Exhaustive proteome analyses of pulmonary neuroendocrine carcinomas

我描述了用不同的蛋白质组技术寻找肺神经内分泌癌标志物的最新结果。穷尽式单克隆抗体的生产：我们利用肺癌细胞作为免疫原产生单克隆抗体。从获得的一组抗体中，存在针对活化C激酶1受体（RACK1）的描述抗体。RACK1在腺癌中表达量高、频率高，但在少数鳞状细胞癌和大细胞癌中几乎未见表达。此外，RACK1的表达也与腺癌患者的病理分期、肿瘤大小和淋巴结状况有显著相关性。这些结果提示RACK1可能是一种新的肺腺癌鉴别诊断标志物。2. 肿瘤细胞株条件培养基中多肽的检测：为了寻找新的肺神经内分泌肿瘤标志物，我们对LCN1和LCNEC细胞株向条件培养基中分泌的多肽进行了综合分析。通过MALDI-TOF/TOF ms鉴定出分别含有40和19个氨基酸残基的肽片段，证实这两个片段是VGF的组成部分。对肺癌细胞系的RT-PCR分析显示，vgf仅在神经内分泌癌源性细胞中表达。我们的数据表明，VGF可以作为肺神经内分泌肿瘤的一种新的血清诊断标志物。3. 二维凝胶电泳的应用：我们使用两种细胞系（来自小细胞肺癌（SCLC）的N231和来自肺大细胞神经内分泌癌（LCNEC）的LCN1）的细胞裂解液，用二维凝胶电泳（2-DE）和MULDI-TOF/TOF ms研究了蛋白质组学方法。在25种鉴定的蛋白质中，与N231细胞相比，LCN1细胞中的细胞角蛋白（CK） 7、8、18和19上调。用免疫组织化学方法研究了81例肺癌组织中它们的表达。LCNEC患者CK7、8、18和19的平均免疫染色评分明显高于SCLC患者。这些数据提示LCNEC和SCLC的生物学特征可能不同，ck的表达可能作为鉴别诊断指标。4. 肺癌患者血清中自身抗体的检测：采用四种细胞系的细胞裂解液免疫印迹法筛选SCLC患者血清中自身抗体（AAs）。采用免疫印迹法进行二维凝胶电泳，并从膜和凝胶上切下靶点，以鉴定抗原。通过该方法，在SCLC患者血清中鉴定出绒毛蛋白1与AA。Villin1可能是区分LCNEC/AD与SCLC/SCC的有用标记物，本方法也可能用于鉴别不同组织学类型肺癌患者血清中特异性肿瘤相关分子。少

I describe recent results of the search for pulmonary neuroendocrine carcinoma markers with diverse proteome techniques.1. Exhaustive monoclonal antibodies production: We generated monoclonal antibodies using cells derived from pulmonary carcinomas as an immunogen. From a group of obtained antibodies, present described antibody for the receptor of activated C kinase 1 (RACK1). The expression RACK1 was significantly high and frequent in adenocarcinomas but was barely detected in a few squamous cell carcinomas and large cell carcinomas. Moreover, RACK1 expression was also significantly associated with the pathological stage, tumor size and lymph node status of adenocarcinoma patients. These results suggest that RACK1 may be a novel differential diagnostic marker for pulmonary adenocarcinomas. 2. Detection of peptides in the conditioned medium from carcinoma cell lines: Aiming to identify new markers of pulmonary neuroendocrine tumors, we analyzed comprehensively analyzed peptides which w … More ere secreted into the conditioned medium by LCN1, an LCNEC line. Two peptide fragments of 40 and 19 amino acid residues were identified by MALDI-TOF/TOF MS. These two fragments were demonstrated to be parts of VGF. RT-PCR analysis of lung cancer cell lines showed that vgf was expressed only in neuroendocrine carcinoma-derived cells. Our data suggest that VGF can be a novel sero-diagnostic marker of pulmonary neuroendocrine tumors. 3. Application of two-dimensional gel electrophoresis: We studied the proteomic approach using cell lysate from two cell lines (N231 derived from small cell lung carcinoma (SCLC) and LCN1 derived from pulmonary large cell neuroendocrine carcinoma (LCNEC)) with two-dimensional gel electrophoresis (2-DE) and MULDI-TOF/TOF MS. Within the 25 identified proteins, cytokeratins (CK) 7, 8, 18 and 19 were up-regulated in LCN1 cells compared with N231 cells. Their expressions were studied immunohistochemically with 81 pulmonary carcinomas. The mean immunostaining scores of CK7, 8, 18, and 19 were significantly higher in LCNEC cases than in SCLC. These data suggest that the biological characteristics of LCNEC and SCLC may be different and the expressions of CKs may serve as differential diagnostic markers. 4. Detection of autoantibodies in sera of patients with lung cancer: To detect autoantibodies (AAs), sera from SCLC patient was screened by immunoblotting using cell lysate of four cell lines. To identify the antigens recognized by AAs, two-dimensional gel electrophoresis was immunoblotted and target spots were cut out from the membrane and gel. By this method, villin1 was identified with AA in sera from patients with SCLC. Villin1 may be useful markers to distinguish LCNEC/AD from SCLC/SCC, and the present method might be useful to identify specific tumor-associated molecules in sera from pulmonary carcinoma patients with different histologic types. Less