CAJ | 학술논문

目的探讨长波紫外线(UVA)对人皮肤成纤维细胞(HSF)自噬水平的影响.方法 HSF慢性UVA照射分组:未照射组、5 J/cm2组、10 J/cm2组和20 J/cm2组,每天照射1次连续4 d;HSF急性照射分组:未照射组、5 J/cm2组、10 J/cm2组、30 J/cm2组和60 J/cm2组,单次照射.采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐分析法(MTT)检测细胞增殖活力.单丹酰戊二胺染色法(MDC)检测细胞自噬体.Western印迹法检测细胞LC3蛋白LC3-Ⅰ向LC3-Ⅱ转化分析(LC3-Ⅱ/LC3-Ⅰ).结果与未照射组HSF比较,5、10、20 J/cm2慢性UVA照射组细胞增殖活力降低(F=155.5,P＜ 0.05).与未照射组HSF比较,5、10、30、60 J/cm2急性UVA照射后1、6和12h,细胞增殖活力均降低(F值分别为1 335、1 649、2 774、均P＜0.05).MDC法标记细胞自噬囊泡,与未照射组比较,5、10、20 J/cm2慢性照射均可上调细胞自噬水平(F=748.62,P＜ 0.05),而5、10、30、60 J/cm2急性照射后1、6和12h对细胞自噬水平无明显影响(F值分别为0.014、0.004、0.002,均P＞0.05).5、10和20 J/cm2慢性照射组LC3-Ⅰ向LC3-Ⅱ转化(LC3-Ⅱ/LC3-Ⅰ)均增加(t值分别为9.002、21.772、18.33,均P＜0.05),细胞自噬水平上调.与未照射组比较,5、10、30、60 J/cm2急性照射后1、6和12h,细胞LC3-Ⅰ向LC3-Ⅱ转化均无明显变化(F值分别为0.13、0.27、0.06,均P＞0.05),对细胞自噬水平无明显影响.结论慢性UVA照射可上调HSF自噬水平,急性UVA照射HSF自噬无明显变化.
목적 탐토장파자외선(UVA)대인피부성섬유세포(HSF)자서수평적영향.방법 HSF만성UVA조사분조:미조사조、5 J/cm2조、10 J/cm2조화20 J/cm2조,매천조사1차련속4 d;HSF급성조사분조:미조사조、5 J/cm2조、10 J/cm2조、30 J/cm2조화60 J/cm2조,단차조사.채용3-(4,5-이갑기새서-2)-2,5-이분기사담서추염분석법(MTT)검측세포증식활력.단단선무이알염색법(MDC)검측세포자서체.Western인적법검측세포LC3단백LC3-Ⅰ향LC3-Ⅱ전화분석(LC3-Ⅱ/LC3-Ⅰ).결과 여미조사조HSF비교,5、10、20 J/cm2만성UVA조사조세포증식활력강저(F=155.5,P＜ 0.05).여미조사조HSF비교,5、10、30、60 J/cm2급성UVA조사후1、6화12h,세포증식활력균강저(F치분별위1 335、1 649、2 774、균P＜0.05).MDC법표기세포자서낭포,여미조사조비교,5、10、20 J/cm2만성조사균가상조세포자서수평(F=748.62,P＜ 0.05),이5、10、30、60 J/cm2급성조사후1、6화12h대세포자서수평무명현영향(F치분별위0.014、0.004、0.002,균P＞0.05).5、10화20 J/cm2만성조사조LC3-Ⅰ향LC3-Ⅱ전화(LC3-Ⅱ/LC3-Ⅰ)균증가(t치분별위9.002、21.772、18.33,균P＜0.05),세포자서수평상조.여미조사조비교,5、10、30、60 J/cm2급성조사후1、6화12h,세포LC3-Ⅰ향LC3-Ⅱ전화균무명현변화(F치분별위0.13、0.27、0.06,균P＞0.05),대세포자서수평무명현영향.결론 만성UVA조사가상조HSF자서수평,급성UVA조사HSF자서무명현변화.
Objective To evaluate the effects of ultraviolet A (UVA) on autophagy in human skin fibroblasts (HSFs).Methods Cultured HSFs were randomly divided into chronic and acute UVA radiation groups.HSFs in the chronic UVA radiation groups were irradiated with UVA at 5,10 and 20 J/cm2 separately once a day for 4 consecutive days,with HSFs receiving no radiation serving as the chronic radiation control group;HSFs in the acute UVA radiation groups received a single session of radiation with 5,10,30 and 60 J/cm2 UVA separately,with HSFs receiving no radiation serving as the acute radiation control group.After additional culture for different durations,methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the proliferative activity of HSFs,monodansylcadaverin (MDC) staining to determine autophagy levels,and Western blot analysis to track the conversion of the microtubule-associated protein 1 light chain-3 (LC3)-Ⅰ to LC3-Ⅱ.Statistical analysis was carried out by using one-way analysis of variance followed by Students-Newman-Keuls (SNK) test for multiple-group comparisons and by the independent sample t test for two-group comparisons.Results The cellular proliferative activity significantly decreased in the 3 chronic radiation groups at 1 hour after the final UVA radiation compared with the chronic radiation control group (F =155.5,P ＜ 0.05),and in the 4 acute radiation groups at 1,6 and 12 hours after UVA radiation compared with the acute radiation control group (F =1 335,1 649,2 774,all P ＜ 0.05).MDC staining showed that the autophagy levels in HSFs significantly increased in the 3 chronic radiation groups after UVA radiation compared with the chronic radiation control group (F =748.62,P ＞ 0.05),but showed no significant changes in any of the acute radiation groups at 1,6 or 12 hours after UVA radiation compared with the acute radiation control group (F =0.014,0.004,0.002,all P ＞ 0.05).The ratio of LC3-Ⅱ to LC3-Ⅰ was significantly elevated in all the 3 chronic radiation groups at 1 hour after UVA radiation compared with the chronic radiation control group (t =9.002,21.772,18.33,all P ＜ 0.05),but experienced no obvious changes in any of the acute radiation groups at 1,6 or 12 hours after UVA radiation compared with the acute radiation control group (F =0.13,0.27,0.06,all P ＞ 0.05).Conclusion Chronic UVA radiation can upregulate autophagy levels in HSFs,but acute UVA radiation has no evident effects on it.