Caspase-14在慢性光化性皮炎中的表达及UVB和去甲基化对其mRNA和蛋白表达的影响

发布时间：2018-07-04 08:32

本文选题：慢性光化性皮炎 + 中波紫外线　；参考：《昆明医科大学》2017年硕士论文

【摘要】：[目的]探讨慢性光化性皮炎的临床和病理特征,以加深对CAD的了解。同时观察Caspase-14在慢性光化性皮炎中的表达情况以及中波紫外线(UVB)和去甲基化对HaCaT细胞Caspase-14mRNA和蛋白表达的影响,初步探讨Caspase-14在CAD中作用以及可能的分子作用机制,以便为CAD的临床防治提供进一步的理论依据。[方法]1.收集昆明地区2008年-2016年昆明医科大学第一附属医院皮肤科确诊的173例慢性光化性皮炎临床样本,从性别、年龄、发病部位、皮损特点、发病季节、职业及组织病理学特点进行统计分析。2.收集昆明医科大学第一附属医院皮肤科确诊慢性光化性皮炎、湿疹和正常人皮肤组织各10例分别作为实验组、对照组和正常对照组。免疫组化方法检测Caspase-14在正常皮肤、CAD及湿疹皮损中的表达情况,比较Caspase-14在正常皮肤、CAD和湿疹中表达情况,并分析Caspase-14在皮肤屏障中的作用。3.96孔板培养HaCaT细胞,实验分成三组:①正常对照组;②UVB照射组(给予30mJ/cm2、60mJ/cm2、90mJ/cm2UVB 照射);③药物干预组(加入 1.25umom/L、2.5umom/L、5umom/L浓度的5-azaC)。MTT检测不同浓度5-azaC干预及不同剂量UVB照射HaCaT细胞生长情况,确定5-azaC最佳浓度。4.取第3-8代HaCaT细胞进行试验,待细胞90%融合时以不同强度中波紫外线照射,其中药物处理组加入5-azaC,继续培养24小时后终止细胞培养,通过实时荧光定量PCR(QRT-PCR)、蛋白免疫印迹比较照光组、药物处理组及正常对照组细胞中Caspase-14mRNA和蛋白表达水平。[结果]1.临床资料分析结果:①173例CAD患者中,男性患者158例(91.33%),女性 15 例(8.67%);②发病年龄 20-85 岁,平均 58.26±12.86 岁,其中男性平均发病年龄60.57岁,女性平均发病年龄49.23;首诊年龄22-88岁,平均 62.94±11.65 岁;20-39 岁 8 例(4.62%),40-59 岁 45 例(26.01%),60-79岁114例(65.90%),80岁及以上者6例(3.47%);③发病最多的是12月份20例(11.56%),四季发病无明显差异,春季稍高51例(29.48%);④光敏实验资料完整59例,UVB敏感的46例(77.97%),对UVA、UVB均敏感的11例(18.64%),对UVA无反应42例(71.19%);⑤行病理组织检查126例(72.83%),表皮角化过度45例(35.71%),角化过度伴角化不全73例(57.94%),有渗出、结痂12例(9.52%),棘层水肿77例(61.11%),基底层完整50例(39.68%),基底层液化变性21例(16.67%),真皮胶原嗜碱变性33例(26.19%),伴嗜酸性粒细胞浸润26例(20.63%),炎细胞外渗23例(18.25%),合并蕈样肉芽肿3例(2.38%),典型 Pautrier 微脓肿 2 例(1.59%)。2.Caspase-14在正常组织中表达在基底层上方,角质层显著,阳性率为90%;在CAD及湿疹中表达在棘层和颗粒层,角质层不表达,阳性率均为50%;CAD与湿疹比较,差异无统计学意义;与正常组织比较,差异有统计学意义(P0.05)。3.MTT实验结果显示,UVB照射对HaCaT细胞增殖活性有抑制作用,照射剂量增大,细胞增殖活性逐渐下降;5-azaC在0-5umom/L的浓度范围内不影响角质形成细胞的增殖活性。4.实时荧光定量PCR及Western印迹检测显示,UVB照射使Caspase-14表达水平下降;给予5-AzaC干预后,Caspase-14mRNA及蛋白表达均增加,与照光组相比差异有统计学意义(P0.05)。[结论]1.CAD好发中老年男性;女性患CAD平均年龄较男性提前约10岁;发病无明显季节差异;大多对UVB敏感,对UVA耐受;病理组织检查似慢性湿疹、皮炎样改变,特征性表现为真皮胶原嗜碱变,伴嗜酸性粒细胞浸润。CAD由PD向AR进展少见。2.慢性光化性皮炎发病与Caspase-14基因异常表达有关。3.UVB照射抑制HaCaT细胞增殖活性,使HaCaT细胞Caspase-14mRNA及蛋白表达下降,可能通过影响Filaggrin的表达而破坏皮肤屏障,从而参与CAD的发病。4.甲基化转移酶抑制剂5-AzaC使基因去甲基化可以上调Caspase-14mRNA及蛋白表达,从而达到修复皮肤屏障功能的作用,进而参与紫外线诱发或加重CAD的病理进程。
[Abstract]:[Objective] to explore the clinical and pathological features of chronic actinic dermatitis in order to deepen the understanding of CAD and to observe the expression of Caspase-14 in chronic actinic dermatitis and the effect of UVB (UVB) and demethylation on the expression of Caspase-14mRNA and protein in HaCaT cells, and to explore the role of Caspase-14 in CAD and possible points. The mechanism of subaction in order to provide further theoretical basis for the clinical prevention and control of CAD. [methods]1. collected 173 cases of chronic actinic dermatitis confirmed by Department of Dermatology, the First Affiliated Hospital of Kunming Medical University, 2008 -2016, from sex, age, location, skin lesion, disease season, occupational and histopathological characteristics. Statistical analysis.2. collected 10 cases of chronic actinic dermatitis, eczema and normal human skin tissue in the Department of Dermatology, the First Affiliated Hospital of Kunming Medical University, respectively as experimental group, control group and normal control group. Immunohistochemical method was used to detect the expression of Caspase-14 in normal skin, CAD and skin lesions, compared with Caspase-14 in normal skin. The expression of normal skin, CAD and eczema, and analysis of the role of Caspase-14 in the skin barrier by.3.96 orifice plate culture HaCaT cells, the experiment was divided into three groups: (1) normal control group; (2) UVB irradiation group (30mJ/cm2,60mJ/cm2,90mJ/cm2UVB irradiation); (3) drug intervention group (1.25umom/L, 2.5umom/L, 5umom/L concentration 5-azaC).MTT detection different Concentration 5-azaC intervention and the growth of HaCaT cells irradiated by different doses of UVB, determine the best concentration of 5-azaC.4. to take the 3-8 generation HaCaT cells to carry out the experiment. When the cell 90% fusion is irradiated with different intensity of ultraviolet light, the drug treatment group joins 5-azaC and continues to culture for 24 hours after the terminal cell culture, through real-time fluorescence quantitative PCR (QRT-PCR). The expression level of Caspase-14mRNA and protein in the treatment group and the normal control group was compared with the protein immunoblotting group. [results the results of]1. clinical data analysis: (1) among 173 CAD patients, 158 cases (91.33%) and 15 women (8.67%) were male patients; the average age of onset was 20-85 years, the average age was 58.26 + 12.86 years, and the average age of male was 6. 0.57 years old, the average age of female onset was 49.23, the age of first diagnosis was 22-88 years old, the average age was 62.94 + 11.65 years, 20-39 years old 8 cases (4.62%), 40-59 year 45 (26.01%), 60-79 years 114 cases (65.90%), the most serious cases were December, there was no obvious difference in the four seasons 59 cases were complete, 46 cases (77.97%) sensitive to UVB, 11 cases (18.64%) sensitive to UVA and UVB, 42 cases (71.19%) without response to UVA, 126 cases (72.83%), 45 cases of epidermis keratinization (35.71%), hyperkeratokeratosis (57.94%), exudation, spinous layer edema, and basal layer complete cases 21 cases (16.67%), 33 cases of dermis collagen basophilic degeneration (26.19%), 26 cases of eosinophil infiltration (20.63%), 23 cases of inflammatory cell infiltration (18.25%), 3 cases of mycosis Mycosis (2.38%), and 2 cases (1.59%) of typical Pautrier micro abscess (1.59%) were expressed in the normal tissue above the basal layer, the horny layer was significant, the positive rate was positive. 90% in CAD and eczema were expressed in the spinous layer and granular layer, the cuticle was not expressed, the positive rate was 50%, and the difference between CAD and eczema was not statistically significant. Compared with the normal tissue, the difference was statistically significant (P0.05).3.MTT experimental results showed that UVB irradiation inhibited the HaCaT fine cell proliferation activity, the irradiation dose increased, and the cell proliferation activity was increased. In the concentration range of 0-5umom/L, 5-azaC did not affect the proliferation activity of keratinocytes in the range of keratinocytes,.4. real-time quantitative PCR and Western blot detection showed that UVB irradiation reduced the expression level of Caspase-14, and the prognosis of 5-AzaC drying, Caspase-14mRNA and protein expression were increased, compared with the light group, the difference was statistically significant (P0.05). [conclusion]1.CAD is good for middle-aged and elderly men; the average age of women with CAD is about 10 years ahead of men; there is no obvious seasonal difference in onset; most of them are sensitive to UVB, tolerance to UVA, pathological examination like chronic eczema, dermatitis like changes, characteristic manifestations of dermophilous collagen eosinophilia, and eosinophil infiltration of.CAD from PD to AR to develop rare.2. chronic light. .3.UVB irradiation inhibits the proliferation activity of HaCaT cells and reduces the expression of Caspase-14mRNA and protein in HaCaT cells, which may destroy the skin barrier by affecting the expression of Filaggrin, and thus participates in the pathogenesis of the.4. methylation transferase inhibitor 5-AzaC so that the gene demethylation can be up regulation of C in the pathogenesis of HaCaT cells. Aspase-14mRNA and protein expression can help repair skin barrier function, and then participate in UV induced or aggravated the pathological process of CAD.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R758.1

【参考文献】