血清C反应蛋白复合物中肺癌早期诊断标志物的筛选和鉴定

Chronic inflammation has been recognized to play a very important role in lung carcinogenesis. C-reaction protein (CRP) is a sensitive marker and mediator of inflammation. The epidemiologic studies indicate that the increased level of circulating CRP is a high risk factor for lung cancer. However, CRP is not a specific biomarker for lung cancer because many factors could affect its serum level. CRP can bind to exogenous and endogenous ligands and results in CRP complexes which regulate the immune function of phagocytic cell. We found that some tumor progression associated molecules, such as CK7、CEACAM5、SAA1 could be detected in serum CRP complexes from patients with lung cancer, while remain undetectable in serum CRP complexes from healthy donors and patients with pneumonia. These results suggested that some specific biomarkers for lung cancer might exist in the circulating CRP complexes. No similar research has been reported. To find more specific and suitable biomarkers for lung cancer, we plan to develop the following researches: (1) We will perform a target proteomic study of circulating CRP complexes using bead-based immunopurification and shotgun proteomics by2D- LC/MS/MS. We would not only identify new tumor markers in serum CRP complexes, but also confirm our previous study by this method. （2）A sandwich ELISA assay will be established to detect the candidates of biomarkers. (3) A large scale of clinical validation of these biomarkers will be carried out to evaluate their clinical values in early diagnosis or prognosis prediction for lung cancer. This study will indentify new specific biomarkers for lung cancer and establish sensitive ELISA test kits, which could be employed in the early diagnosis and prognosis prediction of lung cancer, and provide a novel strategy for prevention and treatment of lung cancer.

慢性炎症与肺癌发生发展密切相关。C反应蛋白(CRP)是炎症反应标志物，血清CRP浓度升高是肺癌的高危因素，但其浓度受多因素影响，用于肺癌早期诊断特异性不高。CRP通过与配体(如凋亡或坏死细胞降解产物) 结合形成CRP复合物，激活补体和单核吞噬细胞系统，清除废物。我们发现肺癌患者血清CRP复合物中存在CK7、CEACAM5、SAA1等与肿瘤相关的分子，而肺炎和正常人血清CRP复合物中则未检测到。这提示肺癌患者血清CRP复合物中存在特异标志物。但目前CRP结合的配体作为肺癌标志物尚无相关报道。本课题拟：①采用靶向CRP复合物的"鸟枪法"定量蛋白质组技术，在前期工作基础上进一步从CRP复合物中筛选出肺癌早期诊断特异标志物；②建立夹心ELISA技术检测筛选的标志物；③大规模临床验证其在肺癌早期诊断和疗效判断的价值。本课题研究将发现新的肺癌特异标志物并建立检测试剂盒，用于肺癌的早期诊断及疗效判断。

865例非小细胞肺癌（NSCLC）患者血清炎症指标和其他临床特征回顾性nomogram分析，显示血清CRP是NSCLC预后不良较好指标。 CRP 可结合多种蛋白，可用于肺癌的早期诊断、病程预测及预后判断。利用CRP抗体微珠分别捕获肺癌患者和正常人血清中CRP复合物，差异蛋白质组技术从肺癌患者血清CRP复合物中筛选出SAA、IL23R、CEACAM5、CK1和Ki67等上调蛋白。体外正反Co-IP验证了 CRP 结合 SAA，建立了ELISA检测血清CRP-SAA（Binding-SAA）。 检测484 例NSCLC病人及 159 例健康人血清Binding-SAA、Total-SAA和CRP，发现肺癌患者血清中 Binding-SAA (0.37±0.58 OD )和 Total-SAA 的含量（0.42±0.15 OD）显著高于正常人（Binding-SAA ：0.03 ± 0.04 OD；Total-SAA：0.13±0.05 OD； P<0.001）。ROC 曲线分析表明Binding-SAA 诊断肺癌的 AUC=0.901(敏感性 81.0%，特异性 80.0%)； Total-SAA 诊断肺癌的 AUC=0.845(敏感性 70.0%，特异性85.0%)。血清 Total-SAA 与 Binding-SAA 的水平与NSCLC患者的性别、TNM 分期、淋巴结转移、远处转移、肿瘤大小等临床特征均密切相关。 回顾性组（HR = 2.181, 95% CI = 1.641-2.897, P < 0.001）和前瞻性组（HR = 2.744, 95% CI = 1.810-4.161, P < 0.001）研究都表明Binding-SAA 水平高的肺癌患者5年总体生存期明显比低水平者短，单因素和多因素COX分析表明 Binding-SAA是肺癌患者独立预后因素。分层预后分析显示Binding-SAA也是早期肺癌患者预后不良指标，而Total-SAA和CRP不能预测早期患者预后。作为肺癌预后指标，Binding-SAA的效能优于CRP和Total-SAA。肺癌组织高表达SAA，而癌旁正常组织SAA表达弱。 体外实验证实CRP结合SAA，能够抵抗Cisplatin对肺癌细胞株A549和95D的毒性，能减弱Cisplatin对肺癌细胞株A549的凋亡作用，可以降低Cap