【作者】 吴迪；

【导师】 郭昊；

【作者基本信息】 中国医科大学 ， 皮肤病与性病学， 2025， 硕士

【摘要】 研究目的:疣是由人乳头瘤病毒（human papillomavirus,HPV）感染皮肤和黏膜引起的常见临床疾病,包括寻常疣、扁平疣、趾疣和尖锐湿疣等。本课题组前期随机对照临床试验发现,对单个疣体施加44℃、30分钟的多次局部温热治疗,可以实现全身疣体的系统清除,提示局部温热可使机体建立对病原体的免疫识别,以启动对含有相同HPV的其他非靶向或远程皮损的免疫清除。与传统治疗方法相比,温热疗法具有无创、复发率低的优势,但起效慢,部分患者无反应限制了其推广,且温热治疗病毒疣的机制尚未完全明晰,明确关键分子机制有助于提升免疫应答率。NLRP3炎症小体作为固有免疫系统的重要调控复合物通过识别病原相关分子模式（pathogen-associated molecular patterns,PAMPs）或损伤相关分子模式（damage-associated molecular patterns,DAMPs）激活下游炎症级联反应。其活化可诱导促炎因子IL-1β/IL-18的成熟释放并触发GSDMD依赖性焦亡。目前研究普遍认为,活性氧（reactive oxygen species,ROS）是NLRP3炎症小体组装的关键分子开关,而钙离子(Ca²⁺)信号则通过双重机制参与调控:1)直接介导NLRP3寡聚化;2)通过内质网-线粒体钙循环紊乱诱发线粒体功能障碍,间接放大炎症信号。值得注意的是,胞内Ca²⁺稳态失衡已被证实是焦亡执行阶段膜孔道形成的必要前提。TRPM2将ROS与NLRP3炎症小体的激活联系起来。ROS通过PARPs途径产生ADPR,后者结合TRPM2的NUDT9-H结构域诱导Ca²⁺内流,进而调节NLRP3炎症小体组装。胞内Ca²⁺与ADPR形成正反馈环路,持续增强TRPM2通道活性。敲除或抑制TRPM2可消除相关细胞中ROS依赖的NLRP3炎症小体激活,减少IL-1β分泌,体外抑制TRPM2也会降低相关细胞中NLRP3表达及细胞因子产生。研究方法:1、本研究首先对37℃恒温培养及44℃、30min温热处理后8h的人角质形成细胞进行m RNA测序分析,获得有效数据后建立基因表达矩阵。2、Pearson相关系数及主成分分析检验测序样本关联性,筛选差异表达基因并进行GO及KEGG富集分析寻找关键通路。3、RT-q PCR及Western Blot实验初步检验通路上关键基因及关键蛋白表达水平。4、利用CRISPR/Cas9技术获得TRPM2缺陷的Ha Cat细胞（Trpm2-/-）。5、在Wt及Trpm2-/-细胞中模拟氧化应激、温热及二者联合作用环境,分别利用CCK-8实验检测细胞活性,LDH释放实验检测细胞毒力,ELISA实验检测IL-1β及IL-18释放,免疫荧光实验检测胞内ASC及caspase-1荧光强度,western blot实验检测NLRP3、IL-1β、IL-18、caspase-1、pro caspase-1蛋白表达含量。6、给予BAPTA-AM以螯合胞内钙离子后,利用流式细胞术检测细胞凋亡,收集患者新鲜疣体组织分别进行温热、氧化应激及联合处理后,利用免疫组织化学实验,检测疣组织NLRP3及caspase-1表达。结果:1、测序数据质量:提取的RNA样本纯度和完整性均达到合格标准。借助Illumina测序平台获取原始数据后,经过严格过滤去除不合格序列,得到了高质量的测序数据并构建基因表达矩阵。2、利用Pearson相关系数和主成分分析对测序样本进行评估,结果显示各样本之间呈现出良好的关联性。在此基础上,成功筛选出一批在37℃和44℃处理组之间存在差异表达的基因,这些基因可能是引发细胞差异反应的关键因素。3、富集分析结果:对鉴定到的差异表达基因注释及表达筛选后,GO＿CC富集结果显示膜系统及质膜系统有显著改变,GO＿MF结果主要集中在蛋白结合及离子结合等功能上调,KEGG富集分析以KEGG Pathway为单位检验到与细胞焦亡相关的NLR信号通路（NOD-like receptor signaling pathway）的改变及与钙调控密切相关的Calcium Signaling Pathway显著上调。TRP通道表达量热图显示TRPM2、TRPV3、TRPV2基因表达量增高,相关性系数图表明NLRP3与TRPM2存在较大程度的功能正相关性。筛选钙调节相关通路并进行GO条目富集,发现相关通路显著上调。4、Rt-q PCR及Western Blot初步验证结果:Rt-q PCR实验结果显示,通路上的关键基因如NLRP3、IL-1β、IL-18等在44℃温热处理组中的表达水平显著上调,与RNA测序分析结果趋势一致,Western Blot实验进一步证实,这些关键基因所编码的蛋白在44℃处理组中的表达量也明显增加,表明温热处理不仅影响了基因的转录水平,还影响了蛋白的表达水平。5、为深入探究TRPM2对温热后Ha Cat细胞生物功能的影响,采用CRISPR/Cas9技术敲除了Ha Cat细胞系上的TRPM2基因序列。6、TRPM2缺陷细胞相关实验结果:CCK-8实验结果提示TRPM2通道的缺失对细胞具有一定的保护作用,减轻了氧化应激和温热刺激对细胞的损伤,LDH释放实验Trpm2-/-细胞LDH释放量低于Wt细胞,进一步证实了TRPM2通道在细胞毒性损伤中的促进作用,ELISA实验表明TRPM2通道的缺失抑制了炎症因子IL-1β及IL-18的释放,减轻了炎症反应,免疫荧光实验发现Wt细胞在处理后胞内ASC及caspase-1的荧光强度显著增强,说明TRPM2通道参与NLRP3炎症小体的激活过程,Western blot实验结果显示,Wt细胞在处理后NLRP3、IL-1β、IL-18、caspase-1、pro caspase-1蛋白表达含量显著上调,联合处理组的上调程度更为明显,进一步证实了TRPM2通道在炎症信号通路激活中的关键作用。7、螯合钙离子及疣体组织的实验结果:给予BAPTA-AM螯合胞内钙离子后,流式细胞术检测细胞凋亡结果显示,Wt细胞在处理后的细胞焦亡比例有所降低,提示细胞内钙离子水平的降低可能抑制了细胞焦亡的发生。Trpm2-/-细胞本身的细胞焦亡比例就低于Wt细胞,在螯合钙离子后,细胞焦亡比例进一步降低。8、对患者新鲜疣体组织分别进行温热、氧化应激及联合处理后,免疫组织化学实验结果显示,温热和氧化应激处理均能导致疣组织中NLRP3及caspase-1表达上调,联合处理的上调效果更为显著。这与细胞实验结果一致,进一步证明了温热和氧化应激刺激能激活NLRP3炎症小体信号通路。同时,在给予TRPM2抑制剂进行类似处理时,NLRP3及caspase-1的表达上调程度减弱,再次强调了TRPM2通道在这一过程中的重要作用。结论:本研究揭示44℃温热联合H₂O₂刺激对Ha Ca T细胞中TRPM2/NLRP3通路作用机制,发现温热与氧化应激协同激活NLRP3炎症小体,诱导细胞焦亡和炎症因子释放,此过程有TRPM2介导的Ca²⁺内流参与,TRPM2缺失或抑制可减轻损伤和炎症反应。该研究为温热疗法在疣体治疗应用提供理论支持,也为开发基于TRPM2的新型治疗策略提供可能。更多还原

【Abstract】 Objective:Warts are common clinical conditions caused by infection of the skin and mucous membranes with Human Papillomavirus（HPV）,encompassing verruca vulgaris,flat warts,plantar warts,and condyloma acuminata.Previous randomized controlled trials conducted by our research group demonstrated that repeated local hyperthermia treatment at 44℃for 30 minutes on a single wart could lead to systemic clearance of warts across the body.This suggests that localized hyperthermia may establish immune recognition of the pathogen,thereby initiating immune clearance of other non-targeted or distant lesions harboring the same HPV.Compared to conventional treatments,hyperthermia therapy offers advantages such as being non-invasive and having a low recurrence rate.However,its slow onset and lack of response in some patients limit its widespread adoption.Moreover,the molecular mechanisms underlying hyperthermia-mediated viral wart clearance remain incompletely understood.Elucidating these key mechanisms is crucial for enhancing immune response rates.The NLRP3 inflammasome,a critical regulatory complex of the innate immune system,consists of the pattern recognition receptor NLRP3,it activates downstream inflammatory cascades by recognizing pathogen-associated molecular patterns（PAMPs）or damage-associated molecular patterns（DAMPs）.Activation of the NLRP3 inflammasome induces the maturation and release of pro-inflammatory cytokines IL-1βand IL-18,and triggers GSDMD-dependent pyroptosis,playing a pivotal role in the pathogenesis of infectious diseases,metabolic syndromes,and autoimmune disorders.Current research widely acknowledges that reactive oxygen species（ROS）serve as a key molecular switch for NLRP3 inflammasome assembly,while calcium ion(Ca²⁺)signaling participates in its regulation through dual mechanisms:1)directly mediating NLRP3 oligomerization,and 2)indirectly amplifying inflammatory signals by disrupting endoplasmic reticulum-mitochondrial calcium cycling,leading to mitochondrial dysfunction.Notably,intracellular Ca²⁺homeostasis imbalance has been identified as a prerequisite for membrane pore formation during the execution phase of pyroptosis.TRPM2 links the activation of ROS and the NLRP3 inflammasome.The ROS signal releases ADPR PARPs pathway.ADPR binds to the NUDT9-H domain at the C-terminus to indirectly activate TRPM2,induce Ca²⁺influx,and regulate the assembly of the NLRP3 inflammasome.Moreover,Ca²⁺in the cytoplasm and ADPR act synergistically to increase the Ca²⁺concentration,and positively feedback to enhance the opening of the TRPM2 channel.Blocking the calcium influx mediated by TRPM2 will damage the activation of caspase-1.Knocking out or inhibiting TRPM2can eliminate the activation of the ROS-dependent NLRP3 inflammasome in related cells and reduce the secretion of IL-1β.In vitro inhibition of TRPM2 will also reduce the expression of NLRP3 and the production of cytokines in related cells.Methods:1.This study first analyzed m RNA sequence on human keratinocytes treated with 37℃constant temperature culture and 44℃,30-minute heat treatment for 8 hours,and established a gene expression matrix after obtaining valid data.2.The correlation of sequencing samples was tested using Pearson correlation coefficients and principal component analysis to screen differentially expressed genes.Subsequently,Gene Ontology（GO）and Kyoto Encyclopedia of Genes and Genomes（KEGG）enrichment analyses were performed to identify key pathways.3.Real-time PCR and Western Blot experiments were conducted to preliminarily verify the expression levels of key genes and key proteins in the pathways.4.TRPM2-deficient Ha Cat cells（Trpm2-/-）were obtained using the CRISPR/Cas9 technology.5.Oxidative stress,hyperthermia,and their combined action environments were simulated in wild-type（Wt）and Trpm2-/-cells.The CCK-8 assay was used to detect cell viability,the LDH release assay was used to detect cell toxicity,the ELISA assay was used to detect the release of IL-1βand IL-18,the immunofluorescence assay was used to detect the fluorescence intensity of intracellular ASC and caspase-1,and the Western blot assay was used to detect the expression levels of NLRP3,IL-1β,IL-18,Caspase-1,and pro caspase-1 proteins.6.After BAPTA-AM was added to chelate intracellular calcium ions,flow cytometry with Annexin V/PI staining was used for detection.Fresh wart tissues from patients were collected and treated with hyperthermia,oxidative stress,and their combination respectively.Then,immunohistochemical experiments were performed to detect the expression of NLRP3 and Caspase-1 in the wart tissues.Results:1.Sequencing data quality:The purity and integrity of the extracted RNA samples both met the qualified standards.After obtaining the raw data using the Illumina sequencing platform,the unqualified sequences were strictly filtered out,resulting in high-quality sequencing data.2.Gene expression analysis:By reconstructing transcripts,the expression levels of all genes in the samples were accurately calculated,and a gene expression matrix was constructed.Further evaluation of the sequenced samples using Pearson correlation coefficient and principal component analysis showed a good correlation among the samples.On this basis,a batch of differentially expressed genes between the 37℃and 44℃treatment groups were successfully screened out.These genes may be the key factors triggering the differential cellular responses.3.Enrichment analysis results:After annotation and expression screening of the identified differentially expressed genes,the GO＿CC enrichment results showed significant changes in the membrane system and plasma membrane system.The GO＿MF results mainly showed an up-regulation of functions such as protein binding and ion binding.In the KEGG enrichment analysis,using KEGG Pathway as the unit,changes in the NOD-like receptor signaling pathway（NLR signaling pathway）related to pyroptosis and a significant up-regulation of the Calcium Signaling Pathway closely related to calcium regulation were detected.The heatmap of TRP channel expression levels showed an increase in the expression levels of TRPM2,TRPV3,and TRPV2 genes.The correlation coefficient diagram indicated a high-degree positive functional correlation between NLRP3 and TRPM2.Combining the significant up-regulation of“metal ion binding”in the GO＿MF enrichment and the up-regulation of the“Calcium Signaling Pathway”in the KEGG enrichment,calcium-regulation-related pathways were screened and subjected to GO term enrichment,revealing a significant up-regulation of the related pathways.4.Preliminary verification results of Real-time PCR and Western Blot:Real-time PCR results showed that the expression levels of key genes in the pathway,such as NLRP3,IL-1β,and IL-18,were significantly up-regulated in the 44℃hyperthermia-treated group compared with the 37℃constant-temperature cultured group,which was consistent with the trend of RNA sequencing analysis results.Western Blot experiments further confirmed that the expression levels of the proteins encoded by these key genes also increased significantly in the 44℃treatment group,indicating that hyperthermia treatment affected not only the transcriptional level of genes but also the protein expression level.5.To further explore the impact of TRPM2 on the biological functions of Ha Cat cells after hyperthermia,the TRPM2 gene sequence in the Ha Cat cell line was knocked out using the crisp/cas9 technology.6.Experimental results of TRPM2-deficient cells:The results of the CCK-8 assay suggested that the absence of the TRPM2 channel had a certain protective effect on cells,reducing the damage to cells caused by oxidative stress and hyperthermia.The LDH release assay showed that the LDH release of Trpm2-/-cells was lower than that of Wt cells,further confirming the promoting effect of the TRPM2 channel in cytotoxic damage.The ELISA assay indicated that the absence of the TRPM2 channel inhibited the release of inflammatory factors IL-1βand IL-18,alleviating the inflammatory response.The immunofluorescence assay found that the fluorescence intensity of intracellular ASC and Caspase-1 in Wt cells increased significantly after treatment,suggesting that the TRPM2 channel is involved in the activation process of the NLRP3 inflammasome.Western blot results showed that the expression levels of NLRP3,IL-1β,IL-18,caspase-1,and pro caspase-1 proteins in Wt cells increased significantly after treatment,and the up-regulation was more obvious in the combined treatment group,further confirming the key role of the TRPM2 channel in the activation of the inflammatory signaling pathway.7.Experimental results of chelating calcium ions and wart tissues:After adding BAPTA-AM to chelate intracellular calcium ions,the results of flow cytometry with Annexin V/PI staining showed that the pyroptosis ratio of Wt cells decreased after treatment,suggesting that the decrease in intracellular calcium ion levels may inhibit the occurrence of pyroptosis.The pyroptosis ratio of Trpm2-/-cells was already lower than that of Wt cells,and it decreased further after chelating calcium ions.After subjecting fresh wart tissues from patients to hyperthermia,oxidative stress,and their combined treatment respectively,immunohistochemical experimental results showed that both hyperthermia and oxidative stress treatment could up-regulate the expression of NLRP3 and Caspase-1in wart tissues,and the up-regulation effect was more significant in the combined treatment group.This was consistent with the cell experiment results,further proving that hyperthermia and oxidative stress stimuli can activate the NLRP3 inflammasome signaling pathway.Meanwhile,when a TRPM2 inhibitor was added during similar treatment,the up-regulation degree of NLRP3 and caspase-1 expression was weakened,highlighting the important role of the TRPM2 channel in this process again.Conclusion:This study systematically elucidates the mechanisms underlying the effects of combined 44℃heat and H₂O₂stimulation on the TRPM2/NLRP3 pathway in Ha Ca T cells.It was found that heat and oxidative stress synergistically activate the NLRP3 inflammasome,inducing pyroptosis and the release of inflammatory factors.This process is mediated by Ca²⁺influx via TRPM2,and the deletion or inhibition of TRPM2 can mitigate damage and inflammatory responses.This research provides theoretical support for the application of heat therapy in the treatment of warts and offers possibilities for developing novel therapeutic strategies based on TRPM2.更多还原