【作者】 刘娟；

【导师】 张庆；

【作者基本信息】 中南大学 ， 皮肤病与性病学， 2025， 硕士

【摘要】 背景:白癜风（vitiligo）是一种常见的慢性自身免疫性皮肤病,以斑片状皮肤色素脱失为特点,可发生于身体的各个部位,发病机制目前尚不明确。其基本病理特征是功能性的黑素细胞（melanocytes,MCs）数量减少。已发现白癜风患者的角质形成细胞（keratinocytes,KCs）受到刺激后,可导致趋化因子CXCL9和CXCL10大量产生,它们能与CD8⁺T细胞上的CXCR3受体相结合,从而募集CD8⁺T细胞至MCs附近,进而杀伤MCs。所以KCs在白癜风发生发展中起到关键作用。因此,探究KCs功能异常的原因,可以为白癜风疾病的治疗提供新的思路。S100A8属于S100家族的Ca²⁺结合蛋白,其通常与S100A9以异二聚体的形式存在。S100A8/A9二聚体中S100A8发挥主要作用,而S100A9主要是维持S100A8蛋白结构的稳定性。S100A8可通过诱导白细胞募集和刺激细胞因子分泌在调节炎症反应中发挥关键作用。既往研究已证明S100A8可通过激活NF-κB信号通路促进炎症因子（如IL-6和TNF-α等）释放,而已知IL-6和TNF-α的增多是白癜风发病的原因之一,且IL-6和TNF-α均可导致细胞衰老。然而,S100A8是否在白癜风患者KCs中起作用尚无报道。目的:检测S100A8在白癜风患者皮损KCs中的表达水平,探索S100A8对NF-κB信号通路和KCs功能的影响,并初步探究S100A8在白癜风发病机制中的作用。方法:（1）S100A8与其下游因子CXCL9、CXCL10、NF-κB信号通路相关基因及细胞衰老通路相关基因在白癜风患者病变皮损中的表达:在GEO数据库下载单细胞数据集GSE203262,分析白癜风患者皮损差异表达基因最丰富的KCs cluster14细胞亚群中S100A8、CXCL9和CXCL10的m RNA表达水平,分析白癜风患者病变皮损KCs cluster14细胞亚群和MCs亚群中高表达的信号通路。通过转录组数据集GSE75819,分析S100A8、CXCL9和CXCL10在白癜风病变皮损表皮中m RNA表达水平和白癜风病变皮损表皮中高表达的信号通路。通过Western Blot和免疫荧光染色实验分析并比较正常人皮肤和白癜风患者病变皮损中S100A8和p65蛋白的表达水平,通过免疫荧光染色实验检测S100A8与CXCL9和CXCL10的分布关系以及p16⁺MCs占MCs的百分比、p21⁺MCs占MCs的百分比。（2）在人原代角质形成细胞（NHEKs）中调控S100A8的表达对NF-κB信号通路和KCs分泌功能的影响:构建S100A8过表达质粒/空白质粒和S100A8-si RNA/对照si RNA。收集正常人的皮肤组织,分离NHEKs,对一组NHEKs转染S100A8过表达质粒/空白质粒,对另一组NHEKs转染S100A8-si RNA/对照si RNA。转染后培养24 h,收取一部分细胞,采用Real-time PCR实验检测S100A8 m RNA水平。转染48 h后,收取其余细胞,行Western Blot实验检测S100A8、p65和p-p65的蛋白水平;收取上清液,行ELISA实验检测CXCL9、CXCL10、IL-6和TNF-a的分泌水平。结果:1.S100A8与其下游因子CXCL9、CXCL10、NF-κB信号通路相关基因及细胞衰老通路相关基因在白癜风患者病变皮损中的表达:（1）在单细胞数据集GSE203262（6个白癜风患者病变和非病变皮肤的表皮样本）分析中发现S100A8、CXCL9和CXCL10均在KCs cluster14细胞亚群中高表达,白癜风患者病变皮损KCs cluster14细胞亚群表达上调的差异基因中富集到了NF-κB、Toll样受体结合、趋化因子受体结合和细胞粘附分子结合等信号通路,白癜风患者病变皮损MCs亚群表达上调的差异基因中富集到了细胞衰老、细胞凋亡和细胞周期等信号通路。同时发现在转录组数据集GSE75819（30个白癜风患者病变和非病变皮肤的表皮样本）中S100A8、CXCL9和CXCL10在白癜风病变皮损中也高表达,还在白癜风患者病变皮损表皮表达上调的差异基因中富集到了NF-κB和细胞衰老等信号通路。（2）白癜风患者病变皮损（n=11）和正常人皮肤组织（n=10）的Western Blot结果显示S100A8在白癜风患者病变皮损中高表达（P<0.001）。白癜风患者病变皮损（n=16）和正常人皮肤组织（n=16）的免疫荧光实验结果亦显示S100A8在白癜风患者病变皮损KCs中高表达（P<0.0001）。白癜风患者病变皮损（n=10）和正常人皮肤组织（n=10）的免疫荧光实验结果还显示S100A8、CXCL9和CXCL10在白癜风患者病变皮损KCs中高表达且部位大致一致,三者形成共染;相对于正常人皮肤,白癜风患者病变皮损中产生S100A8、CXCL9和CXCL10的KCs明显增多（P<0.0001）。（3）白癜风患者病变皮损（n=12）和正常人皮肤组织（n=13）Western Blot实验结果显示p65在白癜风患者病变皮损中高表达（P<0.01）。白癜风患者病变皮损（n=13）和正常人皮肤组织（n=11）的免疫荧光实验结果亦显示p65在白癜风病变皮损KCs中高表达（P<0.0001）。（4）白癜风患者病变皮损（n=8）和正常人皮肤组织（n=6）的免疫荧光实验结果显示在白癜风患者病变皮损中p16⁺MCs占MCs的百分比明显高于正常组（P<0.01）。白癜风患者病变皮损（n=7）和正常人皮肤组织（n=10）的免疫荧光实验结果显示在白癜风患者病变皮损中p21⁺MCs占MCs的百分比（P<0.01）明显高于正常组。2.在NHEKs中调控S100A8的表达对NF-κB信号通路和KCs分泌功能的影响:（1）在NHEKs中转染质粒并培养24 h后,与空白质粒组相比,S100A8过表达质粒组S100A8 m RNA表达明显升高（P<0.01）。在NHEKs中转染质粒并培养48 h后,与空白质粒组相比,S100A8过表达质粒组S100A8（P<0.05）、p65（P<0.05）和p-p65（P<0.05）蛋白表达水平明显升高,IL-6（P<0.05）、TNF-α（P<0.05）、CXCL9（P<0.05）和CXCL10（P<0.05）蛋白分泌水平明显升高,差异具有统计学意义。（2）在NHEKs中转染si RNA并培养24 h后,与对照si RNA组相比,S100A8-si RNA组S100A8 m RNA表达明显下降（P<0.05）。在NHEKs中转染si RNA并培养48 h后,与对照si RNA组相比,S100A8-si RNA组S100A8（P<0.05）、p65（P<0.05）和p-p65（P<0.05）蛋白表达水平明显下降,IL-6（P<0.05）、TNF-α（P<0.05）、CXCL9（P<0.05）和CXCL10（P<0.05）蛋白分泌水平明显降低,差异具有统计学意义。结论:我们的研究发现白癜风患者病变皮损KCs中S100A8高表达,继而导致KCs NF-κB信号通路活化,CXCL9、CXCL10、IL-6和TNF-α过度分泌,IL-6和TNF-α分泌增多可能引起MCs的衰老,CXCL9和CXCL10分泌增多可能趋化CD8⁺T细胞杀伤MCs,这些因素最终引起白癜风疾病的发生发展。图29幅,表37个,参考文献83篇更多还原

【Abstract】 Background:Vitiligo is a common chronic autoimmune skin disease characterized by patchy loss of skin pigmentation,which can occur on any part of the body.Its pathogenesis remains unclear.The fundamental pathological feature is a decrease of functional melanocytes（MCs）.Existing studies have shown that stimulated keratinocytes（KCs）in vitiligo patients can overproduce the chemokines CXCL9 and CXCL10,which bind to the CXCR3 receptor on CD8⁺T cells.The CD8⁺T cells are then recruited to the vicinity of MCs and ultimately led to MCs destruction.Thus,KCs play a critical role in the development and progression of vitiligo.Therefore,investigating the mechanism of abnormal KCs function can provide new insights for the treatment of vitiligo.S100A8,a Ca²⁺binding protein belonging to the S100 family,typically exists as a heterodimer with S100A9.In the S100A8/A9heterodimer,S100A8 plays the dominant functional role,while S100A9primarily structural stabilizes the S100A8 protein.S100A8 exerts a crucial role in regulating inflammatory responses by inducing leukocyte recruitment and stimulating cytokine secretion.Previous studies have demonstrated that S100A8 can promote the release of inflammatory factors（such as IL-6 and TNF-α）by activating the NF-κB signaling pathway.It is well established that increased levels of IL-6 and TNF-αcontribute to the pathogenesis of vitiligo,and they can both induce cellular senescence.However,whether S100A8 plays a role in KCs of vitiligo patients remains unreported.Objective:To investigate the expression of S100A8 in KCs of lesional skin from vitiligo patients,explore the effects of S100A8 on the NF-κB signaling pathway and KCs function,and preliminarily inquiry the role of S100A8 in the pathogenesis of vitiligo.Methods:（1）The expression of S100A8 and its downstream factors（CXCL9,CXCL10）,NF-κB signaling pathway-related genes,and cellular senescence pathway-related genes in lesional skin of vitiligo patients:We downloaded the single-cell RNA-seq dataset GSE203262 from the GEO database and analyzed the m RNA expression levels of S100A8,CXCL9,and CXCL10 in the cluster 14 of KCs,which contained the richest differentially expressed genes（DEGs）,from the lesional skin of patients with vitiligo.Additionally,we investigated the highly activated signaling pathways in both cluster 14 of KCs and MCs subpopulations in vitiligo lesions.We also downloaded the transcriptome dataset GSE75819 and assessed the m RNA expression levels of S100A8,CXCL9 and CXCL10 in the epidermal tissues of vitiligo lesions.Moreover,we evaluated the signaling pathways that were highly expressed in the lesional epidermis of vitiligo patients.We performed western blot and immunofluorescence staining to compare the protein expression levels of S100A8 and p65 in skin tissue of healthy controls with lesional skin from vitiligo patients.In addition,immunofluorescence staining was used to examine the spatial distribution relationship between S100A8 and CXCL9/CXCL10,as well as to quantify the percentages of p16⁺MCs and p21⁺MCs relative to the total MCs population.（2）Effects of interfering S100A8 expression on NF-κB signaling pathway and KCs secretion function in normal human epidermal keratinocytes（NHEKs）.We constructed S100A8 overexpression plasmids and corresponding empty vector controls,along with S100A8-si RNA and control si RNA.Normal human skin tissues were collected,and NHEKs were isolated.NHEKs were divided into two experimental groups:one transfected with either S100A8 overexpression plasmid or empty vector,and the other transfected with either S100A8-si RNA or control si RNA.At 24 hours post transfection,some cells were collected,and S100A8 m RNA levels were quantified by real-time PCR.After 48 hours of transfection,other cells were harvested for western blot to detect the protein expression levels of S100A8,p65,and p-p65.Culture supernatants were collected for ELISA measurement of secreted levels of CXCL9,CXCL10,IL-6,and TNF-α.Results:1.The expression of S100A8 and its downstream factors（CXCL9,CXCL10）,NF-κB signaling pathway-related genes,and cellular senescence pathway-related genes in lesional skin of vitiligo patients:（1）The analysis of the single-cell RNA-seq dataset GSE203262（including epidermal samples from both lesional and non-lesional skin of6 vitiligo patients）revealed high expression of S100A8,CXCL9,and CXCL10 specifically within the cluster 14 of KCs.The analysis of upregulated DEGs in the cluster 14 of KCs from vitiligo lesional skin revealed significant enrichment in the following signaling pathways:NF-κB signaling pathway,Toll-like receptor binding pathway,chemokine receptor binding pathway,and cell adhesion molecule binding pathway.The analysis of upregulated DEGs in MCs from vitiligo lesional skin revealed significant enrichment in signaling pathways related to cellular senescence,apoptosis,and cell cycle regulation.Furthermore,analysis of the transcriptomic dataset GSE75819（epidermal samples from lesional and non-lesional skin of 30 vitiligo patients）revealed that S100A8,CXCL9,and CXCL10 were also highly expressed in vitiligo lesions.Additionally,upregulated DEGs in lesional epidermis showed enrichment in signaling pathways including NF-κB and cellular senescence.（2）The results of western blot demonstrated significantly higher protein expression levels of S100A8 in lesional skin from vitiligo patients（n=11）compared to normal skin controls（n=10）（P<0.001）.The results of immunofluorescence staining also revealed markedly increased S100A8 expression in KCs of vitiligo lesions（n=16）relative to normal skin（n=16）（P<0.0001）.The analysis of immunofluorescence staining of vitiligo lesional skin（n=10）and normal control skin（n=10）demonstrated significantly elevated expression of S100A8,CXCL9,and CXCL10 in lesional KCs of vitiligo patients,with distinct spatial colocalization patterns among the three biomarkers.Compared with normal skin,KCs expressing S100A8,CXCL9 and CXCL10 in the lesions of vitiligo patients increased significantly（P<0.0001）.（3）The results of western blot of vitiligo lesional skin（n=12）and normal human skin controls（n=13）demonstrated markedly higher protein levels of p65 in vitiligo lesions（P<0.01）.The results of immunofluorescence staining further confirmed profoundly increased protein expression of p65 in KCs from vitiligo lesions（n=13）compared with normal control skin（n=11）（P<0.0001）.（4）Immunofluorescence staining of lesions from vitiligo patients（n=8）and normal skin tissues（n=6）showed that the percentage of p16⁺MCs in lesions of vitiligo patients was significantly higher than that in normal group（P<0.01）.Similarly,immunofluorescence test of lesions from vitiligo patients（n=7）and normal skin tissues（n=10）showed that the percentage of p21⁺MCs in lesions of vitiligo patients was significantly higher than that in normal group（P<0.01）.2 Effects of interfering S100A8 expression on NF-κB signaling pathway and KCs secretion function in normal human epidermal keratinocytes（NHEKs）.（1）After transfected into NHEKs and cultured for 24 h,the m RNA expression of S100A8 in the S100A8 overexpression plasmid group was significantly higher than that in the blank plasmid group（P<0.01）.After transfected into NHEKs and cultured for 48 h,the protein expression levels of S100A8（P<0.05）,p65（P<0.05）and p-p65（P<0.05）in S100A8 overexpression plasmid group were greatly increased compared with the blank plasmid group.The protein secretion levels of IL-6（P<0.05）,TNF-α（P<0.05）,CXCL9（P<0.05）and CXCL10（P<0.05）were also higher in S100A8 overexpression plasmid group,and the differences were statistically significant.（2）After transfection of si RNA in NHEKs and culture for 24 h,the m RNA expression of S100A8 in S100A8-si RNA group was significantly decreased compared with that in the control group（P<0.05）.After transfected with si RNA in NHEKs and cultured for 48 h,the protein expression levels of S100A8（P<0.05）,p65（P<0.05）and p-p65（P<0.05）in S100A8-si RNA group were greatly decreased compared with the control group.The protein secretion levels of IL-6（P<0.05）,TNF-α（P<0.05）,CXCL9（P<0.05）,and CXCL10（P<0.05）were also alleviated in S100A8-si RNA group,and the differences were statistically significant.Conclusions:Our study demonstrated that increased expression of S100A8 in KCs of vitiligo lesional skin activates the NF-κB signaling pathway,leading to excessive secretions of CXCL9,CXCL10,IL-6 and TNF-α.The increased IL-6 and TNF-αproductions may induce the cellular senescence of MCs,while overexpressed CXCL9 and CXCL10may recruit CD8⁺T cells to kill MCs.These factors collectively contribute to the occurrence and development of vitiligo.更多还原