GPC3刺激对DC-CTL靶向杀伤肝细胞癌的影响

Effect of GPC3 Stimulation on DC-CTL Targeted Killing of Liver Hepatocellular Carcinoma

目的本研究将磷脂酰肌醇蛋白聚糖3(GPC3)作为肝细胞癌(LIHC)的靶分子,研究其在激活树突状细胞(DC)-细胞毒性T淋巴细胞(CTL)和增强肿瘤杀伤能力中的作用。方法为更系统、更全面地鉴定GPC3在多种恶性肿瘤中的意义,对GPC3在泛癌中的基因表达水平进行评估,并从LIHC与临床特征的相关性角度进一步研究GPC3。分别通过生存预后分析(Kaplan-Meier图)和受试者工作特征(ROC)曲线分析评估GPC3在LIHC中的预后价值和诊断价值。重组GPC3蛋白用于刺激DC,然后与CTL细胞共培养,经酶联免疫吸附试验(ELISA)分析以检测DC-CTL细胞中细胞因子的表达。通过CCK8测定法测定在Huh7细胞上用GPC3培养刺激的DC-CTL细胞的细胞毒性。结果GPC3的基因表达水平在大多数癌症中显著不同,并且在LIHC中与正常组织相比更高。GPC3在血清中与年龄和甲胎蛋白(AFP)水平相关(均P<0.001),而与LIHC的TNM分期、病理分期和组织学分级无关。ROC曲线分析表明,GPC3可用于准确预测LIHC。GPC3的表达水平与LIHC的总生存期无关。联合治疗显著上调了IFN-γ和TNF-α的分泌。用150 ng/ml和200 ng/ml GPC3刺激的DC-CTL细胞杀死Huh7细胞的能力强于不使用GPC3刺激的DC-CTL细胞(均P<0.05)。结论用GPC3刺激DC-CTL细胞通过促进细胞毒性在体外抑制肿瘤生长。GPC3可能是一种潜在的肿瘤标志物,可用于提高DC-CTL细胞的治疗效率。

Objective This study applied GPC3 as a target molecular for liver hepatocellular carcinoma(LIHC)in order to study its role in activating dendritic cell(DC)-cytotoxic T lymphocytes(CTL)and enhancing the tumor killing ability.Methods To provide a more systematic and comprehensive dimension in identifying the diverse significance of GPC3 in a variety of malignant tumors,the gene expression level of GPC3 in pan-cancer was assessed.Furthermore,we focused on LIHC and further investigated GPC3 from the perspective of the correlations with clinical characteristics.The prognostic value and diagnostic value of GPC3 in LIHC were assessed by survival prognosis analysis(Kaplan–Meier plots)and receiver operating characteristic(ROC)curve analysis,respectively.Recombinant GPC3 protein was used to load DCs,and the cells were then coincubated with CTL cells.Enzyme linked immunosorbent assay(ELISA)analysis was performed to detect the expression of cytokines in DCCTL cells.The cytotoxic efficiency of DC-CTL cells stimulated with GPC3-cultured on Huh7 cells was determined by CCK8 assays.Results The gene expression level of GPC3 differs significantly in most cancers and is dramatically higher in LIHC compared with normal tissues.GPC3 is identified to be significantly correlated with age and alpha fetoprotein(AFP)level in serum,while not correlated with TNM stage,histological type,or histologic grade in LIHC.ROC curve analysis showed that GPC3 could be used to accurately predict LIHC.The expression level of GPC3 was no correlated with the overall survival of LIHC.Combined treatment significantly up-regulated the secretion of IFN-γand TNF-α.DC-CTL cells stimulated with 150 ng/ml and 200 ng/ml GPC3 had a stronger ability to kill Huh7 cells than DC-CTL cells stimulated without GPC3(both P<0.05).Conclusions This study demonstrates that stimulation of DC-CTL cells with GPC3 significantly suppressed tumor growth in vitro by promoting cytotoxicity.GPC3 may be a potential tumor marker that could be used to improve the therapeutic efficiency of DC-CTL cells.