本文選題：慢性光化性皮炎 + 中波紫外線�。� 參考：《昆明醫(yī)科大學(xué)》2017年碩士論文

【摘要】：[目的]探討慢性光化性皮炎的臨床和病理特征,以加深對CAD的了解。同時觀察Caspase-14在慢性光化性皮炎中的表達(dá)情況以及中波紫外線(UVB)和去甲基化對HaCaT細(xì)胞Caspase-14mRNA和蛋白表達(dá)的影響,初步探討Caspase-14在CAD中作用以及可能的分子作用機(jī)制,以便為CAD的臨床防治提供進(jìn)一步的理論依據(jù)。[方法]1.收集昆明地區(qū)2008年-2016年昆明醫(yī)科大學(xué)第一附屬醫(yī)院皮膚科確診的173例慢性光化性皮炎臨床樣本,從性別、年齡、發(fā)病部位、皮損特點(diǎn)、發(fā)病季節(jié)、職業(yè)及組織病理學(xué)特點(diǎn)進(jìn)行統(tǒng)計(jì)分析。2.收集昆明醫(yī)科大學(xué)第一附屬醫(yī)院皮膚科確診慢性光化性皮炎、濕疹和正常人皮膚組織各10例分別作為實(shí)驗(yàn)組、對照組和正常對照組。免疫組化方法檢測Caspase-14在正常皮膚、CAD及濕疹皮損中的表達(dá)情況,比較Caspase-14在正常皮膚、CAD和濕疹中表達(dá)情況,并分析Caspase-14在皮膚屏障中的作用。3.96孔板培養(yǎng)HaCaT細(xì)胞,實(shí)驗(yàn)分成三組:①正常對照組;②UVB照射組(給予30mJ/cm2、60mJ/cm2、90mJ/cm2UVB 照射);③藥物干預(yù)組(加入 1.25umom/L、2.5umom/L、5umom/L濃度的5-azaC)。MTT檢測不同濃度5-azaC干預(yù)及不同劑量UVB照射HaCaT細(xì)胞生長情況,確定5-azaC最佳濃度。4.取第3-8代HaCaT細(xì)胞進(jìn)行試驗(yàn),待細(xì)胞90%融合時以不同強(qiáng)度中波紫外線照射,其中藥物處理組加入5-azaC,繼續(xù)培養(yǎng)24小時后終止細(xì)胞培養(yǎng),通過實(shí)時熒光定量PCR(QRT-PCR)、蛋白免疫印跡比較照光組、藥物處理組及正常對照組細(xì)胞中Caspase-14mRNA和蛋白表達(dá)水平。[結(jié)果]1.臨床資料分析結(jié)果:①173例CAD患者中,男性患者158例(91.33%),女性 15 例(8.67%);②發(fā)病年齡 20-85 歲,平均 58.26±12.86 歲,其中男性平均發(fā)病年齡60.57歲,女性平均發(fā)病年齡49.23;首診年齡22-88歲,平均 62.94±11.65 歲;20-39 歲 8 例(4.62%),40-59 歲 45 例(26.01%),60-79歲114例(65.90%),80歲及以上者6例(3.47%);③發(fā)病最多的是12月份20例(11.56%),四季發(fā)病無明顯差異,春季稍高51例(29.48%);④光敏實(shí)驗(yàn)資料完整59例,UVB敏感的46例(77.97%),對UVA、UVB均敏感的11例(18.64%),對UVA無反應(yīng)42例(71.19%);⑤行病理組織檢查126例(72.83%),表皮角化過度45例(35.71%),角化過度伴角化不全73例(57.94%),有滲出、結(jié)痂12例(9.52%),棘層水腫77例(61.11%),基底層完整50例(39.68%),基底層液化變性21例(16.67%),真皮膠原嗜堿變性33例(26.19%),伴嗜酸性粒細(xì)胞浸潤26例(20.63%),炎細(xì)胞外滲23例(18.25%),合并蕈樣肉芽腫3例(2.38%),典型 Pautrier 微膿腫 2 例(1.59%)。2.Caspase-14在正常組織中表達(dá)在基底層上方,角質(zhì)層顯著,陽性率為90%;在CAD及濕疹中表達(dá)在棘層和顆粒層,角質(zhì)層不表達(dá),陽性率均為50%;CAD與濕疹比較,差異無統(tǒng)計(jì)學(xué)意義;與正常組織比較,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。3.MTT實(shí)驗(yàn)結(jié)果顯示,UVB照射對HaCaT細(xì)胞增殖活性有抑制作用,照射劑量增大,細(xì)胞增殖活性逐漸下降;5-azaC在0-5umom/L的濃度范圍內(nèi)不影響角質(zhì)形成細(xì)胞的增殖活性。4.實(shí)時熒光定量PCR及Western印跡檢測顯示,UVB照射使Caspase-14表達(dá)水平下降;給予5-AzaC干預(yù)后,Caspase-14mRNA及蛋白表達(dá)均增加,與照光組相比差異有統(tǒng)計(jì)學(xué)意義(P0.05)。[結(jié)論]1.CAD好發(fā)中老年男性;女性患CAD平均年齡較男性提前約10歲;發(fā)病無明顯季節(jié)差異;大多對UVB敏感,對UVA耐受;病理組織檢查似慢性濕疹、皮炎樣改變,特征性表現(xiàn)為真皮膠原嗜堿變,伴嗜酸性粒細(xì)胞浸潤。CAD由PD向AR進(jìn)展少見。2.慢性光化性皮炎發(fā)病與Caspase-14基因異常表達(dá)有關(guān)。3.UVB照射抑制HaCaT細(xì)胞增殖活性,使HaCaT細(xì)胞Caspase-14mRNA及蛋白表達(dá)下降,可能通過影響Filaggrin的表達(dá)而破壞皮膚屏障,從而參與CAD的發(fā)病。4.甲基化轉(zhuǎn)移酶抑制劑5-AzaC使基因去甲基化可以上調(diào)Caspase-14mRNA及蛋白表達(dá),從而達(dá)到修復(fù)皮膚屏障功能的作用,進(jìn)而參與紫外線誘發(fā)或加重CAD的病理進(jìn)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.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R758.1

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