热损伤对人皮肤成纤维细胞氧化应激的影响  

Oxidate stress changes of human fibroblasts after thermal injury

作　　者：宋晖 石有振[2] 俞为荣[2] 傅秀军[2] 胡逸萍[2] 方勇[2] 姚敏[2] 王莹[2] 

机构地区：[1]深圳市光明新区人民医院外三科,广东省518106 [2]上海交通大学医学院附属第三人民医院烧伤整形科

出　　处：《中华临床医师杂志（电子版）》2013年第13期122-125,共4页Chinese Journal of Clinicians(Electronic Edition)

基　　金：国家自然科学青年基金(81201469);上海市科委基金(10ZR1418400)

摘　　要：目的探讨体外培养人皮肤成纤维细胞热损伤后氧化应激及前列腺素E2(PGE2)分泌的变化及其可能的机制。方法体外培养人皮肤成纤维细胞,制作热损伤模型后分为损伤组、DPI预处理组、NAC预处理组三组,以未受热损伤细胞作为对照组。CCK8比色法检测细胞增殖。荧光探针法及化学发光法观察细胞内活性氧(ROS)含量和NADPH氧化酶(Nox)活性变化,EIA检测上清液中PGE2的变化。半定量RTPCR法检测人皮肤成纤维细胞表达的Nox亚型的mRNA水平。Western blot法检测热损伤后Nox1蛋白水平的变化。结果热损伤明显抑制人皮肤成纤维细胞的增殖;热损伤后即刻细胞内ROS含量和Nox活性明显升高,在4 h后二者均抵达峰值,而DPI预处理组ROS含量和Nox酶活性明显低于损伤组(P<0.01或P<0.05);热损伤后细胞分泌PGE2明显增高(P<0.01),而DPI预处理组和NAC预处理组均显著低于损伤组(P<0.01)。RT-PCR结果提示正常人皮肤成纤维细胞表达Nox1、Nox3和Nox4三种亚型,三者表达量无明显差异(P>0.05)。Western blot结果显示热损伤后不同时间点Nox1的蛋白表达量逐渐升高。结论热损伤可明显抑制人皮肤成纤维细胞增殖并诱导Nox活性升高和Nox1蛋白表达增高,从而使细胞内ROS量升高,进一步引起PGE2分泌增多。Objective To explore oxidative injury and change of PGE2 secretion in human skin fibroblasts after thermal injury and the mechanisms involved. Methods Cultured human skin fibroblasts were undergone the thermal injury model. The injured cells were divided into three groups： heat injury group, DPI （ Nox inhibitor） pretreatment group and NAC（antioxidant） pretreatment group,whereas cells without thermal injury worked as control group. ROS production and Nox activity were detected by fluorescence probe and chemiluminescence respectively. PGE2 from cell supematant was measured by enzyme immunosorbent assay（EIA）. Reverse transcription polymerase chain reaction（RT-PCR） was used to analyze the mRNA level expressed on the membrane of fibroblasts while western blotting analyzing the protein level of the Nox. Results Thermal injury significantly inhibited the proliferation of human skin fibroblasts. The intracellular ROS and Nox activity were largely increased （P 〈 0. 01 ）, and peaked 4 h post injury. They were significantly lower in DP! pretreatment group than injury group（ P 〈 0. 01 or P 〈 0. 05 ）. The concentration of PGE2 in supernatant went up significantly after thermal injury（ P 〈 0.01 ） while DPI pretreatment and NAC pretreatment significantly decreased PGE2 secreting（ P 〈 0.01 ）. RT-PCR showed Noxl, Nox3 and Nox4 were expressed on the membrane of human skin fibroblasts, and no significant difference in expression amount of the three kinds of Nox subtypes. The protein level of Noxl gradually increased after thermal injury. Conclusion Thermal injury may inhibit the proliferation of human skin fibroblasts, and increase Nox activity and expression of Noxl protein,which further induce ROS production and PGE2 secretion.

关 键 词：氧化性应激 创伤与损伤 地诺前列酮 

分 类 号：R644[医药卫生—外科学]