水杨酸对人角质形成细胞丝聚蛋白和激肽释放酶7表达的影响

发布时间：2018-05-12 01:12

本文选题：水杨酸 + 人角质形成细胞　；参考：《大连医科大学》2012年硕士论文

【摘要】：背景和目的：特应性皮炎（atopic dermatitis，AD）是一种较常见的皮肤病，特点为出现不同程度的皮肤瘙痒、干燥脱屑及炎症反应。越来越多的研究表明皮肤屏障功能异常在特应性皮炎的发病机制中具有重要作用。丝聚蛋白（FLG）是完整的皮肤屏障和表皮水化功能必不可少的分化蛋白。FLG的缺乏及基因突变可以破坏皮肤屏障，引起皮肤干燥而导致瘙痒,变应原易于进入。人组织激肽释放酶7（KLK7）的作用是参与皮肤细胞脱落过程，同时在其他的皮肤病理生理学如角化，炎症等过程中发挥作用。特应性皮炎KLK7表达水平明显增高，引起脱屑及瘙痒。本研究通过测定不同浓度的水杨酸对人角质形成细胞中FLG和KLK7的影响，探讨水杨酸对表皮屏障功能的影响，从而为临床上水杨酸对治疗特应性皮炎提供实验依据。方法：不同浓度的水杨酸作用于人角质形成细胞(HKC)24h，采用MTT法检测人角质形成细胞的细胞存活率以确定水杨酸的无毒剂量。在无毒剂量范围内，采用不同浓度的水杨酸（500μg/ml、50μg/ml、5μg/ml、0.5μg/ml）作用于HKC，并设立空白对照组（用培养基代替药物），培养24h后收集细胞或上清液，分别用S-P免疫组化方法检测人角质形成细胞中FLG的表达水平，用ELISA方法检测HKC上清液KLK7的水平，用RT-PCR方法检测FLG和KLK7mRNA的表达水平。各组数据采用单因素方差分析进行统计学比较。结果： 1. MTT法检测水杨酸对HKC生存率的影响：2000μg/ml，1000μg/ml的水杨酸浓度作用HKC24h的存活率分别为28.37%和71.16%，与阴性对照组（100%）相比明显降低（P0.01）；500μg/ml,50μg/ml,5μg/ml，0.5μg/ml的水杨酸作用于HKC24h后的存活率分别为98.04%，97.93%，98.28%，99.1%，均95%，与阴性对照组（100%）相比无显著差异（P0.05）。因此，后续实验水杨酸的浓度确定为500μg/ml,50μg/ml,5μg/ml,0.5μg/ml。 2. S-P免疫组化方法检测水杨酸对HKC中FLG表达的影响：与阴性对照组（430.996±96.979）相比，四个浓度（500μg/ml、50μg/ml、5μg/ml、0.5μg/ml）的水杨酸对HKC细胞FLG的表达均有促进作用（1520.476±37.807，3627.906±228.503,1590.677±166.396,808.073±154.108），差异具有统计学意义（P0.01）。 3. RT-PCR方法检测水杨酸对HKC中FLG mRNA表达的影响：与阴性对照组（7760±74.746）相比，四个浓度（500μg/ml、50μg/ml、5μg/ml、0.5μg/ml）的水杨酸对HKC细胞FLG的mRNA表达均有促进作用（13500±1014.193,16308±2034.382,137770±2255.839,11935±505.433），差异具有统计学意义（P0.01）。 4. ELISA方法检测水杨酸对HKC KLK7表达水平的影响：与阴性对照组（0.2325±0.1516）相比，四个浓度（500μg/ml、50μg/ml、5μg/ml、0.5μg/ml）的水杨酸对HKC中KLK7的蛋白表达均有抑制作用（0.1017±0.01830,0.0787±0.01180,0.9570±0.19140,0.1120±0.02120），差异具有统计学意义（P0.01）。 5. RT-PCR方法检测水杨酸对HKC KLK7mRNA表达的影响：RT-PCR结果表明，与阴性对照组（10570±800.733）相比，四个浓度（500μg/ml、50μg/ml、5μg/ml、0.5μg/ml）的水杨酸对HKC细胞中KLK7的mRNA表达均有抑制作用（9556±512.000,7186±183.567,7571±235.119；8421±211.028），差异具有统计学意义（P0.01）。四个检测的浓度中，，均是浓度为50μg/ml的水杨酸作用效果最明显。结论： 1、浓度为0.5μg/ml~500μg/ml的水杨酸可以促进体外培养HKC中FLG的蛋白和mRNA表达，并且抑制KLK7的蛋白和mRNA表达水平。其中，水杨酸浓度在50μg/ml时作用效果最明显。 2、水杨酸可通过上调FLG和下调KLK7的表达水平，而改善表皮屏障功能，为水杨酸治疗特应性皮炎提供了理论依据。
[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 卤 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 卤 37.807 , 3627.906 卤 228.503 , 155.677 卤 166.396 , 808.073 卤 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 卤 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 卤 1014.193 , 16308 卤 203.382 , 137770 卤 2255.839 , 11935 卤 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 卤 0.01830 , 0.0787 卤 0.01180 , 0.970 卤 0.19140 , 0.1120 卤 0.02120 ) in four concentrations ( 0.1017 卤 0.01830 , 0.0787 卤 0.01180 , 0.970 卤 0.19140 , 0.1120 卤 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 卤 12.000 , 7186 卤 183.567 , 7571 卤 235.119 ) as compared with the negative control group ( 10570 卤 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 .

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R758.2

【参考文献】