【作者】 陈露；

【导师】 卢丹；

【作者基本信息】 扬州大学 ， 妇产科学， 2025， 硕士

【摘要】 第1章双氢青蒿素通过STAT3/GPX4信号通路诱导卵巢癌铁死亡的体外实验研究目的:通过体外细胞实验阐明双氢青蒿素（Dihydroartemisinin,DHA）抑制卵巢癌（Ovarian cancer,OC）细胞增殖的具体作用机制,并探究DHA诱导OC细胞发生铁死亡可能的分子机制。方法:（1）通过细胞计数试剂盒（Cell Counting Kit-8,CCK-8）检测DHA对OC细胞（SKOV3及A2780）活力的影响,并用铁死亡抑制剂（Ferrostatin-1,Fer-1）进行挽救实验,通过CCK-8检测Fer-1对细胞活力的影响;（2）通过细胞划痕实验检测DHA对细胞迁移能力的影响;（3）通过细胞平板克隆形成实验检测DHA对细胞增殖克隆能力的影响;（4）通过活性氧（Reactive oxygen species,ROS）荧光探针DCFH-DA结合荧光显微镜观察DHA对细胞内ROS表达水平的影响;（5）通过微板法检测DHA处理后细胞内谷胱甘肽（L-glutathione,GSH）表达水平的变化;（6）通过亚铁离子(Ferrous iron,Fe²⁺)检测试剂盒评估DHA对细胞内Fe²⁺水平的影响;（7）通过蛋白免疫印迹（Western blot,WB）检测细胞内信号转导与转录激活因子3（Signal transducer and activator of transcription 3,STAT3）、溶质载体家族7成员11（Solute carrier family 7 membrane 11,SLC7A11）和谷胱甘肽过氧化物酶4（Glutathione peroxidase 4,GPX4）蛋白的表达;（8）通过实时荧光定量PCR（Quantitative real-time PCR,q PCR）检测细胞内STAT3、SLC7A11和GPX4 m RNA的表达。结果:（1）CCK-8结果显示,DHA可明显抑制SKOV3和A2780细胞细胞活力,且具有浓度依赖性,经铁死亡抑制剂（Fer-1）处理后,SKOV3和A2780细胞的存活率较前明显提高;（2）细胞划痕实验结果发现,随着DHA浓度的增高,SKOV3和A2780的细胞迁移速度显著减缓;（3）细胞克隆形成实验发现,随着DHA浓度的增加,OC细胞克隆形成能力显著下降;（4）ROS荧光探针实验显示,细胞内ROS绿色荧光的强度随DHA浓度的增加而增加;（5）GSH检测试剂盒检测结果分析显示,随着DHA浓度的升高,细胞内GSH水平消耗增加;（6）亚铁试剂盒检测发现,细胞内Fe²⁺的含量随着DHA浓度的增加而增多;（7）Western blot结果发现,随着DHA浓度的增高,DHA在体外对OC细胞内STAT3、SLC7A11和GPX4的蛋白表达水平均下调;（8）q PCR结果分析显示,随着DHA浓度的增高,DHA在体外对OC细胞内STAT3、SLC7A11和GPX4的m RNA表达水平均下调。结论:DHA能显著抑制卵巢癌SKOV3和A2780细胞的增殖,这可能是通过抑制STAT3/GPX4信号通路,破坏细胞氧化还原稳态、加剧氧化应激,从而诱导卵巢癌细胞铁死亡。第2章双氢青蒿素通过STAT3/GPX4信号通路诱导卵巢癌铁死亡的体内实验研究目的:构建裸鼠皮下异体移植瘤,进一步通过裸鼠体内实验模型,对上述发现进行验证。方法:（1）通过皮下注射卵巢癌SKOV3细胞构建裸鼠皮下异种移植瘤模型,观察DHA对裸鼠皮下移植瘤生长的影响;（2）通过Western blot实验检测DHA对裸鼠移植瘤内p-STAT3、STAT3、SLC7A11和GPX4蛋白表达水平的影响;（3）通过H&E染色法观察DHA对组织细胞形态的影响;（4）通过免疫组织化学法（Immunohistochemistry,IHC）检测DHA对裸鼠移植瘤内STAT3和GPX4蛋白表达水平的影响。结果:（1）裸鼠体内实验发现,DHA对裸鼠皮下移植瘤的生长具有明显的抑制作用;（2）Western blot的结果表明,经DHA治疗组中p-STAT3、STAT3、SLC7A11和GPX4的蛋白水平显著下调;（3）H&E染色结果显示,经DHA治疗后的肿瘤细胞内出现大规模裂解现象,卵巢癌（腺癌）结构消失,细胞间形成诸多空洞;（4）IHC的实验结果显示,经DHA治疗组中STAT3和GPX4的表达水平也同样下调。结论:在体内实验中,DHA仍可以显著抑制裸鼠皮下移植瘤的生长,进一步探究发现,其可能通过调控STAT3/GPX4信号通路,诱导卵巢癌细胞发生铁死亡。该实验结果与前期体外实验结论是一致的。在体内实验中,DHA抑制裸鼠异体移植瘤的生长。更多还原

【Abstract】 Chapter 1 An In Vitro Investigation of Dihydroartemisinin-Induced Ferroptosis in Ovarian Cancer via the STAT3/GPX4 Signalling PathwayObject:In vitro cellular experiments,the specific mechanism underlying the inhibition of ovarian cancer（OC）cell proliferation by dihydroartemisinin（DHA）was elucidated,and the potential molecular mechanisms by which DHA induces ferroptosis in OC cells were also explored.Methods:（1）The effects of DHA on the viability of OC cells（SKOV3 and A2780）were assessed using the Cell Counting Kit-8（CCK-8）assay.A rescue experiment was conducted with the ferroptosis inhibitor Ferrostatin-1（Fer-1）,and the impact of Fer-1 on cell viability was subsequently examined with the CCK-8 assay as well;（2）The effect of DHA on cell migration ability was examined using the cell scratch assay;（3）The impact of DHA on cellular proliferative and colony-forming capabilities was assessed with the colony formation assay;（4）The effect of DHA on intracellular reactive oxygen species（ROS）expression levels was examined by monitoring ROS levels using the fluorescent probe2’,7’-dichlorodihydrofluorescein diacetate（DCFH-DA）in conjunction with fluorescence microscopy;（5）The impact of DHA on intracellular Fe²⁺levels was evaluated using a ferrous ion detection kit;（6）The changes in intracellular L-glutathione（GSH）expression levels following DHA treatment were measured using the microplate-based assay;（7）The expression levels of intracellular proteins,including signal transducer and activator of transcription 3（STAT3）,solute carrier family 7 member 11（SLC7A11）,and glutathione peroxidase 4（GPX4）,were analyzed using Western blot（WB）analysis;（8）The expression levels of intracellular STAT3,SLC7A11,and GPX4 m RNA were quantified using quantitative real-time PCR（q PCR）.Results:（1）The results from the CCK-8 assay demonstrated that DHA significantly suppressed the viability of SKOV3 and A2780 cells in a concentration-dependent manner.Notably,treatment with the ferroptosis inhibitor Fer-1 substantially rescued cell survival rates in both SKOV3 and A2780 cells compared to the DHA-only group;（2）The results from the cell scratch assay revealed that the migration rates of SKOV3 and A2780 cells were significantly reduced with increasing concentrations of DHA;（3）The results from the colony formation assay demonstrated that the clonogenic capacity of OC cells was significantly diminished with escalating concentrations of DHA;（4）The results from the ROS fluorescent probe assay revealed that the intensity of intracellular green fluorescence,indicative of ROS levels,increased proportionally with escalating concentrations of DHA;（5）The results from the ferrous ion detection kit demonstrated that the intracellular Fe²⁺content increased proportionally with escalating concentrations of DHA;（6）The analysis of results from the GSH detection kit revealed that intracellular GSH depletion intensified with escalating concentrations of DHA;（7）The results from Western blot analysis demonstrated that the protein expression levels of STAT3,SLC7A11,and GPX4 in OC cells were downregulated by DHA in vitro in a concentration-dependent manner;（8）The analysis of q PCR results revealed that the m RNA expression levels of STAT3,SLC7A11,and GPX4 in OC cells were downregulated by DHA in vitro in a concentration-dependent manner.Conclusions:DHA can significantly inhibit the proliferation of ovarian cancer SKOV3 and A2780 cells,which may be achieved by inhibiting the STAT3/GPX4 signalling pathway,disrupting cellular redox homeostasis,and exacerbating oxidative stress,thereby inducing ferroptosis in ovarian cancer cells.Chapter 2 An In Vivo Investigation of Dihydroartemisinin-Induced Ferroptosis in Ovarian Cancer via the STAT3/GPX4 Signalling PathwayObject:Subcutaneous xenograft tumors were established in nude mice to further validate the aforementioned findings using an in vivo experimental model within the nude mouse system.Methods:（1）A subcutaneous xenograft tumor model was established in nude mice by injecting ovarian cancer SKOV3 cells,and the effects of DHA on tumor growth were evaluated;（2）Western blot analysis was performed to assess the impact of DHA on the protein expression levels of p-STAT3,STAT3,SLC7A11,and GPX4 within the xenograft tumors of nude mice;（3）Hematoxylin and eosin（H&E）staining was employed to investigate the morphological alterations in tumor and surrounding tissues induced by DHA;（4）Immunohistochemistry（IHC）was utilized to examine the effects of DHA on the protein expression levels of STAT3 and GPX4 within the xenograft tumors of nude mice.Results:（1）In vivo experiments in nude mice demonstrated that DHA significantly inhibited the growth of subcutaneous xenograft tumors;（2）Western blot analysis revealed that the protein levels of p-STAT3,STAT3,SLC7A11,and GPX4 were markedly downregulated in the DHA-treated group compared to the control;（3）H&E staining indicated extensive tumor cell lysis,disruption of ovarian adenocarcinoma architecture,and the formation of numerous vacuoles between cells following DHA treatment;（4）IHC results showed that the expression of STAT3 and GPX4 was also significantly reduced in the DHA-treated group.Conclusions:In vivo experiments,DHA still significantly inhibited the growth of subcutaneous xenograft tumors in nude mice.Further investigation revealed that it might induce ferroptosis in ovarian cancer cells by regulating the STAT3/GPX4 signalling pathway.This experimental result is consistent with the conclusions drawn from previous in vitro experiments.更多还原