~目的：反常性痤疮（acne inversa, AI）是一种主要累及顶泌汗腺分布及间擦皱褶部位的慢性炎症性皮肤病，常反复发作，临床早期主要表现为丘疹、结节，后期可逐渐形成脓肿、窦道、瘢痕，常伴有疼痛。本病是与遗传、环境因素等多种因素有关的免疫相关疾病，且此病极大地影响患者的社交活动、生活质量，易伴发抑郁、焦虑及社交障碍等心理性疾病。反常性痤疮患者中约有30%-40%的患者有家族史，即家族性反常性痤疮；而占60%-70%的散发性反常性痤疮患者中一直未找到γ-分泌酶相关基因突变，其致病原因至今尚未明确。研究散发性反常性痤疮的致病因素和发病机制至关重要，对该病的早期干预有着不可忽视的作用。临床与实验室研究发现，反常性痤疮的发病以及病情加重通常因为其它因素所导致，比如患者肥胖、出汗、摄入高热量饮食、吸烟等，据此推测，环境因素引起表观遗传学改变很可能参与反常性痤疮的发病与病情恶化。其中DNA甲基化是表观遗传学的重要组成部分，已经证实其参与炎症性疾病、免疫性疾病以及肿瘤等多种疾病的发病过程，本研究将着重于研究DNA甲基化在散发性反常性痤疮发病机制中的作用，通过筛查散发性反常性痤疮皮损中的DNA甲基化异常的基因，观察在AI患者中表达的变化，并通过构建反常性痤疮模型小鼠进一步深入研究其在反常性痤疮发病机制中的作用。
方法：获得患者知情同意后，对接受外科手术和植皮治疗的散发性反常性痤疮患者进行取材，切取手术切除的炎性皮损的边缘处皮肤以及植皮修剪所得的边缘正常皮肤组织，去除坏死组织及皮下脂肪组织后分别提取全层皮肤组织的基因组DNA，进行全基因组DNA甲基化测序，对测序结果进行归纳总结，对差异基因DMR经生物信息分析以及文献检索，筛选出目标基因CXCL16，分别采用免疫组化、Western Blot对其编码的蛋白质在患者皮肤中的表达量进行分析和比较，同时检测CXCL16的相应受体CXCR6的表达情况；而后构建NCSTNΔKC模型小鼠，运用他莫昔芬诱导反常性痤疮小鼠模型，对NCSTNΔKC小鼠模型背部皮损处CXCL16的表达情况进行进一步验证，提取NCSTNΔKC小鼠与正常对照组小鼠脾脏的单细胞悬液进行流式分析，检测辅助型T淋巴细胞亚群Th1、Th17、Treg的比例差异。
结果：正常皮肤对照与皮损处皮肤组织经全基因组DNA甲基化测序分析结果分析显示，共检测到10807个DMR差异基因，筛选出差异值较大的DMR基因，经分析，位于启动子区域的CG序列高甲基化的差异基因有2101个，进一步根据GO分析、KEGG分析、蛋白功能定位以及与本病发病机制通路的相关性，并经过文献检索筛选出目的基因CXCL16，CXCL16在皮损处其启动子区域呈高甲基化状态；免疫组化及Western Blot结果均显示反常性痤疮皮损处CXCL16蛋白表达降低，CXCL16的相应受体CXCR6的表达也降低；他莫昔芬诱导NCSTNΔKC小鼠构建反常性痤疮小鼠模型，在第40天左右开始出现典型皮损，且NCSTNΔKC小鼠脾脏明显肿大，皮损处CXCL16的表达降低，与患者皮损处CXCL16的表达情况一致；对正常对照组小鼠、NCSTNΔKC模型组小鼠的脾脏细胞进行流式检测，检测辅助型T淋巴细胞亚群的比例变化，结果显示NCSTNΔKC模型组小鼠脾脏中Th1、Th17、Treg细胞比例降低。
结论：在散发性反常性痤疮皮损中，CXCL16基因启动子区域呈高甲基化状态，CXCL16启动子区域高甲基化导致其编码的CXCL16蛋白表达减少，导致皮损处CXCL16的低表达状态，CXCL16的唯一受体CXCR6在皮损处表达也降低，NCSTNΔKC小鼠模型脾脏中Th1、Th17、Treg细胞比例降低提示反常性痤疮可能存在免疫应答缺陷或其他免疫异常。CXCL16高甲基化可能是反常性痤疮的发病因素之一，其致病机制亟待进一步研究。

Objective:
Acne inversa (AI) is a chronic and recurrent inflammatory skin disease primarily affecting apocrine gland-rich and inframammary fold areas of the body, and it usually presents with initial painful nodules, abscesses and then progress to sinus tracts and scars. AI is a multifactorial immune related disease, which genetic and environmental factors play a trigger role. AI has a destructive impact on the social activities and quality of life of patients, and patients with AI have a higher risk of developing psychological disease. About 30%-40% of AI patients were reported have a family history, while gene mutation was not found in 60%-70% of the sporadic patients with no family history. The exact etiology of AI is still unproven. Clinical and laboratory studies indicated that acne inversa is usually caused by other factors such as obesity, sweating, high fat diet, smoking and so on, so researchers speculated that obesity and smoking are eminent environmental risk or trigger factors in AI development. The study of the pathogenic factors and pathogenesis is important and cannot be neglected in the early intervention of the disease. DNA methylation participated in the pathogenesis of many diseases such as inflammatory, immune, tumor and other diseases, which is an important part in epigenetics. This study focuses on the pathogenesis of DNA methylation in sporadic acne inversa. To study the abnormal DNA methylated genes and its expression in sporadic acne inversa lesions, and further focused its role in the pathogenesis of AI by building a mice model.
Methods
With the informed consent of the patient who treated with surgery, after excising necrotic tissue and subcutaneous fat, the whole genome DNA methylation sequencing of inflammatory skin lesions and non-lesional skin (trimed edge of skin grafts) from sporadic acne inversa patients was conducted. After summarize, literature search and biological analyze of DMR genes, we choose CXCL16 as our target gene. CXCL16 expression in lesion and normal control skin was analyzed and compared by IHC and WB, and the expression of the CXCL16’s specific receptor CXCR6 protein was detected. The acne inversa mice model was build by applying tamoxifen on NCSTNΔKC mice, and the expressions of CXCL16 was analyzed in the skin lesions of NCSTNΔKC mice, and flow cytometry was used to detect the splenic T helper cell such as Th1, Th17 and Treg cell proportion of the NCSTNΔKC mice.

Results
A total of 10807 different genes was analyzed after whole genome DNA methylation sequencing, the DMR (differentially methylated regions) genes with signaficative difference was filtrated. Compared with control, total 2101 genes which CG hypermethylation anchored in promotor area were identified in acne inversa lesion. Bioinformatics analysis was conducted, including gene ontology (G0) analysis, KEGG, protein analysis, function localization and pathogenic pathways relevance, we choose CXCL16 as our target gene after literature review, which the promoter regions of CXCL16 is hypermethylated in skin lesion; IHC and WB results shows that the CXCL16 expression in acne inversa skin lesion was decreased, and the corresponding receptor CXCR6, was also decreased.
The acne inversa mice model was build by applying tamoxifen on NCSTNΔKC mice, the typical lesions appeared in the 40 days, splenomegaly was observed. The CXCL16 expression in skin lesion of mice was identified decreased, consistent with the patients’ lesions. The ratio of Th cell in the spleen of NCSTNΔKC mice was detected by flow cytometry, the results suggested that the proportions of Th1, Th17 and Treg cells in NCSTNΔKC mice were decreased.
Conclusion
In this study, the hypermethylation in CXCL16 gene promoter region and the decreased CXCL16 expression was revealed in acne inversa lesion. And the protein expression of its specific receptor CXCR6 was also decreased. The decreased spleen lymphocyte subsets in NCSTNΔKC mice of Th1, Th17 and Treg cell proportion may suggest the defective immune response. Hypermethylation of CXCL16 may be one of the pathogenesis factors of acne inversa, and the pathogenesis of acne inversa from molecular level needs further study.