| 林川川,于加武,杨振兴,等.生长分化因子15在骨肉瘤细胞放射抵抗中的机制研究[J].中华放射医学与防护杂志,2026,46(6):564-572.Lin Chuanchuan,Yu Jiawu,Yang Zhenxing,et al.Mechanisms behind the influence of growth differentiation factor 15 on the radioresistance of osteosarcoma cells[J].Chin J Radiol Med Prot,2026,46(6):564-572 |
| 生长分化因子15在骨肉瘤细胞放射抵抗中的机制研究 |
| Mechanisms behind the influence of growth differentiation factor 15 on the radioresistance of osteosarcoma cells |
| 投稿时间：2025-06-14 |
| DOI：10.3760/cma.j.cn112271-20250614-00201 |
| 中文关键词: 骨肉瘤|生长分化因子15|放射抗性|活性氧 |
| 英文关键词:Osteosarcoma|Growth differentiation factor 15 (GDF15)|Radioresistance|Reactive oxygen species (ROS) |
| 基金项目:重庆市自然科学基金面上项目(CSTB2022NSCQ-MSX0179, CSTB2023NSCQ-MSX0270);陆军军医大学第二附属医院青年博士人才孵化计划(2022YQB077, 2022YQB064);重庆市自然科学基金重点项目(cstc2020jcyj-zdxmX0013) |
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| 中文摘要: |
| 目的 探索生长分化因子15(GDF15)表达水平对骨肉瘤放射敏感性的影响及其机制。方法 收集骨肉瘤癌和癌旁组织,通过免疫组织化学和Western blot检测GDF15表达水平。将人源骨肉瘤细胞系U2OS和Saos2随机分为阴性对照组、GDF15沉默组、照射对照组、联合处理组,照射对照组接受9 Gy γ射线处理,采用qPCR和Western blot检测照射对U2OS和Saos2中GDF15表达水平的影响。采用流式细胞术、免疫荧光、Western blot等方法检测9 Gy照射剂量下各组的细胞凋亡率及凋亡相关蛋白表达水平。通过转录组测序分析,筛选与GDF15相关的放射应激基因和信号通路。采用qPCR、Western blot、免疫荧光等验证沉默GDF15对放射后关键分子的影响。结果 4组配对的临床样本癌组织中GDF15的mRNA和蛋白表达水平远高于癌旁组织(tmRNA= 5.33 ~ 16.32,P < 0.05;t蛋白= 5.83 ~ 13.49,P < 0.05),且照射可引起U2OS和Saos2中GDF15的mRNA水平随照射后继续培养的时间上调(tU2OS = 12.9 ~ 54.71,P < 0.05;tSaos2= 5.46 ~ 37.69,P < 0.05)。与照射对照组相比,联合处理组的细胞凋亡显著上升(t= 10.93,P ＜ 0.05)、G2/M期比例增加(t= 15.63,P ＜ 0.05)、照射后24 h γ-H2AX焦点数量显著升高(t= 15.54,P ＜ 0.05)。转录组测序分析提示活性氧(ROS)相关信号通路被富集,qPCR验证结果与之相一致,ROS相关基因ACSL4、SLC7A11、GPX4、SLC3A2等均因沉默GDF15发生显著改变。与照射对照组相比,联合处理组的ROS水平升高和线粒体膜电位降低,谷胱甘肽(GSH)水平下降以及丙二醛(MDA)含量升高。此外,添加外源GDF15单克隆抗体珀塞古单抗可导致放射后NRF2蛋白水平降低,γ-H2AX焦点数量和细胞凋亡率增加;反之,添加GDF15重组蛋白可在放射后提高NRF2的蛋白水平,降低γ-H2AX焦点数量、显著减少细胞凋亡率(t= 21.32,P ＜ 0.05)。结论 GDF15在骨肉瘤肿瘤组织中高表达,放射后表达上调,并通过NRF2及ROS相关基因参与氧化应激反应,沉默GDF15可增强骨肉瘤细胞的放射敏感性。 |
| 英文摘要: |
| Objective To explore the effects of the expression level of growth differentiation factor 15 (GDF15) on the radiosensitivity of osteosarcoma and the mechanisms behind the effects. Methods The cancerous and paracancerous tissues of osteosarcoma were collected, and the expression levels of GDF15 in these tissues were detected using immunohistochemistry and Western blot. Human osteosarcoma cell lines U2OS and Saos2 were randomly divided into four groups: negative control, GDF15 knockdown (i.e., siGDF15), irradiation control, and combined treatment groups. Among these, the irradiation control group received 9 Gy of γ-ray irradiation. The effects of irradiation on GDF15 expression levels in U2OS and Saos2 were then detected using quantitative polymerase chain reaction (qPCR) and Western blot. Using method including flow cytometry, immunofluorescence assay, and Western blot, the apoptosis rates and the expression levels of apoptosis-related proteins in various groups were assessed following 9 Gy of irradiation. Subsequently, the radiation stress genes and signaling pathways associated with GDF15 were selected through transcriptome sequencing analysis. Additionally, the effects of siGDF15 on key molecules after irradiation were verified using qPCR, Western blot, and immunofluorescence assay. Results The four groups of clinical samples of paired cancerous tissues exhibited significantly higher mRNA and protein expression levels of GDF15 than the paracancerous tissues (tmRNA = 5.33-16.32, P < 0.05; tprotein = 5.83-13.49, P < 0.05). Furthermore, irradiation could upregulate the mRNA expression levels of GDF15 in U2OS and Saos2 over time of continued culture (tU2OS= 12.9-54.71, P < 0.05; tSaos2= 5.46-37.69, P < 0.05). Compared to the irradiation control group, the combined treatment group showed significantly upregulated apoptosis (t = 10.93, P < 0.05), increased proportions of cells in the G2/M phases (t = 15.63, P < 0.05), and significantly increased number of γ-H2AX foci at 24 h after irradiation (t = 15.54, P < 0.05). Transcriptome sequencing analysis suggested the enrichment in reactive oxygen species (ROS)-related signaling pathways, which was consistent with the qPCR validation result. The analytical result also indicated that ROS-related genes, including ACSL4, SLC7A11, GPX4, and SLC3A2, were significantly altered by siGDF15. Compared to the irradiation control group, the combined treatment group showed elevated ROS level and malondialdehyde (MDA) content, as well as reduced mitochondrial membrane potential (MMP) and glutathione (GSH) level, after radiation. Besides, the addition of ponsegromab, an exogenous GDF15 monoclonal antibody, decreased the protein level of NRF2 and increased the number of γ-H2AX foci and the apoptosis rate after radiation. In contrast, the addition of recombinant GDF15 protein increased the protein level of NRF2, decreased the number of γ-H2AX foci, and significantly reduced the apoptosis rate after radiation (t = 21.32, P < 0.05). Conclusions GDF15 is highly expressed in the cancerous tissues of osteosarcoma. Following radiation, GDF15 manifests an upregulated expression level and participates in oxidative stress response through NRF2- and ROS-related genes. Knockdown of GDF15 can enhance the radiosensitivity of osteosarcoma cells. |
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