發(fā)布時間：2018-05-12 01:12

  本文選題：水楊酸 + 人角質(zhì)形成細胞��； 參考：《大連醫(yī)科大學(xué)》2012年碩士論文

【摘要】：背景和目的： 特應(yīng)性皮炎（atopic dermatitis，AD）是一種較常見的皮膚病，特點為出現(xiàn)不同程度的皮膚瘙癢、干燥脫屑及炎癥反應(yīng)。越來越多的研究表明皮膚屏障功能異常在特應(yīng)性皮炎的發(fā)病機制中具有重要作用。絲聚蛋白（FLG）是完整的皮膚屏障和表皮水化功能必不可少的分化蛋白。FLG的缺乏及基因突變可以破壞皮膚屏障，引起皮膚干燥而導(dǎo)致瘙癢,變應(yīng)原易于進入。人組織激肽釋放酶7（KLK7）的作用是參與皮膚細胞脫落過程，同時在其他的皮膚病理生理學(xué)如角化，炎癥等過程中發(fā)揮作用。特應(yīng)性皮炎KLK7表達水平明顯增高，引起脫屑及瘙癢。 本研究通過測定不同濃度的水楊酸對人角質(zhì)形成細胞中FLG和KLK7的影響，探討水楊酸對表皮屏障功能的影響，從而為臨床上水楊酸對治療特應(yīng)性皮炎提供實驗依據(jù)。 方法： 不同濃度的水楊酸作用于人角質(zhì)形成細胞(HKC)24h，采用MTT法檢測人角質(zhì)形成細胞的細胞存活率以確定水楊酸的無毒劑量。在無毒劑量范圍內(nèi)，采用不同濃度的水楊酸（500μg/ml、50μg/ml、5μg/ml、0.5μg/ml）作用于HKC，并設(shè)立空白對照組（用培養(yǎng)基代替藥物），培養(yǎng)24h后收集細胞或上清液，分別用S-P免疫組化方法檢測人角質(zhì)形成細胞中FLG的表達水平，用ELISA方法檢測HKC上清液KLK7的水平，用RT-PCR方法檢測FLG和KLK7mRNA的表達水平。各組數(shù)據(jù)采用單因素方差分析進行統(tǒng)計學(xué)比較。 結(jié)果： 1. MTT法檢測水楊酸對HKC生存率的影響：2000μg/ml，1000μg/ml的水楊酸濃度作用HKC24h的存活率分別為28.37%和71.16%，與陰性對照組（100%）相比明顯降低（P0.01）；500μg/ml,50μg/ml,5μg/ml，0.5μg/ml的水楊酸作用于HKC24h后的存活率分別為98.04%，97.93%，98.28%，99.1%，均95%，與陰性對照組（100%）相比無顯著差異（P0.05）。因此，后續(xù)實驗水楊酸的濃度確定為500μg/ml,50μg/ml,5μg/ml,0.5μg/ml。 2. S-P免疫組化方法檢測水楊酸對HKC中FLG表達的影響：與陰性對照組（430.996±96.979）相比，四個濃度（500μg/ml、50μg/ml、5μg/ml、0.5μg/ml）的水楊酸對HKC細胞FLG的表達均有促進作用（1520.476±37.807，3627.906±228.503,1590.677±166.396,808.073±154.108），差異具有統(tǒng)計學(xué)意義（P0.01）。 3. RT-PCR方法檢測水楊酸對HKC中FLG mRNA表達的影響：與陰性對照組（7760±74.746）相比，四個濃度（500μg/ml、50μg/ml、5μg/ml、0.5μg/ml）的水楊酸對HKC細胞FLG的mRNA表達均有促進作用（13500±1014.193,16308±2034.382,137770±2255.839,11935±505.433），差異具有統(tǒng)計學(xué)意義（P0.01）。 4. ELISA方法檢測水楊酸對HKC KLK7表達水平的影響：與陰性對照組（0.2325±0.1516）相比，四個濃度（500μg/ml、50μg/ml、5μg/ml、0.5μg/ml）的水楊酸對HKC中KLK7的蛋白表達均有抑制作用（0.1017±0.01830,0.0787±0.01180,0.9570±0.19140,0.1120±0.02120），差異具有統(tǒng)計學(xué)意義（P0.01）。 5. RT-PCR方法檢測水楊酸對HKC KLK7mRNA表達的影響：RT-PCR結(jié)果表明，與陰性對照組（10570±800.733）相比，四個濃度（500μg/ml、50μg/ml、5μg/ml、0.5μg/ml）的水楊酸對HKC細胞中KLK7的mRNA表達均有抑制作用（9556±512.000,7186±183.567,7571±235.119；8421±211.028），差異具有統(tǒng)計學(xué)意義（P0.01）。 四個檢測的濃度中，，均是濃度為50μg/ml的水楊酸作用效果最明顯。 結(jié)論： 1、濃度為0.5μg/ml~500μg/ml的水楊酸可以促進體外培養(yǎng)HKC中FLG的蛋白和mRNA表達，并且抑制KLK7的蛋白和mRNA表達水平。其中，水楊酸濃度在50μg/ml時作用效果最明顯。 2、水楊酸可通過上調(diào)FLG和下調(diào)KLK7的表達水平，而改善表皮屏障功能，為水楊酸治療特應(yīng)性皮炎提供了理論依據(jù)。
[Abstract]:Background and Purpose :

atopic dermatitis ( AD ) is a common skin disease characterized by the occurrence of different degrees of skin itching , drying and demobilizing and inflammatory reaction . More and more studies have shown that the abnormal skin barrier function plays an important role in the pathogenesis of atopic dermatitis .

In this study , the effects of salicylic acid on the function of epidermal barrier were investigated by measuring the effects of salicylic acid on FLG and KLK7 in human keratinocytes .

Method :

The expression levels of FLG and KLK7 mRNA in human keratinocytes were detected by ELISA . The levels of FLG and KLK7 mRNA were detected by ELISA .

Results :

1 . The effect of salicylic acid on the survival rate of HKC was detected by MTT assay : the survival rate of HKC24h was 28.37 % and 71.16 % in 2000 渭g / ml and 1000 渭g / ml , respectively ( P0.01 ) .
The survival rates of salicylic acid at 500 渭g / ml , 50 渭g / ml , 5 渭g / ml , 0.5 渭g / ml for HKC24h were 98.04 % , 97.93 % , 98.28 % , 91.1 % and 95 % , respectively , and the concentrations of salicylic acid were determined to be 500 渭g / ml , 50 渭g / ml , 5 渭g / ml , 0.5 渭g / ml .

2 . The effect of salicylic acid on the expression of FLG in HKC was detected by S - P immunohistochemical method : compared with the negative control group ( 40.996 鹵 96.79 ) , the expression of FLG in the four concentrations ( 500 渭g / ml , 50 渭g / ml , 5 渭g / ml , 0.5 渭g / ml ) promoted the expression of FLG in HKC cells ( 1520.476 鹵 37.807 , 3627.906 鹵 228.503 , 155.677 鹵 166.396 , 808.073 鹵 154.108 ) , with statistical significance ( P0.01 ) .

3 . The effect of salicylic acid on the expression of FLG mRNA in HKC was detected by RT - PCR . Compared with the negative control group ( 7760 鹵 74.746 ) , four concentrations ( 500 渭g / ml , 50 渭g / ml , 5 渭g / ml , 0.5 渭g / ml ) of salicylic acid could promote the expression of FLG mRNA in HKC cells ( 13500 鹵 1014.193 , 16308 鹵 203.382 , 137770 鹵 2255.839 , 11935 鹵 505.433 ) , with statistical significance ( P0.01 ) .

4 . The effect of salicylic acid on the expression level of HKC KLK7 was detected by ELISA . The expression of KLK7 protein in HKC was significantly inhibited by salicylic acid ( 0.1017 鹵 0.01830 , 0.0787 鹵 0.01180 , 0.970 鹵 0.19140 , 0.1120 鹵 0.02120 ) in four concentrations ( 0.1017 鹵 0.01830 , 0.0787 鹵 0.01180 , 0.970 鹵 0.19140 , 0.1120 鹵 0.02120 ) .

5 . The effect of salicylic acid on the expression of KLK7 mRNA was detected by RT - PCR . The results showed that the expression of KLK7 mRNA in HKC cells was inhibited by salicylic acid ( 9556 鹵 12.000 , 7186 鹵 183.567 , 7571 鹵 235.119 ) as compared with the negative control group ( 10570 鹵 800.733 ) .
The difference was statistically significant ( P0.01 ) .

Among the four concentrations tested , the effect of salicylic acid at the concentration of 50 渭g / ml was the most obvious .

Conclusion :

1 . salicylic acid with a concentration of 0.5 渭g / ml ~ 500 渭g / ml can promote the expression of FLG protein and mRNA in HKC in vitro , and inhibit the protein and mRNA expression level of KLK7 . The effect of salicylic acid is the most obvious when the concentration of salicylic acid is 50 渭g / ml .

2 . salicylic acid can improve the epidermal barrier function by up - regulating the expression level of FLG and down - regulating KLK7 , and provide a theoretical basis for the treatment of atopic dermatitis for salicylic acid .

【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R758.2

【參考文獻】

相關(guān)期刊論文 前1條

1 馬琳;申春平;;特應(yīng)性皮炎與皮膚屏障相關(guān)性研究進展[J];皮膚病與性病;2011年02期

本文編號：1876451