization of TNFR1in Fadu cells can significantly increase the therapeutic effect of TNF-ɑ andDDP.
5In hypopharyngeal carcinoma Fadu cells, it is highly likely thatTNFR1mediates proliferation pathway whereas TNFR2mediates apoptosispathway. Regulating the expression levels of TNFR1and TNFR2may shed light on improving the therapeutic effects of TNF-ɑ and DDP throughinterfering with the crosstalk between the two receptors.
引文
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