CAJ | 학술논문

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RT-PCR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。
목적：탐토체외배양인피부성섬유세포열손상후양화응격급전렬선소E2（ PGE2）분비적변화급기가능적궤제。방법체외배양인피부성섬유세포，제작열손상모형후분위손상조、DPI예처리조、NAC예처리조삼조，이미수열손상세포작위대조조。 CCK8비색법검측세포증식。형광탐침법급화학발광법관찰세포내활성양（ ROS）함량화NADPH양화매（ Nox）활성변화，EIA검측상청액중PGE2적변화。반정량RT-PCR법검측인피부성섬유세포표체적Nox아형적mRNA수평。 Western blot법검측열손상후Nox1단백수평적변화。결과열손상명현억제인피부성섬유세포적증식；열손상후즉각세포내ROS함량화Nox활성명현승고，재4 h후이자균저체봉치，이DPI예처리조ROS함량화Nox매활성명현저우손상조（ P＜0.01혹P＜0.05）；열손상후세포분비PGE2명현증고（ P＜0.01），이DPI예처리조화NAC예처리조균현저저우손상조（ P＜0.01）。 RT-PCR결과제시정상인피부성섬유세포표체Nox1、Nox3화Nox4삼충아형，삼자표체량무명현차이（ P＞0.05）。 Western blot결과현시열손상후불동시간점Nox1적단백표체량축점승고。결론열손상가명현억제인피부성섬유세포증식병유도Nox활성승고화Nox1단백표체증고，종이사세포내ROS량승고，진일보인기PGE2분비증다。
Objective To explore oxidative injury and change of PGE 2 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 supernatant 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 DPI 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 Nox1,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 Nox 1 gradually increased after thermal injury .Conclusion Thermal injury may inhibit the proliferation of human skin fibroblasts , and increase Nox activity and expression of Nox 1 protein,which further induce ROS production and PGE 2 secretion.