慢性乙型肝炎外周血单核细胞的蛋白质组学分析筛选肝纤维化诊断标志物

Proteomic analysis of peripheral blood mononuclear cells to identify potential markers of fibrosis in chronic hepatitis B

目的 通过慢性乙型肝炎患者的外周血单核细胞（PBMCs）筛选诊断肝纤维化的标志物. 方法 入组48例慢性乙型肝炎的肝纤维化患者[其中24例为轻度肝纤维化（S1）,另24例为重度（S4）].用密度梯度离心法分离PBMCs且进行纯度测定,提取PBMCs蛋白质进行双向电泳分离,并通过液相色谱串联质谱（LC-MS/MS）鉴定出差异表达的蛋白质.患者年龄、单核细胞和淋巴细胞比例采用非参数检验分析,患者性别采用x2检验分析,血小板及白细胞数目采用t检验分析. 结果 在纯化的PBMCs中,24例S1和24例S4患者的血小板数目（×109/L）分别为19.268±6.413和19.480±6.538,与临床正常参考值（100 ～ 300）× 109/L相比,血小板数目明显减少,而两组之间的差异没有统计学意义（P=0.930）.通过双向电泳和LC-MS/MS分析,共鉴定了12个差异蛋白质. 结论 从PBMCs中筛选出的膜突蛋白、辅酶Q脱氢酶铁硫蛋白3等12个蛋白质对肝纤维化具有诊断潜能.这些蛋白质参与了细胞运动、细胞黏附、激酶和转录等重要生物过程,可能与肝纤维化的形成密切相关.

Objective To identify non-invasive biomarkers for diagnosis and/or prognosis of liver fibrosis in chronic hepatitis B （CHB）.Methods Peripheral blood samples were obtained from 48 patients with CHB,including 24 with mild fibrosis （stage 1,S1） and 24 with severe fibrosis （stage 4,S4）,and subjected to Ficoll density gradient centrifugation in order to obtain enriched samples of peripheral blood mononuclear cells （PBMCs）.The PBMC proteomes of the two groups were assessed by first separating the total proteins by two-dimensional gel electrophoresis （2DE） and then identifying the differentially expressed proteins by liquid chromatography combined with tandem mass spectrometry （LCMS/MS）.Results The enriched PBMC samples from the S1 group and the S4 group had similar amounts ofplatelets [（19.268 ± 6.413）×109/L and（19.480 ± 6.538）×109/L,respectively）; however,for both,the platelet amounts were 5 to 15-fold lower than that of the normal reference （100-300 ×109/L）.There was no significant difference found between the platelet amounts in the S1 patients and healthy controls （P =0.930）.Twelve differentially expressed proteins were identified through 2DE-LC-MS/MS,including proteins such as moesin and NADH dehydrogenase [ubiquinone] ironsulfur protein 3 that are involved in various biological processes like cell movement,cell adhesion,kinase signaling and transcription.Conclusions The 12 proteins with differential expression in S1 and S4 patients with CHB and liver fibrosis may represent markers related to development and/or progression of liver fibrosis.