肝癌患者外周血中抑制树突状细胞分化的蛋白成分筛选及鉴定  

Screening and identification of suppressor proteins targeting dendritic cells in peripheral blood of hepatocellular carcinoma

作　　者：董晓微[1] 郭东 董育清 柳斌 樊瑞军[1] DONG Xiaowei;GUO Dong;DONG Yuqing;LIU Bin;FAN Ruijun(Clinical Laboratory and Diagnosis Center,People′s Hospital of Ningxia Autonomous Region,Yinchuan 750002,China)

机构地区：[1]宁夏回族自治区人民医院临床医学检验诊断中心,宁夏银川750002

出　　处：《宁夏医学杂志》2024年第7期573-577,F0003,共6页Ningxia Medical Journal

基　　金：宁夏自然科学基金项目(2022AAC03366)。

摘　　要：目的肝癌患者外周血中靶向抑制树突状细胞分化成熟的蛋白成分筛选及鉴定。方法利用免疫磁珠分选健康人外周血CD14^(+)单核细胞,诱导分化为未成熟的树突状细胞(iDCs),进一步诱导分化为成熟的树突状细胞(mDCs),鉴定iDCs纯度。酶切健康人和肝癌患者血清孵育的iDCs的结合蛋白,回收酶切后的多肽,筛选对iDCs分化成熟有潜在调控作用的互作候选蛋白。靶向鉴定差异候选蛋白。构建过表达蛋白质粒,转染至正常肝细胞(HL7702),将细胞培养上清液与iDCs共同培养,诱导iDCs成熟,对特异性抑制iDCs成熟的肝癌源性血清蛋白进行特异性筛选和鉴定。结果TNF-α诱导前抗原标志物阳性细胞表达CD86^(+)比例为79.2%、黏附分子CD40^(+)比例为83.3%、MHC-Ⅱ抗原分子HLA-DR^(+)比例为67.6%、CD83^(+)比例为5.5%以及CD1a^(+)比例为13.7%,符合iDCs的抗原表型特征;TNF-α诱导后抗原标志物阳性细胞表达CD86^(+)比例为92.0%、CD40^(+)比例为89.0%、HLA-DR^(+)比例为68.3%、CD83^(+)比例为37%、CD1a^(+)比例为48.3%,提示iDCs已迅速分化成熟为mDCs,符合mDCs的抗原表型特征。初步确认40多种对iDCs分化成熟有潜在调控作用的互作候选蛋白。选择10种差异候选蛋白,对其过表达进行验证,显示AFP蛋白、AGXT蛋白、ATP1A1蛋白过表达能够明显抑制iDCs分化成熟为mDCs。结论共筛选出10种靶向抑制树突状细胞分化的肝癌外周血清蛋白,其中AFP蛋白、AGXT蛋白、ATP1A1蛋白能够显著抑制树突状细胞分化成熟。Objective To screen and identify the inhibitory proteins targeting dendritic cells in peripheral blood of hepatocellular carcinoma.Methods CD14^(+)Monocyte from healthy human peripheral blood were sorted and induced to differentiate into immature dendritic cells(iDCs),and then into mature dendritic cells(mDCs).The purity of iDCs was identified.Enzymatic digestion of binding proteins of iDCs incubated with serum from healthy individuals and liver cancer patients,recovery of peptides after enzymatic digestion,and screening of potential interacting candidate proteins that have potential regulatory effects on the differentiation and maturation of iDCs.Targeted identification of differential candidate proteins.The overexpression protein particles were constructed,transfected into normal hepatocytes(HL7702)and cultured.The supernatant of overexpression protein hepatocytes and normal hepatocytes were co cultured with iDCs to induce the maturation of iDCs.The hepatoma derived serum proteins that specifically inhibit the maturation of iDCs were screened and identified.Results The proportion of pre induction antigen marker positive cells conforms to the antigen phenotype characteristics of iDCs;The proportion of antigen marker positive cells after induction conforms to the antigen indication characteristics of mDCs.Preliminary identification of over 40 potential interacting candidate proteins that have potential regulatory effects on the differentiation and maturation of iDCs.Select 10 differential candidate proteins.Verification of its overexpression showed that overexpression of AFP protein,AGXT protein,and ATP1A1 protein significantly inhibited the differentiation and maturation of iDCs into mDCs.Conclusion Ten inhibitory proteins targeting dendritic cells in peripheral blood of hepatocellular carcinoma were screened.AFP,AGXT and ATP1A1 could significantly inhibit the maturation of dendritic cells.

关 键 词：肝癌 外周血树突状细胞 抑制蛋白 筛选 鉴定 

分 类 号：R735.7[医药卫生—肿瘤]