热损伤角质形成细胞外泌蛋白的差异表达

Different expression of protein in the supernatant of heat injured keratinocytes

目的 初步探索热损伤表皮角质形成细胞（keratinocytes,KC）培养上清蛋白质的整体变化规律.方法 选用人源性表皮角质形成细胞建立KC热损伤模型,收集正常培养及热损伤后12 h的KC培养上清,超滤、冻干,获得蛋白样品.运用固相pH梯度双向凝胶电泳（two-dimensional gel electrophoresis,2-DE）分离正常及热损伤后KC培养上清中的总蛋白质,凝胶银染显色后选用ImageMaster 2D图像分析软件,进行比较分析识别差异表达的蛋白质.结果 ①正常培养及热损伤后KC培养上清凝胶的平均蛋白质点数分别为1 898±113和1 877±97,经匹配每张胶上用于统计学分析的点为1118个.②正常培养及热损伤后KC的双向凝胶电泳图谱中差异表达蛋白点数为26个,其中16个点在正常KC培养上清中高表达,10个点在热损伤后KC培养上清中高表达.③经质谱分析、数据库搜索,成功鉴定了16个差异蛋白质点,包括10种蛋白.热损伤后表达下调的蛋白质点有：alpha-enolase,actin cytoplasmic2,peroxiredoxin-4,phosphoglycerate mutase 1,G proteinregulated inducer of neurite outgrowthl;表达上调的蛋白质点有：purine nucleoside phosphorylase,tumor necrosis factor ligand superfamily member10,proteasome subunit alpha type-7,UDP-glucose 6-dehydrogenase.结论 通过建立正常及热损伤KC培养上清的双向凝胶电泳图谱,发现两者间存在一些差异表达的蛋白,为进一步从中优选调控组织损伤修复的主要作用分子,明晰创面修复过程中修复细胞间的调控机制提供了一定的理论依据.

Objective To compare the difference of protein expression in the supernatant of heat injured keratinocytes （ KC） and normal KC. Methods A model of heat injured KC was produced in vitro. The supernatant of normal KC and heat injured KC was collected after culture for 12 hours, and was ultrafiltered and lyophilized to get the protein. The protein sample was separated by immobilized pH gradient based two dimensional gel electrophoresis （2-DE）. The gel was stained and the different expression of protein was analyzed using ImageMaster 2D analysis software. Results ①Average protein spots were 1 898 ±113, 1 877 ±97 in the supernatant of normal and heat injured KC and 1118 protein spots could be used for statistical analysis. ②Statistical result showed that 26 protein spots were significantly different between the two groups. 16 protein spots were higher in the supernatant of normal KC and then 10 protein spots were lower in the normal group. @16 protein spots, which included 10 kinds of proteins, were identified successfully as different spots. Lower expression proteins were alpha-enolase, actin cytoplasmic2, peroxiredoxin-4, phosphoglycerate mutase 1, G protein-regulated inducer of neurite outgrowth 1 in the supernatant of heat injured KC. Higher expression proteins in heat KC were purine nucleoside phosphorylase, tumor necrosis factor ligand superfamily memberl0, proteasome subunit alpha type-7, UDP-glucose 6-dehydrogenase in the supernatant of heat injuryed KC. Conclusions The result indicated that there are some significant different expression proteins in the supernatant of normal KC and heat injured KC.These findings provide new data for screening major molecules of tissue repair and finding the mechanism of wound repair.