单次大分割放疗在体外对树突状细胞激活T细胞的影响-东南大学学报医学版

单次大分割放疗在体外对树突状细胞激活T细胞的影响

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

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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