单次大分割放疗在体外对树突状细胞激活T细胞的影响

Effects of single fractionation radiotherapy on dendritic cells activation to T cell in vitro

目的:探究单次大分割放疗在体外对树突状细胞(DC)激活T细胞的影响。方法:收集肝癌患者外周血单核细胞(PBMC),在细胞因子刺激下体外培养获得DC;分别在15Gy/1f和5Gy/3f条件下对HepG2细胞进行照射,通过离心获得肿瘤相关抗原;采用流式细胞术及ELISA法,检测负载了这两种抗原的DC细胞表型HLA-DR、CD40、CD80、CD83、CD86以及细胞因子IL-12p70、IL-1β、IL-6、TNF-α分泌变化;通过胞内染色法检测负载这两种抗原的DC对特异性T细胞的激活情况;利用MTT法检测上述被活化的T细胞对HepG2细胞的杀伤活性。结果:15Gy/1f组负载肿瘤相关抗原的DC表达细胞表型HLA-DR、CD40、CD80、CD83及CD86均高于5Gy/3f组(P<0.05);15Gy/1f组负载肿瘤相关抗原的DC分泌细胞因子IL-12p70和IL-6高于5Gy/3f组(P<0.05);胞内染色结果显示,15Gy/1f组负载肿瘤相关抗原的DC相比5Gy/3f组更能刺激CD4^(+)T细胞[(0.392±0.187)%vs(0.315±0.118)%]和CD8^(+)T细胞[(0.362±0.159)%vs(0.119±0.090)%]分泌IFN-γ(P<0.05);MTT实验结果显示,15Gy/1f组激活的T细胞对HepG2细胞的杀伤活性更强(P<0.05)。结论:单次大分割放疗更能刺激DC对特异性T细胞的活化作用,这为今后肝癌的治疗提供了理论依据。

Objective:To investigate the effect of single large fraction radiotherapy on dendritic cells activation to T cell in vitro.Methods:Peripheral blood PBMC were collected from hepatocellular carcinoma patients.Then,DC was obtained in vitro under the stimulation of cytokines.HepG2 cells were irradiated at 15Gy/1f and 5Gy/3f,respectively.Tumor associated antigens were obtained by centrifugation.The changes of cell phenotypes of HLA-DR,CD40,CD80,CD83 and CD86 by 15Gy/1f TAA DC and 5Gy/3f TAA DC were detected with flow cytometry.The concentration of cytokines IL-12p70,IL-1β,IL-6 and TNF-αsecreted by 15Gy/1f TAA DC and 5Gy/3f TAA DC were detected using ELISA.Intracellular staining was used to detect the activation of specific T cells by DC loaded with 15Gy/1f and 5Gy/3f TAA.MTT assay was used to detect the killing activity of the above activated T cells on HepG2 cells.Results:The cell phenotypes of HLA-DR,CD40,CD80,CD83 and CD86 of DC loaded with 15Gy/1f TAA were all higher than those of 5Gy/3f group(P<0.05).The concentration of cytokines IL-12p70 and IL-6 secreted by DC loaded with 15Gy/1f TAA were higher than those in the 5Gy/3f group.Intracellular staining results showed that DC loaded with 15Gy/1f TAA tended to stimulate CD4^(+)T cells[(0.392±0.187)%vs(0.315±0.118)%]and CD8^(+)T cells[(0.362±0.159)%vs(0.119±0.090)%]to secret IFN-γthan 5Gy/3f group(P<0.05).MTT results showed that T cells activated by 15Gy/1f group had stronger killing activity on HepG2 cells than 5Gy/3f group(P<0.05).Conclusion:Single high fractionation radiotherapy can stimulate the activation of specific T cells by DC.This provides a theoretical basis for the treatment of liver cancer in the future.