乙型肝炎病毒相关肝癌血清差异表达蛋白的筛选、鉴定及其诊断价值

Detection,identification and diagnostic value of differential proteins in sera of patients with HBV-related primary hepatic carcinoma

目的:筛选、鉴定乙型肝炎病毒(HBV)相关肝癌的血清差异表达蛋白,探讨其可能的早期诊断价值。方法:采用蛋白芯片及表面增强激光解析电离飞行时间质谱(surface-enhanced laser desorption/ionization time-of-flight mass spectrometry,SELDI-TOF-MS)技术对正常人、HBV相关肝炎、肝硬化患者及原发性肝癌患者术前血清分别进行检测,筛选肝癌血清差异表达蛋白;通过离子交换柱分离纯化其中表达差异最明显的蛋白,并进行质谱鉴定。根据筛选结果,建立肝癌诊断决策树模型,评价其诊断肝癌的准确性和敏感度。结果:与正常组相比,肝癌组有44个差异蛋白质波峰(P<0.05),其中21个上调,23个下调;与肝硬化组相比,肝癌组有51个差异蛋白质波峰(P<0.05),其中47个上调,4个下调。质荷比为5805的蛋白质表达在正常人群<慢性肝炎组<肝硬化组<肝癌组,在肝癌组中表达最高,差异具有统计学意义(P<0.01);对其酶解消化产物进行肽指纹谱(PMF)鉴定发现其中有2条肽段序列与硫酸软骨素合酶Ⅱ部分序列相匹配。根据筛选的差异表达蛋白成功建立肝癌诊断决策树模型,其判断肝癌的准确率为94.82%[(23+32)/58]、敏感性88.46%(23/26)、特异性100%(32/32)。结论:成功筛选HBV相关肝癌血清差异表达蛋白,其中差异最显著蛋白(质荷比为5805)酶解产物中有2条肽段与硫酸软骨素合酶Ⅱ部分序列相匹配;根据筛选结果建立的肝癌诊断决策树模型有助于早期诊断HBV相关肝癌。

Objective：To detect and identify differentially expressed proteins in the sera of patients with HBV-related primary hepatic carcinoma and discuss their possible role in early diagnosis of primary hepatic carcinoma. Methods： The surface-en- hanced laser desorption/ionization time-of-flight mass spectroscopy （SELDI-TOF-MS） was used to screen for the differentially expressed serum proteins in patients with HBV-related primary hepatic carcinoma,liver cirrhosis,chronic hepatitis,and healthy adults. Then the most differential protein sample was isolated and purified by ionic exchange chromatography and was subjected to mass spectrometry analysis. The screening results were used to establish a diagnosis model for hepatic carcinoma and the accu- racy and sensitivity of the model were assessed. Results： CM10（weak cation exchange）chip found 44 differentially expressed pro- tein peaks （P〈0.05） in hepatic carcinoma group compared with those in the healthy adult group, with 21 up-regulated and 23 down-regulated. Fifty-one differential peaks were identified in hepatic carcinoma group compared with those in the liver cirrhosis group （P〈0.05）, with 47 up-regulated and 4 down-regulated. The expression of protein with a mass-to-charge ratio （m/z） of 5 805 gradually increased in order in the healthy adult, chronic hepatitis, liver cirrhosis and hepatic carcinoma patients; and the expression in hepatic carcinoma group was significantly higher than that in the healthy adult group （P〈0.01）; peptide mass fingerprint （PMF） after enzyme hydrolysis showed that 2 of the peptides were partially identical to chondroitin sulfate synthase 2. A diagnosis model of hepatic carcinoma was successfully established based on the differentially expressed proteins, with an accuracy of 94.82%（ [23＋32]/58 ）,a sensitivity of 88.46% （23/26） ,and a specificity of 100% （32/32）. Conclusion： We have successfully screened out the differentially expressed proteins in the sera of patients with HBV-related primary hepatic carcinoma： the most differentially expressed protein （5 805） has 2 peptides partially identical to chondroitin sulfate synthase 2 in sequence. The established model may help to diagnose HBV-related primary hepatic carcinoma.