摘要目的:探讨在敏感性皮肤患者皮损中紧密连接蛋白(CLDN)5降低是否通过影响人永生化角质形成细胞系HaCaT的炎症参与敏感性皮肤的发病机制。方法:2018年1月至2020年3月，昆明医科大学附属第一医院皮肤科实验室收集敏感性皮肤女性患者面部皮损组织5份、正常面部皮肤组织5份，石蜡包埋切片后进行免疫组织化学染色确定claudin-5基因在敏感性皮肤和正常皮肤的表达。组织实验分为敏感性皮肤组和正常皮肤组2组，细胞实验分为CLDN5敲减组(sh-CLDN5)和CLDN5正常组(sh-NC)。细胞实验中，用CLDN5和空载体(NC)慢病毒shRNA转染HaCat细胞，收集sh-CLDN5和sh-NC各3份，进行转录组测序，在测序结果中筛选出差异表达的促炎性细胞因子mRNA；对筛选出的差异表达的促炎性细胞因子mRNA，在HaCat细胞中用Western印迹法或ELISA验证其蛋白水平的表达，并用Western印迹法检测sh-CLDN5组和sh-NC组MAPK通路和NF-κB通路关键蛋白的表达情况。结果:免疫组织化学染色显示，claudin-5基因定位于正常皮肤颗粒层细胞间质中，敏感性皮肤患者表皮较正常皮肤薄且claudin-5基因表达减少。在HaCaT细胞中，sh-CLDN5组中促炎性细胞因子白细胞介素(IL)-8升高[sh-CLDN5组(272.91±30.25) pg/ml，sh-NC组[(55.58±7.07 ) pg/ml， P<0.01)]，但sh-NC组IL-1β分泌量[(8.04±1.34) pg/ml]与sh-CLDN5组分泌量[(12.15±3.07) pg/ml]比较，差异无统计学意义( P>0.01)。与sh-NC组比较，sh-CLDN5组MAPK通路P-JNK、p-ERK、P-p38表达增加(均 P<0.01)]。 结论:敏感性皮肤表皮中，claudin-5基因表达降低，CLDN5在HaCaT细胞下调可激活MAPK通路并上调IL-8，参与敏感性皮肤早期炎症过程。

abstractsObjective:To clarify whether CLDN5 is involved in the pathogenesis of sensitive skin (SS) through inflammation.Methods:From January 2018 to March 2020, in the Dermatology Laboratory of the First Affiliated Hospital of Kunming Medical University, facial tissues were collected from 5 patients diagnosed with sensitive skin and 5 cases of normal facial skin. The organizational experiment was divided into two groups: SS group and normal skin (NS) group. The cell experiment was divided into two groups: sh-CLDN5 group in which CLDN5 was knocked down and sh-NC group with normal expression of CLDN5. After paraffin embedding and sectioning, immunohistochemical staining was performed to determine the expression of claudin-5 in sensitive skin and normal skin. In cell experiments, lentivirus shRNA was transfected into HaCaT cells. Three cases in sh-CLDN5 group and three cases in sh-NC group were collected for transcriptome sequencing, and differentially expressed pro-inflammatory factor mRNA was screened from the sequencing results. Western blot or ELISA was used to verify the protein level expression of differentially expressed pro-inflammatory cytokines mRNA in HaCaT cells, and Western blot was used to detect the expression of key proteins in MAPK pathway and NF-κB in the sh-CLDN5 group and sh-NC group.Results:Immunohistochemical staining showed that claudin-5 was localized in the interstitial cells of the normal skin granular layer. Patients with sensitive skin had thinner epidermis than normal skin and significantly reduced claudin-5 expression. In HaCaT cells, the expression of pro-inflammatory cytokine IL-23A was increased, and IL-8 was elevated in the sh-CLDN5 group [(272.91±30.25) pg/ml, n=3] compared with sh-NC group [(55.58±7.07) pg/ml, n=3] ( P<0.01). There was no statistically significant difference in secretion volume between sh-NC group [(8.04±1.34) pg/ml, n=3] and sh-CLDN5 group [(12.15±3.07) pg/ml, n=3]. The expression of P-JNK, p-ERK, and P-p38 in the MAPK pathway was significantly increased ( P<0.01). Conclusions:CLDN5 is an important gene involved in the pathogenesis of SS. CLDN5 upregulates IL-23A and IL-8 in HaCaT and activates the MAPK pathway, which is involved in the process of early SS inflammation.