TNF-α和TNFR1在宫颈鳞癌组织中的表达及临床意义

目的 探讨不同病理类型宫颈组织中TNF-α和TNFR1的表达及其与宫颈鳞癌之间的关系。方法 应用实时定量RT-PCR法检测正常宫颈（正常组,48例）、宫颈上皮内瘤样病变（CIN组,47例）和宫颈鳞癌（SCC组,39例）组织中TNF-α和TNFR1、TNFR2 mRNA表达水平,采用免疫组织化学SP法检测TNF-α蛋白表达,Western blot法检测TNFR1、TNFR2蛋白表达,将TNF-α和TNFR1 mRNA 表达结果与临床病理结果进行相关性分析。结果 TNF-α和TNFR1的mRNA表达水平随宫颈癌变程度的增加而升高,组间两两比较差异均有统计学意义（P<0.05）,而TNFR2在各组中的表达差异无统计学意义（P>0.05）;TNF-α在宫颈组织中的表达随病理程度的增加有显著升高,正常组为6.25%、CIN 组为58.82%、SCC组为71.79%,组间比较差异有统计学意义（P<0.01）;TNFR1蛋白表达水平随宫颈癌变程度的增加而升高,差异均有统计学意义（P<0.05）,TNFR2在各组中的表达变化不明显,均与其mRNA在各组间的表达结果一致;SCC组中的TNF-α和TNFR1 mRNA的表达量与肿瘤大小、临床分期、浸润深度以及淋巴结转移情况呈正相关性,差异均有统计学意义（P<0.05）,与患者年龄以及细胞分化程度无关。结论 TNF-α和TNFR1的激活与宫颈鳞癌的发生、发展相关,参与肿瘤微环境的变化,两者将有望成为治疗宫颈鳞癌的靶标。     

TNFR1与PDC-E2在胶质瘤中表达的相关性及其临床意义

目的:研究TNFR1和PDC-E2在胶质瘤组织中的表达情况,分析TNFR1和PDC-E2异常表达与胶质瘤发生发展的关系。方法:采用Western blot技术检测9例不同级别胶质瘤新鲜组织中TNFR1和PDC-E2的表达情况;运用免疫组化方法检测75例胶质瘤组织石蜡切片中TNFR1和PDC-E2的表达情况,并结合临床病理资料分析TNFR1和PDC-E2与患者年龄、性别、肿瘤位置、手术切除范围以及肿瘤分级等因素之间的关系,并对其进行随访,应用Kaplan-Meier法分析TNFR1和PDC-E2与胶质瘤预后的关系,对两者的表达情况做Spearman等级相关性分析。结果:Western blot分析显示,TNFR1和PDC-E2在胶质瘤组织中的表达随级别增高而增高。免疫组化结果显示,TNFR1和PDC-E2的表达与胶质瘤组织学分级之间有统计学意义（P值均<0.001）。Kaplan-Meier分析显示胶质瘤中TNFR1和PDC-E2的高表达与患者较差的预后相关（P值均<0.01）;对两者的表达情况做Spearman等级相关性分析,结果发现TNFR1和PDC-E2的表达呈正相关（r=0.740,P<0.01）。结论:TNFR1和PDC-E2在胶质瘤中随着肿瘤级别的增加表达升高,高表达TNFR1和PDC-E2的患者预后较差,TNFR1和PDC-E2表达呈正相关。     

TNFR1与PDC-E2对胶质瘤细胞凋亡影响的相关性研究

目的:胶质瘤是一种最常见的中枢神经系统恶性肿瘤,生长迅速,恶性程度高.研究胶质瘤细胞凋亡过程中肿瘤坏死因子受体1(TNFRl)和丙酮酸脱氢酶复合物E2(PDC-E2)蛋白的表达变化,探讨TNFR1和PDC-E2在此过程中的作用.方法:培养TNFR1的小干扰细胞,将干扰掉TNFR1的质粒转染入U87MG细胞,检测TNFR1和PDC-E2对胶质瘤细胞凋亡的影响.通过免疫共沉淀方法检测TNFR1和PDC-E2在体外的相互作用;通过免疫荧光检测两者在U87MG细胞内的共定位;在转染干扰掉TNFR1的胶质瘤细胞中,检测TNFR1和PDC-E2的相互作用对凋亡标记物Caspase-3蛋白表达量的调节作用.结果:将TNFR1的小干扰质粒转染入U87MG细胞,结果显示未干扰掉的TNFR1对胶质瘤细胞凋亡有抑制作用,干扰掉TNFR1时对胶质瘤细胞凋亡的抑制作用减弱.体外HEK293T细胞及胶质瘤细胞中的免疫共沉淀以及免疫荧光显示TNFR1和PDC-E2存在相互作用;在胶质瘤细胞中检测到TNFR1和PDC-E2的相互作用协同促进Caspase-3的表达.结论:TNFR1和PDC-E2抑制胶质瘤细胞的凋亡,协同作用比单独对凋亡的影响更加明显;TNFR1和PDC-E2存在相互作用,两者协同作用更能增高作用底物Caspase-3的表达.     

热疗联合人肿瘤坏死因子对TNFR1高表达胶质瘤的细胞周期、F-肌动蛋白及其侵袭性的影响

目的探讨热疗联合重组人肿瘤坏死因子(recombinant human tumor necrosis factor,rhTNF)对肿瘤坏死因子受体1(tumornecrosis factor receptor 1,TNFR1)高表达的胶质瘤细胞的细胞周期和F-肌动蛋白(F-actin)的影响及其与胶质瘤侵袭性的关系。方法建立TNFR1高表达胶质瘤细胞株,RT-PCR和Western blot法检测胶质瘤细胞TNFR1的表达水平;碘化丙啶染色后用流式细胞术检测胶质瘤细胞细胞周期的变化;WST-8法检测细胞增殖;免疫荧光技术检测胶质瘤细胞内F-actin的表达水平;Transwell小室法检测胶质瘤细胞侵袭性改变。结果与对照组相比,TNFR1高表达胶质瘤细胞株的TNFR1 mRNA水平增加了78.5%,其蛋白质的表达水平增加了89.7%(P<0.05);经热疗联合rhT-NF处理后细胞增殖受抑制,S和G2/M期的TNFR1高表达胶质瘤细胞数之和明显增多,而F-actin的荧光强度和胶质瘤侵袭性分别降低了72.3%和83.10%。结论热疗联合rhTNF可能是通过阻滞TNFR1高表达胶质瘤细胞的细胞周期和降低F-actin的表达来实现降低胶质瘤侵袭性的作用。     

益气清热养阴中药对晚期肺癌恶病质小鼠TNF-α及其受体表达的影响

目的：研究益气清热养阴中药对晚期肺癌恶病质小鼠肿瘤坏死因子-α（TNF-α）及其受体（TNFR1、TNFR2）表达的影响。方法：建立小鼠Lewis肺癌胸腔移植恶病质模型,随机分为6组：正常对照组（NC）、模型对照组（MC）、中药组（TCM）、消炎痛组（IND）、顺铂组（DDP）、甲羟孕酮组（MPA）。ELISA法检测恶病质小鼠血清中TNF-α水平变化;免疫组织化学法（IHC）检测肿瘤组织中TNF-α、TNFR1、TNFR2的表达情况。结果：除正常对照组外,中药组中TNF-α的血清浓度明显低于其他组（P〈0.05）。肿瘤组织IHC：中药组TNF-α和TNFR1表达较其他组有不同程度减少（P〈0.05）;而TNFR2表达较其他组增加（P〈0.05）。结论：益气清热养阴中药可能通过调节TNF-α、TNFR1和TNFR2的表达发挥抗癌性恶病质的作用。     

TNFRⅡ基因-196M/R多态性与乳腺癌易感性的相关性

目的探讨TNF-α受体Ⅱ(TNFR Ⅱ)基因第6外显子-196M/R多态性与中国北方女性乳腺癌发病的相关性。方法采用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)方法检测TNFR Ⅱ基因-196M/R多态性,分析212例乳腺癌患者(实验组),218例乳腺良性病变患者(良性组)和220例健康对照者(对照组)的TNF-α受体Ⅱ(TNFR Ⅱ)基因多态性与乳腺癌易感性的关系。结果(1)650例研究对象中存在TNFR Ⅱ基因-196M/R多态性。(2)-196M/R位点的TG+GG基因型及G等位基因频率在良性病变组与对照组间差异无统计学意义,而在乳腺癌组则显著高于良性病变组和对照组(均P(0.05)。结论TNFR Ⅱ基因-196M/R在乳腺癌组和对照组存在多态性,第6外显子-196M/R位点T/G多态性可能为乳腺癌发病的易感基因位点,G等位基因可能为中国北方女性乳腺癌发病的易感基因。     

热疗降低胶质瘤侵袭性的作用与肿瘤坏死因子受体亲和力的关系

目的探讨热疗降低胶质瘤侵袭性的作用与肿瘤坏死因子受体(tumor necrosis factor receptor,TNFR)亲和力的关系。方法免疫组织化学法观察肿瘤坏死因子受体在胶质瘤组织中的分布;免疫荧光法检测肿瘤坏死因子受体在热疗后胶质瘤组织中的分布;放射配基受体结合分析法测定胶质瘤细胞经热疗联合重组人肿瘤坏死因子(recombinant human tumor necrosis factor,rhTNF）作用后,胶质瘤细胞中rhTNF结合肿瘤坏死因子受体水平的改变;通过荧光分光光度计分析Caspase3/7的活性来检测胶质瘤细胞的凋亡水平;结晶紫染色法测定热疗后胶质瘤侵袭性的变化。结果TNFR存在于胶质瘤细胞和胶质瘤血管内皮细胞,但以TNFR1分布于胶质瘤细胞为主;热疗后的胶质瘤组织中TNFR1的表达明显高于TNFR2,且于热疗后的120 min时TNFR1的表达达到高峰;胶质瘤细胞经热疗联合rhTNF作用后,rhTNF与TNFR1的结合率和Caspase3/7的活性呈上升趋势,两者均于120 min时达高峰后下降,与此同时,胶质瘤侵袭性降至最低水平。结论 热疗后,rhTNF可能是通过增加胶质瘤细胞TNFR1的表达并与之结合,并增强了rhTNF与TNFR1的亲和力,进而引起胶质瘤细胞凋亡导致胶质瘤侵袭性降低的。     

TRAF2在TNF诱导细胞凋亡过程中的作用及其信号转导途径

TNF诱导细胞凋亡的作用是通过许多信号分子共同参与完成的一个网络工程,其中TRAFs家族蛋白,尤其是TRAF2,是这一信号调节网络的中心环节,本文介绍了TRAF2在TNF诱导细胞凋亡过程中的作用及其信号转导途径,TNFR在与TNF结合后发生聚集,并募集TRAF2直接与TNFR2胞内段结合,或通过结合其他接头分子与TNFR1间接相连,TRAF2主要通过激活NIK和JNK／SAPK2个独立的信号转导途径对细胞发挥凋亡调节作用,具有凋亡保护功能,深入研究TRAF2的结构与功能,有利于加深对凋亡调节机理的认识。     

胃癌细胞肿瘤坏死因子受体的研究

目的　探讨人胃癌细胞肿瘤坏死因子受体 ( TNFR)的数目与胃癌细胞分化程度以及与肿瘤坏死因子突变体 ( TNF- m)细胞毒效应之间的关系。方法　以12 5I- TNF- m为配体 ,用放射配体结合分析法 ,检测了高、中、低不同分化程度的体外培养胃癌细胞 ( MKN2 8、SGC790 1、MKN4 5)的 TNFR,同时用 MTT比色法研究了 TNF- m对三株胃癌细胞的细胞毒效应。结果　三株胃癌细胞 TNFR数目分别为每细胞 9.8× 10 -12 nmol、5.6× 10 -12 nmol、3.2× 10 -12 nmol,三者之间比较 ,TNFR数目差异有显著性 ( P<0 .0 5)。解离常数基本一致 ,同一温度时三株胃癌细胞 TNF- m的内化率几乎相等 ,且呈温度依赖关系。TNFR的半衰期大约为 90分钟 ,胞膜与胞浆 TNFR数目之比约为 1∶ 2 ,TNF- m对三株胃癌细胞的最大杀伤率分别为 86%、60 %、34 % ,差异有显著性 ( P<0 .0 5) ,且 39℃时的杀伤率高于37℃时的杀伤率。结论　胃癌细胞表面 TNFR数目与胃癌分化程度相关。 TNF- m的细胞毒效应与TNF数目及 TNF- m的内化量有关。     

4-1BB在肿瘤治疗中的作用

4-1BB是肿瘤坏死因子受体(TNFR)超家族成员，具有影响T细胞扩增和存活的功能特性。通过4-1BBL或者抗4-1BB单克隆抗体干预4-1BB协同刺激途径可能对肿瘤有治疗作用。最近提出的s4-1BBL即4-1BBL可溶性形式，对恶性血液病的诊断和治疗以及判断预后有一定价值。现综述有关4-1BB在肿瘤治疗中的研究进展。     

CD40信号通过TNFRⅠ途径抑制肺癌细胞增殖的研究

背景与目的：CD40活化信号可抑制多种肿瘤细胞体外生长,但其分子机制尚不明确,本研究旨在探讨激发CD40信号对肺癌细胞株NCI—H460、A549的增殖及肿瘤坏死因子受体（tumor necrosis factor receptor,TNFR）、膜型TNF-α（mTNF-α）表达的影响及相关机制。方法：免疫荧光标记和流式细胞术检测肺癌细胞表面CD40的表达以及激发CD40对细胞表面TNFR和mTNF-α表达谱的影响：Western blot检测激发CD40对细胞TNFR和mTNF-α蛋白含量表达的影响;采用四氮唑盐（MTT）比色法抗体中和实验分析阻断TNFRI及TNF-α对CD40激发效应的影响：酶联免疫吸附（ELISA）法检测激发CD40对肺癌细胞培养上清中可溶性TNF-α（mTNF-α）含量的影响。结果：（1）肺癌细胞株NCl-H460、A549表面CD40表达分别为（89．0±3．2）％、（62．2±4．5）％。（2）免疫荧光和流式细胞术示,激发NCl—H460和A549细胞表面CD40分子48h后．其表面TNFRⅠ表达率分别为（36．2±4．6）％、（38．5±5．9）％,较对照组分别为（7．2±1．9）％、（15．2±3．1）％明显升高（P〈0．05）;而其表面TNFRⅡ表达率分别为（5．8±1．2）％、（18．0±1．6）％,较对照组分别为（38．8±4．3）％、（58．1±3．6）％明显降低（P〈0．05）;其表面TNF-α表达率分别为（7．0±0．9）％、（8．7±1．1）％,较对照组分别为（15．0±2．1）％、（26．5±3．2）％也明显降低（P〈0．05）。（3）Westernblot示,激发CD40可使NCl—H460和A549细胞TNFRI蛋白表达增强,TNFRII蛋白表达减弱．而mTNF-α蛋白表达无明显变化。（4）CD40激发前后肺癌细胞培养上清中未检测到sTNF-α的存在。（5）激发CD40能明显抑制NCl-H460、A549细胞的增殖（P〈0．05）,而阻断TNFRⅠ后CD40信号的细胞增殖抑制效应消失。（6）阻断TNF-α亦可明显抑制两肺癌细胞株增殖（P〈0．05）,而同时激发CD40未见两者?     

Mechanisms of tumor vascular shutdown induced by 5,6-dimethylxanthenone-4-acetic acid (DMXAA): Increased tumor vascular permeability

The novel vascular targeting agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) has completed phase 1 clinical trial and has shown tumor antivascular activity in both mice and humans. We have investigated its ability to change tumor vascular permeability, relating it to tumor vascular perfusion and other responses. The murine colon 38 adenocarcinoma was grown in C57Bl wild-type mice and mice lacking expression of either tumor necrosis factor receptor-1 (TNFR1(-/-)) or TNF (TNF-/-). Tumor vascular permeability, as measured by extravasation of albumin-Evans Blue complexes 4 hr after DMXAA treatment, was significantly increased in tumor tissue in C57Bl, TNFR1-/- and TNF-/- mice but not in normal (skin) tissue. Significant linear relationships were found between increased tumor vascular permeability, decreased functioning tumor blood vessels (measured by Hoechst 33342 staining at 4 hr), increased plasma 5-hydroxyindole-3-acetic acid concentrations (as a measure of serotonin release by platelets) and the degree of induced tumor hemorrhagic necrosis. The results support the hypothesis that DMXAA increases tumor vascular permeability both directly and through the induction of other vasoactive mediators, including TNF. DMXAA might be useful clinically to potentiate the vascular permeability of other anticancer modalities such as cytotoxic drugs, antibodies, drug conjugates and gene therapy.     

p53 transdominance but no gain of function in mouse brain tumor model

Although p53 mutations in tumors typically result in loss of transactivation of p53 target genes some mutants display gain-of-function activity. The latter has important implications for the design of rational cancer therapy. We previously described a germ-line p53 mutation (deletion of codon 236, Y236delta) associated with a familial brain tumor syndrome. To determine whether this tissue-specific tumor predisposition reflects a gain-of-function activity of Y236delta or an effect of genetic background we have developed a mouse brain tumor model. Primary neuroectodermal cells deficient for p53 (+/- or -/-) and transduced with Y236delta using a retroviral vector were transplanted into the brain of adult wild-type mice. This neurografting paradigm circumvents the problem of early lethal tumors at extracerebral sites associated with germ-line p53 deficiency. Brain tumors arising in this mouse model were highly invasive, reflecting an important feature of the human disease. Tumors arose from p53+/- cells only when transduced with Y236delta. In keeping with in vitro data showing that Y236delta has dominant-negative activity, these tumors retained the endogenous wild-type p53 allele but accumulated high levels of Y236delta. However, the presence of Y236delta in transplanted p53-/- cells had no effect on the tumor frequency, 15% versus 27% without the mutant. In conclusion, Y236delta is transdominant but exerts no gain-of-function activity mediating a more penetrant tumor phenotype.     

Selective blood-brain barrier permeabilization of brain metastases by a type 1 receptor-selective tumor necrosis factor mutein

BackgroundMetastasis to the brain is a major challenge with poor prognosis. The blood-brain barrier (BBB) is a significant impediment to effective treatment, being intact during the early stages of tumor development and heterogeneously permeable at later stages. Intravenous injection of tumor necrosis factor (TNF) selectively induces BBB permeabilization at sites of brain micrometastasis, in a TNF type 1 receptor (TNFR1)-dependent manner. Here, to enable clinical translation, we have developed a TNFR1-selective agonist variant of human TNF that induces BBB permeabilization, while minimizing potential toxicity.MethodsA library of human TNF muteins (mutTNF) was generated and assessed for binding specificity to mouse and human TNFR1/2, endothelial permeabilizing activity in vitro, potential immunogenicity, and circulatory half-life. The permeabilizing ability of the most promising variant was assessed in vivo in a model of brain metastasis.ResultsThe primary mutTNF variant showed similar affinity for human TNFR1 than wild-type human TNF, similar affinity for mouse TNFR1 as wild-type mouse TNF, undetectable binding to human/mouse TNFR2, low potential immunogenicity, and permeabilization of an endothelial monolayer. Circulatory half-life was similar to mouse/human TNF and BBB permeabilization was induced selectively at sites of micrometastases in vivo, with a time window of ≥24 hours and enabling delivery of agents within a therapeutically relevant range (0.5-150 kDa), including the clinically approved therapy, trastuzumab.ConclusionsWe have developed a clinically translatable mutTNF that selectively opens the BBB at micrometastatic sites, while leaving the rest of the cerebrovasculature intact. This approach will open a window for brain metastasis treatment that currently does not exist.     

Recombinant single-chain antibody fusion construct targeting human melanoma cells and containing tumor necrosis factor

Fusion constructs targeting tumor cells have significant potential applications against both solid tumors and hematologic malignancies. We developed a fusion construct of tumor necrosis factor (TNF) and a single-chain antibody (scFvMEL) recognizing the gp240 antigen on human melanoma cells. The scFvMEL/TNF construct, like TNF itself, was found to exist in solution primarily as a trimer of 45 kDa monomers (trimeric molecular weight = 135 kDa). The fusion construct bound specifically to gp240 antigen-positive but not to antigen-negative cells. The TNF component of the construct was biologically active (specific activity = 1 x 10(7) U/mg) compared with free TNF (specific activity = 2.6 x 10(7) U/mg) and was more cytotoxic to antigen-positive A375-M melanoma cells (IC(50) = 100 pM) than TNF alone (IC(50) = 1,000 pM) and, additionally, was active against AAB-527 melanoma cells (IC(50) = 20 nM) resistant to TNF itself (IC(50) > 1,000 nM). The augmented cytotoxicity was mediated by antibody-specific binding to the cell surface. Both A375-M and AAB-527 cells were shown to express TNFR1 and TNFR2 on the cell surface. The TNF moiety of the fusion construct was efficiently delivered into cells in time-dependent increase in cytosol as assessed by immunofluorescent staining of human melanoma cells. Radiolabeled scFvMEL/TNF localized effectively in human melanoma xenografts in nude (nu/nu) mice with a tumor:blood ratio of approximately 8 at 72 hr after administration. Our studies suggest that because of its unique biologic activity and low antigenic potential, scFvMEL/TNF makes an excellent cytotoxic protein for potential clinical treatment of human melanoma.     

TNF Receptors in Chronic Lymphocytic Leukemia

Two transmembrane receptors for tumor necrosis factor (TNF) with different molecular weight, 55 kD (p55) and 75 kD (p75), have been identified. In addition, the extracellular part of both receptors are shedded by proteolytic cleavage and exist as soluble receptors which bind to TNF in plasma and other biological fluids. Normal B cells produce TNF and express TNF receptors (R), mainly p75, upon stimulation. TNF costimulates DNA synthesis via the p75 receptor in normal B cells. The B cells from patients with chronic lymphocytic leukemia (CLL) produce TNF and have the capacity to express both receptor types. Also in malignant B cells the p75 receptor is dominant. TNF induce DNA synthesis in CLL cells via both receptors. CLL patients have elevated serum levels of soluble (s) TNFR and this is more pronounced in advanced disease. In conclusion, there is considerable support for TNF as an autocrine growth factor in CLL. However, the net effect of TNF is determined by the quan titative relationship between TNFR on the cell surface, TNF in plasma and sTNFR in plasma, and the affinities between TNF-p55 and TNF-p75. Due to increasing serum levels of sTNFR the significance of TNF as a growth factor is probably less important in advanced disease.     

Reduced latency but no increased brain tumor penetrance in mice with astrocyte specific expression of a human p53 mutant

p53-germline mutations located in the core DNA-binding domain have been associated with a more dominant tumor penetrance especially for breast cancer and brain tumors. We previously reported an unusual accumulation of CNS tumors associated with a unique p53 germline mutation, Y236delta (deletion of codon 236). To test whether this tissue-specific tumor predisposition reflects a gain-of-function activity of Y236delta, we generated transgenic mice expressing Y236delta in astrocytes using the regulatory elements of the glial fibrillary acidic protein (GFAP) gene. After transplacental exposure to N-ethyl-N-nitrosourea (25 mg/kg BW) brain tumors developed in 18% (7/39) of GFAP-Y236delta transgenic p53-/- mice, while in p53+/- mice the incidence was 28% (11/40) (P>0.3). However, the mean tumor latency for GFAP-Y236delta/p53+/- mice was significantly shorter than for p53+/- mice, with 19.9 weeks vs 31.6 weeks (P=0.039), respectively. Taken together, cell specific expression of Y236delta results in an acceleration of tumor progression but does not confer a higher tumor penetrance. Conceivably, the transdominant effect of Y236delta provided a growth advantage early in the progression of neoplastic cells, since the endogenous p53 wild-type allele was lost in all brain tumors independent of the genotype. This reflects well observations from human astrocytic neoplasms with p53 mutations.