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单次大分割放疗在体外对树突状细胞激活T细胞的影响
作者:郑声琴1  范璟2  郑勤1  童金龙1  田小强1 
单位:1. 南京中医药大学附属南京医院/南京市第二医院 肿瘤科, 江苏 南京 210003;
2. 南京中医药大学附属南京医院/南京市第二医院 中心实验室, 江苏 南京 210003
关键词:树突状细胞 放疗 肿瘤细胞 抗原负载 
分类号:R459.9
出版年·卷·期(页码):2021·40·第一期(46-52)
摘要:

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-α secreted 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-γ than 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.

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