目的  在非小细胞肺癌中，放射治疗抵抗降低患者生存率，是NSCLC治疗的重要挑战。已有研究表明，自分泌会影响肿瘤的辐射敏感性，但在非小细胞肺癌中，自分泌物的主要作用目前尚不明确。本研究通过观察NSCLC细胞系（H460、H1299、A549）自分泌物对其辐射敏感性的影响，并从整体角度探寻自分泌因子增强细胞辐射抵抗作用的主要机制。
方法  采用人肺癌细胞系H460、H1299、A549体外培养作为研究对象，采用克隆形成实验联合Transwell共培养比较自分泌对非小细胞肺癌细胞（H460、H1299、A549）辐射敏感性的影响及293T细胞对非小细胞肺癌H460细胞辐射敏感性的影响；克隆形成实验比较自分泌条件培养基对H460细胞辐射敏感性的影响；MTT法观察条件培养基对照射后H460细胞增殖情况的影响；流式细胞术检测条件培养基对照射后H460细胞活性氧的表达情况和细胞周期的影响；qRT-PCR检测细胞内SOD1mRNA水平的变化，蛋白免疫印迹实验检测细胞内SOD1蛋白水平的变化；采用细胞免疫荧光法观察照射后H460细胞经条件培养基培养不同时间的γH2AX变化；蛋白免疫印迹实验检测H460细胞经条件培养基培养不同时间的γH2AX和DNA修复相关蛋白的变化；差速离心法收集H460细胞分泌的外泌体；蛋白免疫印迹和透射电镜验证外泌体；克隆形成实验检测外泌体对H460细胞辐射敏感性的影响。
结果  非小细胞肺癌细胞系H460、H1299细胞的自分泌因子能够增强自身的辐射抵抗作用；A549细胞系的自分泌物对其自身的辐射敏感性没有明显影响；293T细胞系的旁分泌作用对H460细胞的辐射敏感性无明显影响；自分泌促进照射后H460细胞的增殖作用、增加细胞内ROS的表达水平和DNA修复能力；照射后的H460出现明显的G2/M期细胞周期阻滞，同源重组修复能力明显增强，但自分泌外泌体对H460细胞的辐射敏感性无明显影响。
结论  非小细胞肺癌细胞能够通过自分泌作用增强DNA修复能力来提高自身的辐射抵抗作用。

Objective: Radiotherapy resistance in patients with non-small cell lung cancer (NSCLC) reduces patient survival and remains a significant challenge for the treatment of NSCLC. Studies have shown that autocrine affects the radiosensitivity of tumors, but in non-small cell lung cancer, the main role of autocrine is not yet clear. In this study, we investigated the effects of autocrine secretions on radiosensitivity of NSCLC cell lines (H460, H1299, and A549) and explore the main mechanism of autocrine secretions to enhance cell radiation resistance from the overall perspective.

Methods: Human lung cancer cell lines（H460, H1299, and A549） were used as research objects, and the effects of autocrine secretions on the radiosensitivity of non-small cell lung cancer cells (H460, H1299, A549) were compared using a colony formation assay and Transwell co-culture. Secretion of 293T cells effects on the radiosensitivity of H460 cells. Clone formation experiments were performed to compare the effects of autocrine conditioned medium on the radiosensitivity of H460 cells; MTT assay was used to observe the effect of conditioned medium on the proliferation of H460 cells after irradiation; Flow cytometry was used to detect the effect of conditioned medium on the expression of reactive oxygen species and cell cycle in H460 cells after irradiation; The level of SOD1 mRNA in cells was detected by qRT-PCR, and the level of SOD1 protein in cells was detected by Western blotting; The changes of γH2AX in irradiated H460 cells cultured in conditioned media at different times were observed by immunofluorescence; Changes of γH2AX and DNA repair related proteins in H460 cells were detected by Western blotting; Differential centrifugation to collect exosomes secreted by H460 cells; Western blotting and transmission electron microscopy experiments were performed to verify the exosomes; Clone formation assays were used to examine the effects of exosomes on radiosensitivity of H460 cells.

Results: In this study, the NSCLC cell lines, H460 and H1299 cells, the autocrine factors enhance their radiation sensitivity; the autocrine effect of A549 cell line has no obvious effect on their radiation sensitivity; Paracrine effect of 293T cell line had no significant effect on radiosensitivity of H460 cells; Autocrine promotes the proliferation of H460 cells after irradiation, increases the expression of intracellular ROS and DNA repair capacity. H460 cells showed obvious G2/M cell cycle arrest after irradiation, and the ability of homologous recombination repair was obviously enhanced, while exosomes secreted by H460 cells had no influence on the radiation resistance.

Conclusions：Non-small cell lung cancer cells can enhance DNA repair ability to increase their own radiation resistance through autocrine.