乙肝相关肝细胞肝癌血清蛋白标志物的筛选

The selection of serum protein markers for HBV-associated hepatocellular carcinoma

目的筛选肝细胞肝癌(hepatocellular carcinoma,HCC)早期诊断特异血清蛋白标志物。方法选取2013年3月至2014年6月在我院肝胆外科进行根治性切除手术的乙型肝炎病毒(hepatitis B virus,HBV)相关性HCC患者20例,同期选取30例健康志愿者、20例慢性乙肝患者、20例HBV肝硬化患者做为对照组。采集受试者血清,采用胰蛋白酶酶解后,以强阳离子交换与反相C18二维一体色谱柱分离并以超高分辨质谱对多肽液进行检测,并对检测结果进行统计学分析。结果检测结果鉴定出3578种蛋白,筛选出富组氨酸糖蛋白(histidine-rich glycoprotein,HRG)、血红蛋白β亚基、血红蛋白α亚基、铜蓝蛋白(ceruloplasmi,CP)、α-1B-糖蛋白(alpha-1B-glycoprotein,A1BG)、抗凝血酶Ⅲ(antithrombin-Ⅲ,ATⅢ)和甲胎蛋白(alpha-fetoprotein,AFP)7种HCC血清分子标志物。HBV相关性HCC组与HBV肝硬化组相比,A1BG及AFP相对表达量增加,ATⅢ相对表达量减少;与慢性乙肝组相比,CP及AFP相对表达量增加,血红蛋白α亚基相对表达量减少;与健康对照组相比,AFP相对表达量增加,HRG及血红蛋白β亚基相对表达量减少,差异均有统计学意义(P均<0.05)。结论 7种血清蛋白标志物全部由肝脏合成,与HCC的发病机制密切相关,血清蛋白指纹图谱及多标志物联合检测可以为肝脏肿瘤疾病临床诊断提供新的思路。

Objective To select specific serum protein markers for the early diagnosis of hepatocellular carcinoma（HCC）. Methods 20 cases hepatitis B virus（HBV） associated HCC patients, 30 cases healthy patients, 20 cases chronic hepatitis B（CHB） patients, and 20 cases HBV associated hepatic cirrhosis from March 2013 to June 2014 in department of hepatobiliary surgery of our hospital were collected. The serum samples of all the subjects were collected and digested by trypsin and then analyzed in homemade strong cation exchange and reverse-phase two-dimensional capillary chromatographic column, and the results were analyzed statistically. Results 3578 kinds proteins were selected and 7 kinds HCC related serum markers including histidine-rich glycoprotein（HRG）, hemoglobin subunit alpha, hemoglobin subunit beta, ceruloplasmi（CP）,alpha-1B-glycoprotein（A1BG）, antithrombin-Ⅲ（ATⅢ）and alpha-fetoprotein（AFP） were identified. Compared with HBV associated hepatic cirrhosis group, the expression levels of A1 BG and AFP were higher and ATⅢ was lower in HBV associated HCC group, and the differences all had statistical significance（Pall〈 0.05）.Compared with CHB group, the expression levels of CP and AFP were higher and hemoglobin subunit alpha was lower in HBV associated HCC group, and the differences all had statistical significance（Pall〈 0.05）.Compared with healthy control group, the expression level of AFP was higher and the HRG and hemoglobin subunit beta were lower, and the differences all had statistical significance（Pall〈 0.05）. Conclusion All these 7 kinds serum protein markers which synthesized in liver are all closely related with HCC incidence and pathogenesis. The proteomic fingerprint and detection of multi-markers provide new ideas for clinical liver tumor diagnosis.