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背景与目的:癌细胞分泌蛋白是潜在的血清标志物,而且对肿瘤的早期筛选也具有重要意义。因此,系统、全面地研究肿瘤细胞分泌蛋白对于肿瘤血清标志物的筛选具有重要意义。本研究旨在鉴定肺癌细胞分泌蛋白,为肺癌血清标志物筛选提供实验依据。方法:用含胎牛血清的RPMI-1640培养液培养非小细胞肺癌细胞系A549细胞,收集A549细胞培养液中的蛋白及胎牛血清培养液中的蛋白(对照)进行蛋白双向电泳,应用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)及生物信息学鉴定A549凝胶上特有的蛋白质点。筛选出人属的蛋白质点后,应用RT-PCR方法检测其在肺癌组织及配对远处肺组织中的表达差异;并检测培养液及血清中锰-超氧化物歧化酶(manganesesuperoxidedismutase,Mn-SOD)水平。结果:鉴定出人属的蛋白质点14个,包括肌动蛋白、!烯醇酶(alphaenolase,ENO1)、Mn-SOD、谷胱苷肽S转移酶P(glutathioneS-transferaseP,GSTP1-1)、二氢二醇脱氢酶(dihydrodioldehydrogenase,DDH)、磷酸甘油酸变位酶1(phosphoglyceratemutase1,PGAM1)、葡萄糖依赖性胰岛素释放肽受体(glucose-dependentinsulinotropicproteinreceptor,GIPR)、肽基脯氨基顺反异构酶(peptidyl-prolylcis-transisomeraseA,PPIA)、磷脂酰乙醇胺结合蛋白(phosphatidylethanolaminebindingprotein,PEBP)、蛋白基因产物9.5(ubiquitincarboxyl-terminalhydrolaseisozymeL1,PGP9.5)、peroxiredoxin1(PDX1)、galectin-1及专利蛋白WO0222660等。RT-PCR检测示ENO1、DDH、PEBP、PGP9.5、PDX1、PGAM1、PPIA在肺癌组织中高表达。酶活性检测发现A549培养液中存在Mn-SOD,且发现非小细胞肺癌患者血清中有高水平的Mn-SOD。结论:本研究首次对非小细胞肺癌细胞的分泌蛋白进行了鉴定;发现多种高表达的非小细胞肺癌分泌蛋白基因,其中首次发现ENO1及PEBP基因在非小细胞肺癌中高表达;Mn-SOD是非小细胞肺癌的分泌型血清标志物。该研究为非小细胞肺癌血清标志物的筛选提供了新的方法和候选分子。
BACKGROUND & OBJECTIVE: The secretory proteins of cancer cells are potential serum markers, and also have important significance for the early screening of tumors. Therefore, systematic and comprehensive study of tumor cell secretory proteins is of great significance for the screening of tumor serum markers. The aim of this study was to identify the secreted proteins of lung cancer cells and provide experimental evidence for the screening of lung cancer serum markers. Methods: A549 cells were cultured in RPMI-1640 culture medium containing fetal bovine serum. Protein in A549 cell culture medium and fetal bovine serum culture medium (control) were collected for protein two-dimensional electrophoresis. Assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) and bioinformatics identification of A549 gel-specific protein spots. After screening the human protein spots, RT-PCR was used to detect the expression differences in lung cancer tissues and matched lung tissues. The contents of Mn-SOD and Mn-SOD )Level. Results: Fourteen human protein spots were identified including actin, ENO1, Mn-SOD, glutathione S-transferase P (GSTP1-1) Dihydrodiol dehydrogenase (DDH), phosphoglyceratemutase 1 (PGAM1), glucose-dependent insulinotropic protein receptor (GIPR), peptidylprolyl cis-trans isomerase peptidyl-prolylcis-transisomerase A (PPIA), phosphatidylethanolamine binding protein (PEBP), protein gene product 9.5 (PGP9.5), peroxiredoxin 1 (PDX1), galectin-1 and the patent protein WO0222660. RT-PCR showed that ENO1, DDH, PEBP, PGP9.5, PDX1, PGAM1 and PPIA were highly expressed in lung cancer tissues. Enzyme activity assay found Mn-SOD in A549 culture medium, and found that patients with non-small cell lung cancer serum levels of Mn-SOD. Conclusions: This study, for the first time, identified the secreted proteins of non-small cell lung cancer cells. We found a variety of highly expressed non-small cell lung cancer secreted protein genes, of which ENO1 and PEBP genes were first found to be highly expressed in non-small cell lung cancer and Mn-SOD Is a secreted serum marker for non-small cell lung cancer. This study provides new methods and candidate molecules for the screening of serum markers for non-small cell lung cancer.