【作者】 刘琳；

【导师】 周金华；

【作者基本信息】 苏州大学 ， 妇产科学， 2024， 硕士

【摘要】 宫颈癌的免疫抑制微环境明显阻碍了免疫治疗的有效性。基于此,本研究开发聚乙二醇化锰掺杂硫化钙纳米颗粒（MCSP NPs）,通过硫化氢（H₂S）气体增强金属离子免疫干预机制,有效增强宫颈癌的抗肿瘤免疫应答。具有生物活性的MCSP NPs在酸性肿瘤微环境中表现出快速释放Ca²⁺、Mn²⁺和H₂S的能力。释放的H₂S作为Mn²⁺和Ca²⁺的重要增效剂,通过调节双重信号机制来桥接先天和适应性免疫反应。一方面,H₂S通过抑制烟酰胺腺嘌呤二核苷酸（NADH）活性干扰氧化磷酸化途径,破坏癌细胞的钙缓冲系统,导致钙超载引发焦亡。另一方面,H₂S介导的线粒体功能障碍进一步促进线粒体DNA（mtDNA）的释放,增强Mn²⁺对cGAS-STING信号轴的激活作用,从而激活树突状细胞。MCSP NPs通过同时激活钙超载介导的焦亡和cGAS-STING途径,有效增强抗肿瘤免疫应答。同时,MCSP NPs与PD-1免疫治疗联合使用可实现协同抗肿瘤作用,有效抑制肿瘤生长。本研究揭示了H₂S气体治疗与金属免疫治疗的潜在协同作用,为纳米免疫调节剂的设计和肿瘤免疫抑制微环境的合理调控提供了新的思路。目的:开发聚乙二醇化锰掺杂硫化钙纳米颗粒（MCSP NPs）,通过H₂S气体增强金属离子免疫干预机制,同时与免疫检查点阻断疗法相结合,有效增强宫颈癌的抗肿瘤免疫应答。方法:1.首先合成材料并进行表征。使用高温油相法合成了MCSP NPs,通过聚乙二醇修饰（MCSP）,提高了生物相容性跟稳定性。通过TEM、水和粒径（DLS）对MCSP NPs的形貌颗粒大小进行了表征;通过STEM、Mapping和XPS对MCSP NPs的元素组成进行了表征;通过电感耦合等离子体质谱分析、醋酸铅试纸、WSP-1探针对MCSP的体外释放性能进行了定性跟定量检测。2.通过Cy5.5标记的MCSP共聚焦成像、WSP-1荧光染色、WST-8法、DCFH-DA流式分析、JC-1流式分析、胞内ATP检测、微量法Ca²⁺ATPase检测、Fluo-4钙离子探针染色等实验验证了MCSP细胞水平的入胞能力、细胞内H₂S释放性能、NADH抑制作用、诱导氧化应激、促进线粒体损伤、阻碍ATP生成等的能力以及抑制钙泵活性从而实现细胞内钙超载的能力。3.通过MTT、AM-PI染色、光学显微镜成像、细胞膜染色成像、细胞内乳酸脱氢酶检测、Western-Blot、ATP释放、CRT和HMGB1染色对MCSP体外宫颈癌细胞杀伤和焦亡诱导能力、促进癌细胞免疫原性死亡的能力进行了检测。4.Western-Blot、q-PCR、流式分析、Elisa检测验证了MCSP激活cGAS-STING通路和促进DCs成熟的能力。5.构建U14宫颈癌皮下模型,通过肿瘤监测、H&E染色、Ki67染色、TUNEL染色、WSP-1染色、cleaved-caspase-3染色、GSDME-N染色验证了MCSP的体内抗肿瘤特性。6.构建U14宫颈癌皮下模型,通过对肿瘤组织流式分析和Elisa检测验证MCSP对小鼠的免疫激活能力。7.U14宫颈癌皮下模型,与αPD-1抗体联合,通过肿瘤监测、肿瘤组织T细胞染色验证了联合治疗有效确保免疫治疗的持久性跟有效性。结果:我们采用高温有机溶液法成功合成了具有生物活性的锰掺杂硫化钙纳米颗粒（Mn-Ca S_x NPs）。经过PEG修饰后,获得的Mn-Ca S_x-PEG（MCSP）在肿瘤微环境下表现出良好的生理稳定性和良好的Ca²⁺、Mn²⁺、H₂S释放性能。释放的H₂S通过抑制烟酰胺腺嘌呤二核苷酸（NADH）的活性诱导氧化应激,从而引起线粒体损伤和能量代谢紊乱。因此,ATP依赖性钙泵被抑制,细胞内钙缓冲系统被破坏,导致细胞内外源性Ca²⁺的异常积累,引发焦亡。H₂S增强的焦亡过程进一步促进mtDNA的释放,促进Mn²⁺激活cGAS-STING通路,促进TIDCs的成熟以及CXCL10、IFN-β、IL-6等cGAS-STING通路相关细胞因子的释放。最终,MCSP通过钙超载介导的焦亡和cGAS-STING途径的激活有效激活了先天和适应性抗肿瘤免疫反应。这明显抑制了U14皮下瘤的生长,改善了宫颈肿瘤的免疫抑制微环境,促进成熟DCs、T细胞、细胞毒性T细胞、M1等抗肿瘤免疫细胞的浸润,抑制了Treg、M2等促肿瘤免疫细胞的浸润,同时增加了TNF-α、IL-1β、IFN-γ等炎症因子的释放。最后与αPD-1连用,有效阻断肿瘤的免疫逃逸,确保MCSP激活的T细胞在肿瘤中更好浸润,提高了免疫治疗疗效。结论:生物活性MCSP被成功开发用于气体放大金属免疫治疗,通过双重激活焦亡和STING途径重塑免疫,抑制宫颈癌。MCSP释放H₂S和Ca²⁺,通过干扰ATP的产生,抑制钙外排,促进钙超载,引发U14细胞焦亡和ICD。释放的H₂S还导致线粒体功能障碍,促进mtDNA的释放,并与Mn²⁺协同激活cGAS-STING通路,导致树突状细胞成熟。这种双向激活策略下调免疫抑制Tregs的比例,促进M1和T细胞的浸润,并通过激活适应性免疫促进促炎因子的释放。此外,MCSP联合aPD-1治疗通过克服免疫逃逸和促进T细胞浸润有效抑制肿瘤生长。这项研究强调了H₂S扩增金属免疫治疗作为一种有前途的增强免疫治疗策略的潜力。更多还原

【Abstract】 Cervical cancer is a major global health concern for women.Recently,immunotherapy has shown promise in the fight against cervical cancer.However,its effectiveness is constrained by the immunosuppressive microenvironment that prevails within cervical cancer lesions,which is characterized by insufficient T cell infiltration,low activation of dendritic cells（DCs）,and an excessive concentration of regulatory T cells（Tregs）.The use of gas adjuvant to enhance metal immunotherapy provides a promising way to improve the effectiveness of immunotherapy for cervical tumors.Objective:To develop the mechanism of metal ion immune intervention enhanced by hydrogen sulfide（H₂S）gas with manganese glycol doped calcium sulfide nanoparticles（MCSP NPs）,and to effectively enhance the anti-tumor immune response of cervical cancer in combination with immune checkpoint blocking therapy.Methods:1.Firstly,the materials were synthesized and characterized.Manganese doped calcium sulfide nanoparticles were synthesized by high temperature oil phase method and modified by polyethylene glycol to improve their biocompatibility and stability.The morphology and particle size of manganese doped calcium sulfide nanoparticles were characterized by TEM,water and particle size.The elemental compositions of manganese doped calcium sulfide nanoparticles were characterized by STEM mapping and XPS.The release properties of MCSP in vitro were tested qualitatively and quantitatively by inductively coupled plasma mass spectrometry,WSP-1probe and lead acetate test paper.The stability of MCSP was measured by UV-visible absorption spectrum and dynamic light scattering.2.Confocal imaging of Cy5.5-labeled MCSP,WSP-1 fluorescence staining,WST-8 method,DCFH-DA flow analysis,JC-1 flow analysis,intracellular ATP detection,micromethod Ca²⁺ATPase detection,Fluo-4 calcium probe staining were used to verify the cytocytosis ability and intracellular H₂S release of MCSP cells Performance,inhibition of NADH,induction of oxidative stress,promotion of mitochondrial damage,inhibition of ATP production,inhibition of calcium pump activity and the ability to achieve intracellular calcium overload.3.MTT,AM-PI staining,optical microscope imaging,cell membrane staining,intracellular lactic acid detection,Western-Blot,ATP release,CRT and HMGB1 staining were used to detect MCSP’s ability to kill cervical cancer cells in vitro,induce pyrodeath,and promote the immunogenic death of cancer cells.4.Western-Blot,q-PCR,flow analysis and Elisa tests verified the ability of MCSP to activate cGAS-STING pathway and promote DC maturation.5.A subcutaneous model of U14 cervical cancer was established,and the in vivo anti-tumor properties of MCSP were verified by tumor monitoring,H&E staining,Ki67 staining,TUNEL staining,WSP-1 staining,cleaved-caspase-3 staining,and GSDME-N staining.6.The subcutaneous model of U14 cervical cancer was constructed,and the immunoactivation ability of MCSP on mice was verified by flow analysis of tumor tissue and Elisa detection.7.The subcutaneous model of U14 cervical cancer was combined withαPD-1 antibody.Through tumor monitoring and tumor tissue T cell staining,it was verified that the combination therapy effectively ensured the durability and effectiveness of immunotherapy.Results:The bioactive manganese doped calcium sulfide nanoparticles（Mn-Ca S_xNPs）were successfully synthesized by high temperature organic solution method.After peg modification,the obtained Mn-Ca S_x-PEG（MCSP）showed good physiological stability and good Ca²⁺,Mn²⁺,H₂S release performance in the tumor microenvironment.Release of H₂S by inhibiting the nicotinamide adenine dinucleotide（nicotinamideadeninedinucleotide,NADH）activity induced oxidative stress,causing mitochondrial damage and energy metabolism disorders.Therefore,the ATP-dependent calcium pump is inhibited and the intracellular calcium buffer system is destroyed,leading to abnormal accumulation of intracellular and extracellular Ca²⁺and triggering pyrodeath.The pyrodeath process enhanced by H₂S further promotes the release of mtDNA,promotes the activation of the cGAS-STING pathway by Mn²⁺,promotes the maturation of TIDCs and the release of CXCL10,IFN-β,IL-6 and other cGAS-STING pathway-related cytokines.Finally,MCSP effectively activated innate and adaptive anti-tumor immune responses through calcium overload-mediated pyrodeath and cGAS-STING pathways.This significantly inhibited the growth of subcutaneous tumors of U14,significantly improved the immunosuppressive microenvironment of cervical tumors,promoted the invasion of anti-tumor immune cells such as mature DCs,T cells,cytotoxic T cells and M1,and inhibited the invasion of pro-tumor immune cells such as Treg and M2.It also increased the release of TNF-α,IL-1β,IFN-γand other inflammatory factors.Finally,when combined withαPD-1,it can effectively block the immune escape of the tumor,ensure the better infiltration of Mc Sp-activated T cells in the tumor,and improve the efficacy of immunotherapy.Conclusion:In summary,bioactive MCSP were successfully developed for gas-amplified metalloimmunotherapy via dual activation of pyroptosis and STING pathway to remodel immunosuppressive cervical cancers.MCSP released H₂S and Ca²⁺,which triggered U14 cell pyroptosis and ICD by interfering with ATP production,inhibiting calcium efflux,and promoting calcium overload.The released H₂S also led to mitochondrial dysfunction,promoted the release of mtDNA,and collaborated with Mn²⁺to activate cGAS-STING pathway,resulting in dendritic cell maturation.This bidirectional activation strategy down-regulated the proportion of immunosuppressive Tregs,promoted the infiltration of M1 and T cells,and facilitated the release of proinflammatory factors by activating adaptive immunity.Additionally,MCSP combined withαPD-1 therapy effectively inhibited the tumor growth by overcoming immune escape and promoting T cell infiltration.This study highlighted the potential of H₂S-amplified metalloimmunotherapy as a promising strategy to enhance immunotherapy.更多还原