异常岩藻糖基化特定蛋白在HBV相关肝癌中的诊断价值和分子机制研究

Most of tumor markers in clinical application are glycoproteins, which are reported to be associated with the tumor progression. Serum glycoproteins are synthesized by the liver or B-lymphocytes, so liver cancer has special connection with abnormal glycosylation. In our previous researches supported by two items of NSFC, we identified the abnormal changes and pathological significance of N-glycan markers on liver diseases. Based on our previous findings, this study we will clarify which types and how the abundances of the abnormal glycosylation exist in serum haptoglobin (HP) and alpha-2-HS-glycoprotein (AHSG) during the progression of liver cancer; Meanwhile, we developed a fucosylated HP and AHSG (Fuc-HP and Fuc-AHSG) detection kit as an ELISA using lectin and the anti-HP/ anti-AHSG antibody for HCC and examined the diagnostic value of Fuc- - HP and Fuc-AHSG in HCC patients; Further more, we investigated the mechanisms associated with the Fuc-HP and Fuc-AHSG production in HCC at a cellular and molecular level, so as to provide laboraotry evidences for clinical explanation. This study is strongly anticipated to develop a brand-new liver disease marker with our independent intellectual property right, supplementing the defects of alpha-fetoprotein in low sensitivity and providing new means for the early diagnosis of HCC.

目前临床应用的肿瘤标志物以糖蛋白为主,研究发现糖蛋白与肿瘤演进密切相关。外周血糖蛋白主要来源于肝脏及B淋巴细胞,提示肝脏肿瘤与异常糖基化有关。本实验室前期在两项NSFC资助下,阐述了肝病外周血N糖组异常改变规律及其病理意义,本课题在此基础上,重点探究肝脏源性特定糖蛋白结合珠蛋白(HP)及胎球蛋白A(AHSG)分子在肝癌中的异常糖基化结构特征,采用凝集素及抗体夹心的lectin-ELISA建立血清特殊糖基化-HP及AHSG(Fuc-HP/AHSG)的检测方法,并评价、验证其作为肝癌标志物的诊断价值；同时针对异常糖代谢相关酶进行干预表达,在细胞水平上探讨肝癌中岩藻糖基化特定蛋白产生机制及病理意义,为未来临床应用诠释提供依据。本研究建立并评价验证的lectin-ELISA检测Fuc-HP及Fuc-AHSG有望形成具有自主知识产权的肝癌标志物,补充甲胎蛋白敏感性不足的缺陷,为肝癌早诊提供新手段。

本研究在本实验室前期大量有关肝病N糖组学系列研究的基础上，以血清中单个特定蛋白胎球蛋白A为主要研究靶分子，在临床水平研究其蛋白水平及其岩藻糖基化水平在HBV相关肝病中的表达改变和潜在诊断价值。.本研究首先通过对原发性肝细胞癌（HCC）、肝硬化和健康对照组的血清胎球蛋白A水平进行分析。收集144例肝癌、肝硬化、正常组血清，用ELISA检测各病例组的胎球蛋白A血清水平。研究发现肝硬化组的血清胎球蛋白A 蛋白明显低于肝癌组和健康对照组（p=0.003）。其中胎球蛋白A 血清蛋白水平和实验室指标TBA、PT、APTT、PLT、IBIL、TBIL、DBil以及Child分级等呈负相关，与实验室指标RBC、TP、 ALB、A/G、AFU、CA199、HGB等呈正相关关系。.本研究自行建立凝集素酶联免疫吸附法（Lectin-ELISA方法）进行血清岩藻糖基化胎球蛋白A水平的检测。本方法根据抗体夹心ELISA原理基础上开发，使用胎球蛋白A抗体包被ELISA板，抓取样本中的抗原后，用特异性的凝集素识别抗原上的岩藻糖结构，以用于检测抗原岩藻糖基化水平。通过条件优化确定包被抗体、标本稀释和特异性识别凝集素的最适工作浓度；采用混合血清的一定倍比稀释梯度为样本标准品建立标准曲线；以重复性、最低检测限、交叉反应、血清胆红素、乳糜等对血清检测的干扰实验评价该Lectin-ELISA方法。.最后，应用Lectin-ELISA方法检测各组血清岩藻糖基化胎球蛋白A水平，通过分析肝癌、肝硬化、HBV无症状携带者、健康对照各组的血清岩藻糖基化胎球蛋白水平，探讨血清岩藻糖基化胎球蛋白A水平对肝癌、肝硬的诊断价值。建模组收集144例肝癌、肝硬化、健康对照组血清，研究发现肝癌组、肝硬化组血清岩藻糖基化胎球蛋白A水平显著高于健康对照组。 验证组研究发现肝癌组、肝硬化组血清岩藻糖基化胎球蛋白A水平显著高于HBV无症状携带者、健康对照组，差异有统计学意义（P<0.05）。采用Cutoff值为1.12，鉴别肝硬化组与HBV无症状携带者、健康对照组时，其ROC曲线下面积为0.906。