人原发性肝癌相关抗原的蛋白质组学初步研究

Detection and validation of hepatocelluar carcinoma associated antigens by proteomics

目的检测肝细胞癌(hepatocellular carcinoma,HCC)的相关抗原,并予以验证。方法分别提取15例HCC组织及其癌旁组织总蛋白。Western blot分别检测各总蛋白与其HCC患者自体血清的杂交情况。双向电泳(two-dimensional electrophoresis,2-DE)技术将15例混合总蛋白中的各蛋白进行分离与染色。图像分析法比对染色胶上HCC组织及其癌旁组织蛋白的表达差异,基质辅助激光解吸/电离飞行时间二级质谱(matrix-assisted laser desorption/ionization-time-of-flight with two mass analyzers for tandem mass spectrometry,MALDI-TOF MS/MS)技术及生物信息学网络数据库分别对表达差异蛋白予以分析鉴定与检索匹配,免疫组织化学与Western blot分别检测该差异蛋白在人HCC组织及其癌旁组织中的变化情况。结果 2-DE染色胶图像分析比对结果显示,HCC及其癌旁组织蛋白表达差异斑点共90个,MALDI-TOF MS/MS与数据库对其中19个蛋白斑点分析鉴定与检索匹配发现15个蛋白表达上调,4个蛋白表达下调。免疫组织化学与Western blot检测结果显示,与对照比较,上调蛋白中的Dna J同源B亚家族成员11(dna J homolog subfamily B member 11,DNAJB11)在HCC组织中的表达明显增强,下调蛋白中的蛋白酶体亚基α3(proteasome subunit alpha type 3,PSMA3)在HCC组织中的表达则相反。结论人原发性肝癌相关抗原DNAJB11在HCC组织中的表达上调,PSMA3在HCC组织中的表达下调。

Objective To detect and validate the new specific antigens associated with hepatocelluar carcinoma （HCC）. Methods Total proteins from 15 HCC tissues and their adjacent tissues were extracted respectively. Western blotting was used to detect the interactions between the total proteins and autologous serums of the 15 HCC patients. Various proteins of the 15 mixed total proteins were separated and dyed by two-dimensional electrophoresis （2-DE）. Image analysis was employed to compare the expression differences of proteins extracted from HCC tissues and those from their adjacent tissues. The obtained proteins with different expression levels were analyzed and identified with matrix-assisted laser desorption/ionization-time-of-flight with two mass analyzers for tandem mass spectrometry （MALDI-TOF MS/MS）, and searched for their sequences in bioinformatics network database, and then confirmed by immunohistochemical assay and Western blotting respectively. Results The results of image comparison from 2-DE dyeing proteins revealed that a total of 90 protein spots with different expression levels were found. And among the 19 protein spots eventually identified by MALDITOF MS/MS, 15 proteins were up-regulated, and 4 down-regulated. Immunohistochemical assay and Western blotting confirmed that compared with the proteins from the adjacent tissues, DnaJ homolog subfamily B member 11 （DNAJB11） was significantly enhanced in these up-regulated proteins, and proteasome subunit alpha type 3 （PSMA3） was the most obviously down-regulated protein from HCC tissues. Conclusion Hepatocelluar carcinoma associated antigen, DNAJB11 is up-regulated, while PSMA3 is down-regulated in the HCC tissue.