【作者】 郭卉；

【导师】 章玲玲；

【作者基本信息】 中南大学 ， 临床检验诊断学， 2025， 硕士

【摘要】 目的:课题组前期单细胞测序发现,ES-CuCl₂可显著逆转免疫抑制性肿瘤微环境,其中调节性T细胞（Treg）变化尤为显著。本研究旨在深入探究ES-CuCl₂靶向调控Treg细胞的具体分子机制,并基于此开发长效、靶向、低毒的结直肠癌治疗新策略。方法:（1）发现铜死亡激活免疫并抑制Treg活性:（1）CCK-8、WB检测ES-CuCl₂处理后结直肠癌细胞活力及铜死亡标志蛋白（DLAT、LIAS和FDX1）的表达水平;（2）流式细胞术及克隆形成实验检测与ES-CuCl₂处理的癌细胞共培养后,DC细胞及T细胞的活化水平;（3）流式细胞术检测ES-CuCl₂治疗后,小鼠外周血中DC、T、M1、M2和Treg细胞的水平;（3）筛选关键分子:（1）醋酸铅法、流式细胞术检测ES-CuCl₂处理后胞内H₂S水平及Treg活性改变;（2）改良生物素开关法联合质谱筛选ES-CuCl₂处理后硫酰化水平发生显著变化的候选蛋白,并与调控Treg的功能基因FOXP3的潜在转录因子取交集,筛选出调控Treg的关键分子是THAP11;（4）鉴定关键位点:（1）根据JASPR预测构建点突变质粒,并通过ChIP和双荧光素酶报告实验确认FOXP3启动子上THAP11的结合位点;（2）基于THAP11氨基酸序列分别构建半胱氨酸点突变体,并通过ChIP等实验确认硫酰化的具体位点;（5）开发治疗策略:构建铜配位水凝胶共同负载PD-L1抗体和ES（ES/αPD-L1@Cu·Gel）,并采用小鼠双侧肿瘤模型和复发模型评估其对侧肿瘤及二次接种肿瘤的生长的影响。结果:（1）（1）ES-CuCl₂显著降低结直肠癌细胞活力,铜死亡标志蛋白DLAT寡聚化,FDX1和LIAS表达下调;（2）ES-CuCl₂处理的癌细胞在体外有效激活DC细胞和T细胞;（3）ES-CuCl₂治疗显著抑制肿瘤的生长,增加DC、CD8⁺T和M1浸润,并抑制Treg和M2活性;（2）（1）ES-CuCl₂显著抑制肿瘤细胞内H₂S的水平,进而下调Treg的活性;（2）筛选出铜死亡过程中调控Treg活性的关键硫酰化蛋白:THAP11;（3）（1）FOXP3启动子上与转录因子THAP11结合的位点为-527 bp～-509 bp;（2）THAP11在Cys48位点发生硫酰化修饰;（4）ES/αPD-L1@Cu·Gel能够显著抑制小鼠对侧肿瘤和二次种植肿瘤的生长,并激活全身CD8⁺T细胞的浸润和记忆T细胞的增殖能力。结论:（1）ES-CuCl₂诱导的铜死亡在激活抗肿瘤免疫的同时,通过下调胞内H₂S水平抑制Treg活性;（2）ES-CuCl₂抑制Treg的具体分子机制:ES-CuCl₂通过下调H₂S,抑制转录因子THAP11的硫酰化,从而减少THAP11与FOXP3启动子的结合,最终抑制FOXP3的转录及其介导的Treg细胞活化;（3）铜配位水凝胶同时负载αPD-L1和ES,协同激活全身性抗肿瘤免疫应答并建立长效免疫记忆,为结直肠癌的免疫治疗提供新型高效的联合治疗策略。图11幅,表5个,参考文献68篇更多还原

【Abstract】 Objective:Based on our previous single-cell sequencing revealed that ES-CuCl₂treatment effectively counteracts tumor immunosuppression,with particularly marked depletion of regulatory T cells（Tregs）.This study aims to elucidate the precise molecular mechanisms by which ES-CuCl₂selectively targets and modulates Treg function,thereby facilitating the development of novel,long-lasting,targeted,and low-toxicity therapeutic strategies for colorectal cancer.Methods:（1）Cuproptosis-mediated immune activation and Treg suppression:（1）CCK-8 and WB were used to assess changes in colorectal cancer cell viability and the expression of cuproptosis-related proteins（DLAT,LIAS,and FDX1）after ES-CuCl₂treatment;（2）Flow cytometry and colony formation assays were performed to evaluate the activation of dendritic cells（DCs）and T cells co-cultured with ES-CuCl₂treated cancer cells;（3）Flow cytometry was used to analyze changes in DCs,T cells,M1/M2 macrophages,and Tregs in the peripheral blood of ES-CuCl₂treated mice;（2）Screening of key molecules:（1）Lead acetate assay and flow cytometry were employed to measure intracellular H₂S levels and Treg activity following ES-CuCl₂treatment;（2）Modified biotin-switch assay coupled with mass spectrometry was used to screen differentially persulfidated proteins,followed by intersection analysis with potential FOXP3-regulating transcription factors to identify THAP11 as the key molecule;（3）Identification of key binding sites:（1）Based on JASPAR predictions,point-mutated plasmids were constructed,and ChIP and dual-luciferase reporter assays confirmed the binding site of THAP11 on the FOXP3 promoter;（2）Cysteine point mutants were generated based on the THAP11 amino acid sequence,and ChIP assays were performed to pinpoint the specific sulfhydrated residue;（4）Development of therapeutic strategy:a copper-coordinated hydrogel co-loaded withαPD-L1 and ES（ES/αPD-L1@Cu·Gel）was developed for combination therapy.Its efficacy was validated in bilateral and recurrent tumor models by monitoring contralateral/relapsed tumor growth and systemic CD8⁺T cell infiltration.Results:（1）（1）Following ES-CuCl₂treatment,the viability of colorectal cancer cells decreased,accompanied by oligomerization of the cuproptosis marker protein DLAT and downregulation of FDX1 and LIAS expression;（2）ES-CuCl₂treated cancer cells effectively activated dendritic cells（DCs）and T cells in vitro;（3）In vivo,ES-CuCl₂treatment significantly inhibited tumor growth,increased infiltration of DCs,CD8⁺T cells,and M1 macrophages,while suppressing the activity of Tregs and M2 macrophages.（2）（1）ES-CuCl₂markedly reduced intracellular H₂S levels in tumor cells,leading to significant downregulation of Treg activity;（2）Screen for the key persulfidated protein THAP11 that regulates Treg activity during cuproptosis;（3）（1）The binding site of THAP11 on the FOXP3 promoter was located at-527 bp to-509 bp;（2）THAP11 underwent persulfidation modification at the Cys48 residue;（4）The ES/αPD-L1@Cu·Gel strategy significantly suppressed the growth of bilateral and secondary implanted tumors in mice,while activating systemic CD8⁺T cell infiltration and memory T cell proliferation.Conclusions:（1）ES-CuCl₂induced cuproptosis exhibits dual immunomodulatory effects:activating antitumor immunity while selectively suppressing Treg activity via intracellular H₂S depletion;（2）Mechanistically,H₂S deficiency inhibits THAP11 persulfidation,attenuating its binding to the FOXP3 promoter,thereby disrupting Treg transcriptional circuitry;（3）By combiningαPD-L1 with cuproptosis inducer and developing a copper-coordinated hydrogel delivery platform（ES/αPD-L1@Cu·Gel）for synergistic co-delivery,we successfully triggered a durable systemic anti-tumor immune response.更多还原