胱天蛋白酶14在慢性光化性皮炎患者皮损中的表达及中波紫外线对其mRNA和蛋白在HaCaT细胞中表达的影响

doi:10.3760/cma.j.issn.0412-4030.2019.07.008

摘要/Abstract

摘要： 【摘要】目的观察胱天蛋白酶14（caspase-14）在慢性光化性皮炎（CAD）患者皮损中的表达以及中波紫外线（UVB）对HaCaT细胞caspase-14 mRNA和蛋白表达的影响。方法 2016年在昆明医科大学第一附属医院皮肤科门诊收集慢性光化性皮炎、湿疹患者皮损各10份作为试验组和阳性对照组，以10例健康人整形手术后的正常皮肤组织作为阴性对照。免疫组化染色检测caspase-14在正常皮肤、CAD及湿疹皮损中的表达。培养HaCaT细胞，分别给予0、30、60、90 mJ/cm2 UVB照射，加或不加甲基化酶抑制剂（5-AzaC）培养24 h后提取细胞，实时荧光定量PCR、Western印迹法检测UVB照射及加入5-AzaC 前后HaCaT细胞caspase-14 mRNA和蛋白表达水平。采用SPSS22.0软件，率的比较采用卡方检验，组间均数比较采用t检验和双因素方差分析。结果在CAD和湿疹皮损内，caspase-14主要表达于棘层和颗粒层，角质层不表达；正常皮肤组织角质层中caspase-14表达显著。10例CAD患者中5例皮损caspase-14阳性，10例健康人中9例阳性，两组阳性比例差异有统计学意义（χ2 = 7.30，P < 0.05）。实时荧光定量PCR及Western印迹检测显示，不同剂量UVB照射后caspase-14 mRNA、蛋白的表达差异均有统计学意义（F值分别为87.54、23.46，均P < 0.05），caspase-14 mRNA、蛋白的表达随UVB剂量升高有下降趋势。UVB剂量为0、30、60、90 mJ/cm2时，5-AzaC组的caspase-14 mRNA、蛋白的表达均高于UVB照射不加5-AzaC的对照组（均P < 0.05）。结论 CAD皮损中caspase-14表达降低，角质层不表达；UVB照射可下调HaCaT细胞caspase-14 mRNA及蛋白表达水平。

关键词: 光过敏疾患, 湿疹, 紫外线, 半胱氨酸天冬氨酸蛋白酶14, 角蛋白细胞, 慢性光化性皮炎

Abstract: 【Abstract】 Objective To determine the expression of caspase-14 in skin lesions of patients with chronic actinic dermatitis （CAD）, and to explore the effect of ultraviolet B （UVB） radiation on its mRNA and protein expression in HaCaT cells. Methods In 2016, skin samples were collected from lesions of 10 patients with CAD (test group), 10 patients with eczema （positive control group） and from normal skin of 10 healthy controls after cosmetic surgery （negative control group） in the Department of Dermatology, First Affiliated Hospital of Kunming Medical University. Immunohistochemical staining was performed to determine the expression of caspase-14 in the normal skin, CAD and eczema lesions. Cultured HaCaT cells were divided into several groups: UVB groups irradiated with 0, 30, 60, 90 mJ/cm2 UVB separately, and 5-AzaC groups irradiated with 0, 30, 60, 90 mJ/cm2 UVB separately followed by the treatment with the methylase inhibitor 5-AzaC for 24 hours. Then, the cells were collected, and real-time fluorescence-based quantitative PCR （RT-PCR） and Western blot analysis were conducted to determine the mRNA and protein expression of caspase-14 respectively in HaCaT cells in the UVB groups and 5-AzaC groups. Statistical analysis was carried out with SPSS22.0 software by using chi-square test for the comparison of rates, and t test and two-factor analysis of variance for the comparison of means. Results In the CAD and eczema lesions, caspase-14 was mainly expressed in the spinous and granular layers, but not in the stratum corneum. However, caspase-14 was markedly expressed in the stratum corneum of the normal skin tissues. Of the 10 CAD samples, 5 were positive for caspase-14, and 9 of 10 normal skin samples were positive for caspase-14. The positive rate of caspase-14 significantly differed between the two above groups （χ2 = 7.30, P < 0.05）. RT-PCR and Western blot analysis showed significant changes in the mRNA and protein expression of caspase-14 in HaCaT cells after irradiation with different doses of UVB （F = 87.54, 23.46, both P < 0.05）, which showed a decreasing trend along with the increase in the dose of UVB. After exposure to 0, 30, 60 and 90 mJ/cm2 UVB, the mRNA and protein expression of caspase-14 was significantly higher in the 5-AzaC groups than in the UVB groups （all P < 0.05）. Conclusions In CAD lesions, the expression of caspase-14 markedly decreased, and was absent in the stratum corneum. UVB radiation can down-regulate the mRNA and protein expression of caspase-14 in HaCaT cells.

Key words: Photosensitivity disorders, Eczema, Ultraviolet rays, Caspase 14, Keratinocytes, Chronic actinic dermatitis

李凌佳刘彤云. 胱天蛋白酶14在慢性光化性皮炎患者皮损中的表达及中波紫外线对其mRNA和蛋白在HaCaT细胞中表达的影响[J]. 中华皮肤科杂志, 2019, 52(7): 486-490.

Li Lingjia, Liu Tongyun . Expression of caspase-14 in skin lesions of patients with chronic actinic dermatitis and effect of ultraviolet B radiation on its mRNA and protein expression in HaCaT cells [J]. Chinese Journal of Dermatology, 2019, 52(7): 486-490.

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