CN111323590B - 抗TIF1γ-IgA与抗TIF1γ-IgG作为联合诊断标志物在肺癌诊断中的应用 - Google Patents
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Abstract
本发明提供了抗TIF1γ‑IgA与抗TIF1γ‑IgG作为联合诊断标志物在肺癌早期诊断中的应用，属于医学诊断技术领域。本发明首次发现TIF1γ蛋白在肺癌早期患者癌组织中存在异常高表达；同时发现肺癌早期患者体内存在针对TIF1γ的强烈体液免疫反应，其中血清抗TIF1γ‑IgA与TIF1γ‑IgG自身抗体可成为肺癌早期诊断的标志物，且抗TIF1γ‑IgA与抗TIF1γ‑IgG的联合检测可在保证特异性的基础上，明显提高肺癌患者早期诊断的敏感性，适合开发成具有临床应用价值的联合检测试剂盒。因此，抗TIF1γ‑IgA与抗TIF1γ‑IgG的联合检测有助于进一步改进肺癌患者的早期血清学筛查质量，从而提高肺癌的治愈率与5年生存率。
Description
技术领域
本发明属于医学诊断技术领域，具体而言，涉及抗TIF1γ-IgA与抗TIF1γ-IgG作为联合诊断标志物在肺癌早期诊断中的应用。
背景技术
肺癌是当今世界上发病率和致死率高居首位的恶性肿瘤，对人类健康危害重大。早期诊断、及时的手术根治性切除对于肺癌患者的预后意义重大，研究显示，Ⅰ期接受手术的肺癌患者5年生存率可高达92％[Goldstraw P,Chansky K,Crowley J,et al.The IASLCLung Cancer Staging Project:Proposals for Revision ofthe TNM Stage Groupingsin the Forthcoming(Eighth)Edition ofthe TNM Classification for LungCancer.JThorac Oncol.2016；11(1):39-51.]；但肺癌早期多无特异临床症状，难以被察觉，大多数患者就诊时已处于疾病的晚期，从而失去了手术的最佳治疗时期，致5年生存率仅5％-15％[Jantus-Lewintre E,Uso M,Sanmartin E,Camps C.Update on biomarkersfor the detection oflung cancer.Lung Cancer(Auckl).2012；3:21-29.]。因此，提高肺癌的早期诊断效率对于改善患者的预后具有重要作用。
血清学指标具有检测方便、安全等优点，但目前常用的肿瘤抗原类标志物不同程度存在检测敏感度或特异性问题，临床实际应用效果欠理想，尤其早期诊断价值有限。近年来的研究发现在癌症发生发展的过程中，少量异常表达的肿瘤相关抗原(TAAs)即可刺激B细胞产生大量的自身抗体，这使得血清自身抗体标志物的检测有助于癌症的早期诊断。文献报道肺癌患者在影像学改变出现的前5年即可在血清中检测到针对TAAs的自身抗体[Zhong L,Coe SP,StrombergAJ,Khattar NH,Jett JR,Hirschowitz EA.Profilingtumor-associated antibodies for early detection ofnon-small cell lungcancer.JThorac Oncol.2006；1(6):513-519.]。
越来越多的研究证明，癌症早期异常表达的TAAs可诱导机体的体液免疫应答，产生针对TAAs的自身抗体并释放到外周血中，通过检测外周血中针对TAAs的自身抗体可以区分正常人和癌症患者。即使TAA水平很低，免疫系统仍能监测到该特异蛋白的存在，产生的大量抗体对抗原信号起到一定的放大作用，因此自身抗体与抗原类标志物相比，在癌症早期具有更高的敏感性；其次，产生的肿瘤相关自身抗体半衰期长，能稳定存在于血清中相当长的一段时间，易于临床检测。TAAs相关自身抗体的这些特点，使其有望成为肺癌早期诊断的理想血清学标志物。因此，发现新的早期肺癌生物标志物作为非侵入性筛查试验以提高患者的存活率是至关重要的。
转录中介因子1γ(Transcriptional intermediary factor-1γ，TIF1γ)，是一种TGF-β/Smad通路的调控因子，具E3泛素蛋白连接酶活性。研究表明TIF1γ可通过抑制Smad4调节TGF-β信号通路的方式参与细胞增殖、分化与迁移侵袭的调控。近年来TIF1γ与肿瘤的相关性引起关注。Jain S等的研究表明TIF1γ在肠癌组织具异常高表达，表现为相比正常的肠粘膜组织，在Ⅰ和Ⅱ期肠癌组织TIF1γ蛋白的表达明显上调，且与患者的病程进展相关[Jain S,Singhal S,Francis F,et al.Association of overexpression of TIF1gamma with colorectal carcinogenesis and advanced colorectaladenocarcinoma.World J Gastroenterol.2011；17(35):3994-4000.]。TIF1γ的异常高表达也见于乳腺癌，Kassem L等通过对248例乳腺癌组织的免疫组化检测，发现核TIF1γ、细胞质TGF-β1的阳性染色率高达35.9％、30.4％，且TIF1γ、TGFβ1的高表达与患者的远端转移、复发等不良预后密切相关[Kassem L,Deygas M,Fattet L,et al.TIF1gammainterferes with TGFbeta1/SMAD4 signaling to promote poor outcome in operablebreast cancer patients.BMC Cancer.2015；15:453.]。以上研究表明TIF1γ的异常高表达可能参与了肠癌、乳腺癌的发生发展进程，但文献报道TIF1γ在单核细胞、胰腺癌的表达明显下调[Aucagne R,Droin N,Paggetti J,et al.Transcription intermediary factor1gamma is a tumor suppressor in mouse and human chronic myelomonocyticleukemia.J Clin Invest.2011；121(6):2361-2370.]、[Vincent DF,Gout J,Chuvin N,etal.Tif1gamma suppresses murine pancreatic tumoral transformation by a Smad4-independent pathway.Am JPathol.2012；180(6):2214-2221.]，提示TIF1γ参与不同肿瘤发生发展进程的作用方式可能不同，表现具复杂性。
另外，Wang L等采用Real-time PCR检测13例非小细胞肺癌(NSCLC)患者癌组织与配对癌旁组织中TIF1γmRNA的表达，结果显示TIF1γ在癌组织中表达减弱[Wang L,Lei Z,Liu X,Liu R,Zhang H.[Association of mutation and methylation in the promoterregion of TIF1gamma with non-small cell lung cancer].Zhongguo Fei Ai ZaZhi.2013；16(5):227-232.]。
发明内容
本发明人通过对60例肺癌早期患者癌组织及配对癌旁组织的免疫组化检测，结果显示TIF1γ在癌组织的阳性表达率83.33％(50/60)，明显高于配对癌旁组织25.00％(15/60)(P<0.01)，提示在肺癌早期患者血清可能存在高水平的针对TIF1γ的自身抗体，从而在蛋白质层面证明了TIF1γ在肺癌患者的癌组织存在异常高表达，克服了现有技术的偏见。
鉴于TIF1γ蛋白在肺癌早期患者癌组织的异常高表达，发明人推测TIF1γ可刺激机体产生针对TIF1γ的自身抗体。进一步地，发明人想到相同抗原会激发机体产生不同Ig类型的自身抗体，为此我们构建了针对四种Ig型抗TIF1γ抗体的ELISA法，检测结果显示，肺癌早期患者组血清抗TIF1γ-IgM与TIF1γ-IgE的水平与阳性率与LBL、HC组相比较无明显差异(P>0.05)；但是令人激动的是，抗TIF1γ-IgG与TIF1γ-IgA的水平与阳性率均明显高于LBL、HC组(P<0.05)，尤其是抗TIF1γ-IgA对LC早期诊断的敏感性达28.2％(特异性95.93％)、AUC 0.704，高于抗TIF1γ-IgG的敏感性18.54％(特异性94.25％)与AUC 0.622。此外，经western blot法鉴定，ELISA检测抗TIF1γ-IgA、TIF1γ-IgG抗体(+)的肺癌早期患者血清均只与目的蛋白结合，不与标签蛋白GST结合；ELISA检测自身抗体(-)的LBL患者、HC的血清均不与目的蛋白和标签蛋白结合。以上结果进一步证明肺癌早期患者血清中存在着大量针对TIF1γ的IgA型与IgG型自身抗体。
由于IgA型、IgG型抗TIF1γ抗体的单独检测对肺癌早期诊断的敏感性有限，本发明人进一步开展了抗TIF1γ-IgA与TIF1γ-IgG联合检测的分析，结果显示二者的联合可在保证特异性(92.34％)的基础上，将对肺癌早期患者的诊断敏感性提高至38.31％，AUC提高至0.734，这说明抗TIF1γ-IgA与TIF1γ-IgG自身抗体的联合检测非常有助于进一步提高对肺癌的早期诊断性能。
与现有技术相比，本发明的优点和进步性在于：首次发现TIF1γ蛋白在肺癌早期患者癌组织中存在异常高表达；同时发现肺癌早期患者体内存在针对TIF1γ的强烈体液免疫反应，其中血清抗TIF1γ-IgA与TIF1γ-IgG自身抗体可成为肺癌早期诊断的标志物，且抗TIF1γ-IgA与抗TIF1γ-IgG的联合检测可在保证特异性的基础上，明显提高肺癌患者早期诊断的敏感性，适合开发成具有临床应用价值的联合检测试剂盒。因此，抗TIF1γ-IgA与抗TIF1γ-IgG的联合检测有助于进一步改进肺癌患者的早期血清学筛查质量，从而提高肺癌的治愈率与5年生存率。
附图说明
图1：早期肺癌患者癌组织和配对癌旁组织中TIF1γ蛋白的免疫组织化学检测(×400)；(a、c)癌组织中TIF1γ强表达，分别为3+、2+；(b、d)配对癌旁组织中TIF1γ阴性或弱表达。
图2：各组血清抗TIF1γ不同亚型自身抗体水平的比较；(a)TIF1γ-IgA；(b)TIF1γ-IgG；(c)TIF1γ-IgM；(d)TIF1γ-IgE(**表示P<0.01，Early LC：早期肺癌，LBL：肺部良性病变，HC：健康对照)。
图3：抗TIF1γ-IgA与IgG血清自身抗体单独及联合检测对早期肺癌患者诊断的ROC曲线比较；(a)TIF1γ-IgA与TIF1γ-IgG单独检测的ROC曲线；(b)TIF1γ-IgA与TIF1γ-IgG联合检测的ROC曲线。
图4：血清TIF1γ-IgA与TIF1γ-IgG的western blot鉴定；(a)TIF1γ-IgA；(b)TIF1γ-IgG。
具体实施例
下面通过具体实施方式对本发明作进一步详细说明。但本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。另外，实施例中未注明具体技术操作步骤或条件者，均按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1
1材料和方法
1.1研究对象
肺癌早期患者248例，肺部良性病变(LBL)患者200例，健康对照(HC)218例，均来自2016年9月-2018年10月期间的福建省立医院。所有患者均严格满足国际或专业学会公认的诊断标准，且之前未曾接受过任何形式的抗肿瘤治疗，患者临床及病理资料见表1。HC为来自福建省立医院体检中心的健康体检者，排除肺部与他部位的恶性肿瘤，以及肺炎、肺脓肿等非恶性肿瘤性质的肺部良性病变。所有研究对象均于手术前采集空腹外周静脉血5ml，4℃3000rpm离心10min，收集血清，-80℃冻存备用；248例肺癌早期患者中随机选取60例患者的癌组织与配对癌旁组织用于IHC检测。以上所有研究对象均予以知情并获得知情同意。
表1早期肺癌、LBL及HC组临床病理资料
LBL：肺部良性病变；HC：健康对照
1.2免疫组织化学检测(IHC)
TIF1γ的IHC检测试剂盒为UltraSensitiveTM SAP(Mouse/Rabbit)IHC Kit(福州迈新生物技术开发公司)，操作严格按照说明书进行。肺癌患者的癌组织及配对癌旁组织切片标本经过水浴展平后37℃烘烤24h，随后行脱蜡、水化及抗原修复，血清封闭，滴加1:1000稀释抗TIF1γ抗体(Cell Signaling Technology，USA)、生物素标记的二抗以及链霉菌抗生物素蛋白-过氧化物酶溶液，最后通过DAB显色，苏木素复染，脱水至透明，封片。每个切片随机观察5个高倍镜视野，由两位有经验的病理科医师阅片。染色强度分级为：0＝阴性,1+＝弱阳性,2+＝中阳性,3+＝强阳性。
1.3酶联免疫吸附试验(ELISA)
GST标记的重组TIF1γ蛋白50ng/孔包被于96酶标孔板，100μl/孔，4℃过夜；PBST洗涤3次，3％BSA于37℃水浴封闭60min；PBST洗涤1次后加入稀释血清样本(抗TIF1γ-IgG、抗TIF1γ-IgM抗体为1:500稀释，抗TIF1γ-IgA、抗TIF1γ-IgE抗体为1:100稀释)，37℃孵育1h；PBST洗涤4次，加入稀释的HRP标记羊抗人IgG、IgM抗体(1:40000)(Jackson Immuno，USA)，抗IgA抗体(1:20000)(Jackson Immuno,USA)及抗IgE抗体(1:20000)(Thermofisherscientific，USA)，37℃孵育30分钟后PBST洗涤4次；加入四甲基联苯胺(TMB)与3％H2O2各50μl，避光显色10分钟；2mol/LH2SO450μl终止显色，酶标仪测定450nm波长处每孔吸光度值(OD)。
1.4免疫印迹试验(Western blot)
GST标记的重组蛋白TIF1γ(150kD)200ng及GST(26kD)50ng煮沸变性后，在8％SDS-PAGE，100V垂直电泳；将分离的蛋白转移到PVDF膜上；5％BSA阻断非特异性结合位后，用相关血清(抗TIF1γ-IgA 1:100稀释，抗TIF1γ-IgG 1:500稀释)或GST抗体(1:2000稀释，北京康为世纪有限公司)4℃孵育过夜；TBST洗涤，分别加入相对应的1:5000稀释的HRP标记的羊抗人IgA(Jackson Immuno，USA)及IgG二抗(北京康为世纪有限公司)，室温避光摇床孵育2h，TBST洗涤后使用ECL显色试剂盒进行显色，拍照。
1.5统计学分析
实验数据应用SPSS 22.0统计软件进行统计分析。以“HC组检测的OD均值+2倍标准差”作为指标的Cut-off值。组间定性资料的比较采用卡方检验，组间定量资料的比较采用Mann-Whitney U-检验。Graphpad Prism5制作图表，Medcalc v18.11制作ROC曲线，计算AUC值，以P<0.05为差异有统计学意义。
2结果
2.1 TIF1γ蛋白在肺癌组织的表达
免疫组化结果显示，TIF1γ蛋白定位于细胞核与细胞浆，在肺癌早期患者癌组织呈现灶状或弥漫性分布的棕黄色或棕褐色颗粒(图1a，c)，而在配对癌旁组织细胞中不表达或弱表达(图1b，d)。TIF1γ在癌组织的阳性表达率83.33％(50/60)，明显高于配对癌旁组织25.00％(15/60)(P<0.01)；此外，腺癌、鳞癌及小细胞肺癌TIF1γ的阳性表达率无显著性差异(P>0.05)(表2)。
表2 60例早期肺癌患者癌组织与配对癌旁组织TIF1γ表达的阳性率比较
2.2抗TIF1γ抗体在肺癌早期患者血清中的表达
ELISA法检测结果显示，早期肺癌组的抗TIF1γ-IgA、抗TIF1γ-IgG水平明显高于LBL组及HC组(P<0.01,图2a,b)，而抗TIF1γ-IgM、抗TIF1γ-IgE在各组间的表达无显著性差异(P>0.05，图2c,d)。抗TIF1γ-IgA对肺癌早期诊断的敏感性28.2％(特异性95.93％)、AUC 0.704，抗TIF1γ-IgG对肺癌早期诊断的敏感性18.54％(特异性94.25％)、AUC 0.622，二者的联合检测对于肺癌早期诊断的敏感性38.31％(特异性92.34％)、AUC 0.734(表3，图3)。
表3血清TIF1γ-IgA与TIF1γ-IgG对肺癌早期诊断性能的比较
**表示P<0.01。
2.3 Western blot验证ELISA结果
我们使用在酵母中表达的GST标记的重组蛋白TIF1γ进行western blot检测，以验证ELISA检测中观察到的血清反应性。如图4a,b所示，ELISA检测抗TIF1γ-IgA、抗TIF1γ-IgG(+)的早期肺癌患者血清均只与目的蛋白结合，不与GST标签蛋白结合；ELISA检测抗TIF1γ-IgA、抗TIF1γ-IgG(-)的LBL患者、HC的血清均不与目的蛋白和GST标签蛋白结合。
Claims (2)
1.由血清抗TIF1γ-IgA自身抗体与抗TIF1γ-IgG自身抗体组成的联合诊断标志物在制备早期诊断肺癌的试剂盒中的应用。
2.根据权利要求1所述的应用，其特征在于，所述的试剂盒为基于ELISA技术的多重检测试剂盒。
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