聚糖蛋白抗体的制备及其在肝癌组织及细胞系蛋白表达检测中的应用

目的制备聚糖蛋白3(glypican-3,GPC3)抗体并应用其对肝癌组织及细胞系中GPC3蛋白表达进行研究,探讨GPC3蛋白作为肝癌潜在的新的肿瘤标志物的可能性。方法利用原核表达系统诱导表达了GPC3抗原并制备了特异性好的兔抗GPC3多克隆抗体,应用该纯化的抗体对40例组织(正常肝7例,肝细胞型肝癌26例,胆管细胞型肝癌7例)及4个细胞系(肝癌细胞系HepG2、HuH-7、Hep3B,非肝癌细胞系Hela)进行了蛋白印记杂交试验。结果Western blot结果显示,肝癌组织及肝癌细胞系HepG2、HuH-7、Hep3B细胞系中有相对相对分子质量约70000的蛋白主带,与GPC3的核心蛋白带相对分子质量大小相一致;正常肝组织不表达GPC3蛋白,肝细胞型肝癌中癌组织(HCC)及癌旁组织表达GPC3蛋白的比例分别为84.6%(22/26)及30.8%(8/26),7例胆管细胞型肝癌(ICC)中有2例癌组织及其旁组织表达GPC3。结论GPC3蛋白广泛存在于肝癌组织中,很可能成为一种新的肝癌肿瘤标志物。

Preparation of anti-GPC3 polyclonal antibody and its application in expression detection of GPC3 protein in liver cancer and HCC cell lines

Objective To study the expression of GPC3 protein in liver cancer and HCC cell lines by anti-GPC3 polyclonal antibody,and to evaluate the possibility of GPC3 protein as a novel tumor marker for liver cancer. Methods By E. coli expressing system, the GPC3 protein as antigen was induced, and the rabbit anti-GPC3 polyclonal antibody was prepared. The purified antibody was used to do Western blot analysis in 40 liver tissues (7 normal liver tissues,26 HCC liver tissues and 7 ICC liver tissues) and 4 cell lines ( HepG2,HuH-7,Hep3B, Hela). Results A main protein band of 70 kD which corresponding to GPC3 core protein was found both in liver cancer tissues and HCC cell lines HepG2, HuH-7,Hep3B by Western blot test. It was found that normal liver tissues did not express GPC3 protein, but in 26 HCC patients, the percentage of GPC3 protein expressed in liver cancer tissues and its corresponding non-cancerous liver tissues was 84. 6% (22/26) and 30. 8% (8/26) .respectively. Among 7 ICC patients, only 2 patients had GPC3 protein expression. Conclusion GPC3 protein is distributed widely in the tissues of liver cancer,which may be a novel tumor marker for liver cancer.