小剂量X射线对人外周血树突状细胞功能影响研究

发布时间：2018-06-11 19:29

本文选题：X射线 + 辐射　；参考：《辽宁医学院》2012年硕士论文

【摘要】：目的观察小剂量X射线照射对体外培养不同时间的人外周血树突状细胞（dendriticcell, DC）凋亡、成熟能力、抗原递呈能力和致敏T细胞能力的影响并探讨其初步机制，为系统研究小剂量照射对人免疫功能影响提供实验依据。方法分离人外周血单个核细胞（PBMC），以细胞因子GM-CSF及IL-4诱导分化、肿瘤抗原及TNF-α刺激DC成熟，以LIN抗体纯化，HLA-DR和CD11C抗体鉴定。实验分为三组：A组：DC培养至第2天接受X线照射；B组：DC培养至第6天接受X线照射，A、B组细胞受照剂量分别为0.05Gy、0.1Gy、0.2Gy和0.5Gy；对照组为假照射组。照射后24h及48h分别以Annexin V联合PI双染法检测DC凋亡率，流式细胞仪检测细胞膜表面分子标记物CD80、CD83表达量；各组DC培养至第8天时，以DC致敏T细胞，CCK-8(Cell Counting Kit-8)法检测混合淋巴细胞反应（MLR，mixed lymphocyte reaction)；MTT法检测A549细胞数量；ELISA检测DC细胞培养液中白介素12（IL-12）、淋巴细胞培养液中γ-干扰素（IFN-γ）水平。结果 1.按本实验方法培养的DC成熟度高，纯度高，可用于本研究。 2.0.5Gy照射培养不同时间DC，A、B两组24、48h检测凋亡率显著高于对照组(A组24、48h, t值分别2.88，2.54；B组24、48h，t值分别为2.97，3.73,P 0.05)，其它剂量组无显著差异（P0.05）。 3.0.2Gy、0.5Gy照射后，A组CD80、CD83表达量显著下降（P0.05），B组CD80、CD83表达量显著上升（P0.05）；其它剂量组无显著差异（P0.05）。 4.受到0.2Gy、0.5Gy照射的DC，培养至第8天时其致敏T细胞增殖的数量，A组显著低于对照组（t值分别为2.79、3,71，P0.05），B组明显高于对照组（t值分别为3.60、3.11，P0.05），，其它剂量组无显著差异（P0.05）；0.2Gy、0.5Gy照射，A组DC分泌IL-12水平明显下降（t值分别为4.44、6.93，P0.05），B组DC分泌IL-12水平明显上升（t值分别为3.51、4.12，P0.05），其它剂量组无显著差异（P0.05）。 5. DC致敏T淋巴细胞对A549的杀伤能力：0.2Gy、0.5Gy照射后，A组明显低于对照组（t值分别为2.89、2.91, P0.05），B组显著高于对照组（t值分别为2.91、2.82, P0.05）；0.05Gy和0.1Gy照射后，A组及B组与对照组比较，所测各指标无明显变化(P0.05)。0.2Gy、0.5Gy照射后，A组DC致敏的T细胞分泌IFN-γ水平下降，而B组DC致敏的T细胞分泌IFN-γ水平明显上升(P0.05)；0.05Gy和0.1Gy照射后，A组及B组与对照组比较，所测各指标无明显变化(P0.05)。结论 0.5Gy X射线照射后24h及48h，DC的凋亡率显著增加。培养早期(2d，A组)接受0.2Gy及0.5Gy X射线照射的DC，在培养至第8天时，其成熟能力、抗原递呈能力、分泌IL-12的能力以及致敏T细胞能力均显著降低；培养晚期(6d，B组)接受0.2Gy及0.5Gy X射线照射的DC，在培养至第8天时，其成熟能力、抗原递呈能力、分泌IL-12的能力以及致敏T细胞的能力显著增强。该发现对研究小剂量核辐射对人免疫系统功能影响、探索一种体外提高DC功能的新方法提供了实验依据。
[Abstract]:Objective to observe the effects of low dose X-ray irradiation on apoptosis, maturation, antigen presentation and sensitized T cells of human peripheral blood dendritic cells (DCs) cultured in vitro for different time and to explore its mechanism. Methods PBMCs were isolated from human peripheral blood mononuclear cells (PBMC) and differentiated by cytokines GM-CSF and IL-4. Tumor antigen and TNF- 伪 stimulated DC maturation. HLA-DR and CD11C antibodies were purified by Lin antibody. The experiment was divided into three groups: group A: DC was cultured to the second day after X ray irradiation, group B: DC was cultured to the 6th day, the irradiation dose of cells in group A was 0. 05 Gy 0. 1 Gy and 0. 5 Gy, respectively, and the control group was sham irradiation group. The apoptotic rate of DC was detected by Annexin V combined with Pi double staining at 24 h and 48 h after irradiation, and the expression of CD80 CD83, a molecular marker on cell membrane, was detected by flow cytometry. The mixed lymphocyte reaction (MLR mixed lymphocyte reactionation) assay was used to detect the number of A549 cells. Elisa was used to detect the levels of IL-12 and IFN- 纬 in DC cell culture medium. The DC cultured by this method has high maturity and purity. It can be used in the present study. The apoptotic rate in the two groups was significantly higher than that in the control group A at 2448 hours, and the t value in group B was 2.97 ~ 3.73 渭 g / h, respectively. There was no significant difference between the other dose groups in the expression of CD80CD83 in group A after irradiation with 0.5 Gy of 0.2Gy or 0.5 Gy, and the expression of CD80CD83 in group A was significantly higher than that in group A after irradiation with 2.0.5 Gy irradiation at different time. The expression of CD80CD83 in group A was significantly higher than that in group A (2.88 卤2.54). In group B, the expression of CD80 and CD83 increased significantly (P 0.05), but there was no significant difference in other dose groups (P 0.05). The number of T cell proliferation in group A was significantly lower than that in group A (2.79 渭 g / kg), respectively, and the t value of group B was significantly higher than that of group B (3.60 卤3.11g / ml), respectively. There was no significant difference between other dose groups (P0.05Gy) and 0.2Gy (0.2Gy) in DC cells of group A, and the number of T cell proliferation in group A was significantly lower than that in group B (P < 0.05). The level of IL-12 secreted by DC was significantly increased in group B (4.44 卤6.93), respectively. The level of IL-12 secreted by DC was 3.51 卤4.12, P 0.05, respectively, but there was no significant difference in other dose groups. The cytotoxicity of DC sensitized T lymphocytes to A549 was significantly lower in group A than that in group A (2.89 卤2.91) after 0.5 Gy irradiation, and significantly higher in group B than that in group B (2.91 卤2.82, P 0.05Gy 0.05Gy and 0.1 Gy, respectively). The levels of IFN- 纬 in DC sensitized T cells in group A decreased, while those in group B increased significantly after irradiation with P0.05Gy, 0.2Gy, 0.5Gy, respectively, and the levels of IFN- 纬 in group A and group B were significantly increased after irradiation with P0.05Gy and 0.1 Gy, and compared with those in group B and group B, respectively, in comparison with those in group B, and that in group B was significantly higher than that in group B, and the level of IFN- 纬 in group A was significantly higher than that in group B. Conclusion the apoptosis rate of DC increased significantly at 24 h and 48 h after 0.5 Gy X-ray irradiation. At the 8th day, the ability of maturation, antigen presentation, IL-12 secretion and sensitized T cells were significantly decreased in group A (group A) exposed to 0.2 Gy and 0.5 Gy X-ray. At the 8th day, the ability of maturation, antigen presentation, IL-12 secretion and sensitized T cells were significantly enhanced in group B (group B) exposed to 0.2Gy and 0.5Gy X-rays at the end of the incubation period, and the ability of antigen presentation, secretion of IL-12 and the ability of sensitized T cells were significantly enhanced at the 8th day of culture. The findings provide an experimental basis for the study of the effects of low dose nuclear radiation on the function of human immune system and the exploration of a new method to improve the function of DC in vitro.
【学位授予单位】：辽宁医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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