目的：探讨铁死亡在结直肠肿瘤再生细胞放疗抵抗中的功能和机制。方法：采用二维常规条件培养结肠癌HCT116细胞（Control细胞），同时通过机械力方法在三维纤维蛋白软胶中培养并筛选出具有高致瘤能力的肿瘤再生细胞（TRC）；用不同剂量（2、4、6、8 Gy）的 X-射线照射 Control 细胞和 TRC，并通过 MTS、细胞克隆实验分别检测各组细胞的存活率和增殖能力；Control细胞和TRC在采用铁死亡诱导剂（Erastin）和X-射线分别处理后，用C11-BODIPY试剂进行染色，通过共聚焦显微镜和流式细胞术观察并测定细胞的脂质过氧化水平。用qPCR法检测X-射线照射、Erastin处理对Control细胞和TRC中铁死亡相关基因谷胱甘肽过氧化物酶4（GPX4）和酰基辅酶a合成酶长链家族成员4（ACSL4）表达的影响，用WB法检测对细胞中铁死亡相关蛋白GPX4、ACSL4表达的影响。结果：从纤维蛋白软胶中培养并筛选出高干性的TRC；用不同剂量（2、4、6、8 Gy）的X-射线照射后，Control细胞存活率较TRC显著降低（P<0.05），且Control细胞的克隆大小和数量较TRC显著降低（均P<0.05）；Control细胞采用不同剂量X-射线（4、8 Gy）照射及用Erastin处理后，X-射线处理组细胞脂质过氧化水平较未处理组细胞显著升高（P<0.05），Erastin处理组细胞脂质过氧化水平较DMSO处理组细胞显著升高（P<0.05），而TRC各处理组间无显著差异（均P>0.05）。机制研究发现，TRC中GPX4和ACSL4在铁死亡诱导条件（X-射线照射或Erastin处理）下较Control细胞呈现出有助于抵抗的表达模式，即GPX4持续上调、ACSL4持续下调，且表现为Erastin剂量依赖性。结论：结直肠TRC可能通过高表达GPX4、低表达ACSL4来抵抗铁死亡，从而抵抗放疗。

Objective: To investigate the function and mechanism of ferroptosis in the radiation resistance of colorectal tumor-repopulating cells. Methods: Human colorectal tumor cells HCT116 (defined as Control cells) were cultured in two-dimensional normal conditions, and tumor regenerative cells with high tumorigenicity (defined as TRCs) were cultured and screened in threedimensional fibrin soft gels by the mechanical force method. Both the control group and TRC group cells were exposed to X-rays with different doses (2, 4, 6, 8 Gy) and MTS and the clone formation assay were used tomeasure the cell viability rate and proliferation ability. After the Control cells and TRCs were treated with ferroptosis inducer (Erastin) and X-rays respectively, they were stained with C11-BODIPY reagent, and the lipid peroxidation level of the cells was observed and determined by confocal microscopy and flow cytometry. qPCR was used to determine the effects of Erastin and X-rays treatments on the expressions of ferroptosis-related genes glutathione peroxidase 4 (GPX4) and acyl-coenzyme A synthetase long-chain family member 4 (ACSL4) in the Control cells and TRCs; WB assay was performed to determine the effects on the expressions of ferroptosis-related proteins GPX4 and ACSL4. Results: Colorectal TRCs with high stemness were cultured and screened out from soft fibrin gels. After irradiation with different doses (2, 4, 6, 8 Gy) of X-rays, the viability rate, the clone sizeand the number of clones in the control group were significantly lower than those in the TRC group (all P<0.05). After the cells in the control group were irradiated with different doses of X-rays (4, 8 Gy) and treated with Erastin, the lipid peroxidation level of the cells in the X-ray treated group was significantly higher than that in the untreated group (P<0.05). The lipid peroxidation level of the cells in the Erastin-treated group was significantly higher than that in the DMSO-treated group (P<0.05). There was no statistical difference among all treatment subgroups in the TRC group (all P>0.05). The mechanism study indicated that compared with those in control cells, GPX4 and ACSL4 in TRCs under ferroptosis-inducing conditions (X-ray radiation and Erastin treatment) presented expressions that contributed more to radiation resistance, i. e., continued upregulation of GPX4 and downregulation of ACSL4 and their expressions were dependent on the doses of Erastin. Conclusion: Colorectal TRCs may resist ferroptosis through a high expression of GPX4 and a low expression of ACSL4, which in turn induces radiation resistance.

R735.3；R730.2

国家自然科学基金资助项目（No. 81773240）