HPV-16 E7负载的DC-CIK对宫颈癌细胞的杀伤效应研究

Study the killing effect of HPV-16 E7-loaded DC-CIK on cervical carcinoma cells

目的研究HPV-16 E7抗原肽负载的树突状细胞(DC)与细胞因子诱导杀伤(CIK)细胞共培养Ag-DC-CIK对人宫颈癌细胞siha的杀伤效应。方法采集健康人外周血,分离提取单个核细胞(PBMC),分别诱导生成DC和CIK细胞,观察细胞形态,流式细胞仪检测DC摄取能力及其表面分子CD83、CD86和CIK细胞CD3、CD8、CD56的表达。酶联免疫吸附试验(ELISA)法检测白细胞介素-12(IL-12)、干扰素-γ(IFN-γ)的水平。实验分为5组:效应细胞CIK、DC-CIK、Ag^1-DC-CIK、Ag^2-DC-CIK、Ag^M-DC-CIK,分别与靶细胞siha共培养,细胞计数试剂盒(CCK8)法检测效应细胞对siha活性的影响,乳酸脱氢酶(LDH)释放法检测效应细胞对siha的杀伤效应。结果DC成熟后,摄取能力减弱,CD86、CD83表达上调,上清液中IL-12水平增高;与DC共培养的CIK细胞,CD3^+CD8^+、CD3^+CD56^+细胞比例提高,分泌IFN-γ能力增强;与CIK细胞、DC-CIK组相比,Ag^1-DC-CIK、Ag^2-DC-CIK、Ag^M-DC-CIK组均表现出对siha细胞更强的杀伤效应。结论成功构建Ag-DC-CIK共培养体系,获得两组有效的HPV-16 E7蛋白抗原肽,为治疗宫颈癌提供了新思路。

Objective To study the killing effect of HPV-16 E7-loaded dendritic(DC)cells co-cultured with cytokine-induced killing(CIK)cells on cervical carcinoma cells.Methods The peripheral blood of healthy adults was collected,and the peripheral blood mononuclear cells(PBMC)were separated,then DC and CIK were induced and cultured.The cell morphology was observed under microscope.Flow cytometry was used to analyze the uptake capacity and surface markers CD86 and CD83 of the DC,and CD3,CD8 and CD56 of the CIK.Levels of IL-12 and IFN-γwere determined by enzyme-linked immunosorbent assay(ELISA).The effector cells were divided into five groups:CIK,DC-CIK,Ag^1-DC-CIK,Ag^2-DC-CIK and Ag^M-DC-CIK,which were co-cultured with target cells siha respectively.The viability of siha was detected by CCK-8 and the killing effects of effector cells were detected by LDH release method.Results The uptake capacity of mature DC was weaken,but the surface markers CD86,CD83 were up regulated,and the levels of secreted IL-12 in the supernatants were higher than that of immature DC.The proportions of CD3^+CD8^+,CD3^+CD56^+were significantly elevated in DC-CIK,and the levels of IFN-γin the supernatants were higher than that of CIK group.The killing effects of Ag^1-DC-CIK,Ag^2-DC-CIK and Ag^M-DC-CIK were obviously higher than CIK and DC-CIK.Conclusion The co-culture system of DCs-CIKs was successfully constructed,and two effective HPV-16 E7 antigen peptides were obtained,which provides an experimental basis for therapy of cervical carcinoma.