中华皮肤科杂志
中華皮膚科雜誌
중화피부과잡지
Chinese Journal of Dermatology
2014年
11期
776-779
,共4页
侯巍%许庆芳%刘晨%郑跃%赖维
侯巍%許慶芳%劉晨%鄭躍%賴維
후외%허경방%류신%정약%뢰유
成纤维细胞%紫外线%辐射损伤,实验性%组织蛋白酶B
成纖維細胞%紫外線%輻射損傷,實驗性%組織蛋白酶B
성섬유세포%자외선%복사손상,실험성%조직단백매B
Fibroblasts%Ultraviolet rays%Radiation injuries,experimental%Cathepsin B
目的 探讨组织蛋白酶B(CatB)在急性光损伤人皮肤成纤维细胞(HDF)中的表达变化及意义.方法 体外培养原代HDF,选取第4～8代细胞进行实验.实验设长波紫外线(UVA)照射组和不照射的对照组.CCK8法分别检测5、10、15、20和25 J/cm2 UVA照射后HDF的增殖率.用10 J/cm2 UVA单次照射致HDF急性光损伤,Western印迹及实时定量逆转录(RT)-PCR分别检测急性光损伤组HDF及对照组HDF照射后24、48、72 h CatB蛋白及mRNA表达;另外分别用10、15、20、25 J/cm2 UVA照射HDF,Western印迹及实时定量RT-PCR分别检测急性光损伤组及对照组HDF照射后48 h CatB蛋白及基因表达.数据采用SPSS 13.0统计软件行方差分析,两组间比较采用最小显著差异法(LSD).结果 UVA照射导致HDF增殖率下降;当UVA剂量≤10 J/cm2时,细胞存活率均保持在85％以上,各照光组分别与对照组24、48、72 h时比较,均P＜0.05.Western印迹结果显示,急性光损伤组(10 J/cm2 UVA照射)CatB蛋白灰度值在照射后24 h(0.76±0.14)、48 h(1.34±0.38)、72 h(0.82±0.09)均较对照组(分别为0.35±0.01、0.45±0.12、0.61±0.06)升高(均P＜0.05).实时定量RT-PCR显示,在急性光损伤组CatB的mRNA表达在照射后24 h(0.149±0.009)、48 h(0.173±0.009)、72 h(0.185±0.158)也较对照组(分别为0.089±0.015、0.091±0.010、0.111±0.017)上调(均P＜0.05).10、15、20、25 J/cm2 UVA照射HDF后48 h,CatB的蛋白灰度值分别为0.99±0.07、1.49±0.14、1.89±0.08、2.07±0.06,均较对照组(0.60±0.05)升高(均P＜0.05),CatB的mRNA表达也较对照组升高.结论 UVA致急性光损伤的皮肤成纤维细胞中CatB蛋白及mRNA表达均上调.
目的 探討組織蛋白酶B(CatB)在急性光損傷人皮膚成纖維細胞(HDF)中的錶達變化及意義.方法 體外培養原代HDF,選取第4～8代細胞進行實驗.實驗設長波紫外線(UVA)照射組和不照射的對照組.CCK8法分彆檢測5、10、15、20和25 J/cm2 UVA照射後HDF的增殖率.用10 J/cm2 UVA單次照射緻HDF急性光損傷,Western印跡及實時定量逆轉錄(RT)-PCR分彆檢測急性光損傷組HDF及對照組HDF照射後24、48、72 h CatB蛋白及mRNA錶達;另外分彆用10、15、20、25 J/cm2 UVA照射HDF,Western印跡及實時定量RT-PCR分彆檢測急性光損傷組及對照組HDF照射後48 h CatB蛋白及基因錶達.數據採用SPSS 13.0統計軟件行方差分析,兩組間比較採用最小顯著差異法(LSD).結果 UVA照射導緻HDF增殖率下降;噹UVA劑量≤10 J/cm2時,細胞存活率均保持在85％以上,各照光組分彆與對照組24、48、72 h時比較,均P＜0.05.Western印跡結果顯示,急性光損傷組(10 J/cm2 UVA照射)CatB蛋白灰度值在照射後24 h(0.76±0.14)、48 h(1.34±0.38)、72 h(0.82±0.09)均較對照組(分彆為0.35±0.01、0.45±0.12、0.61±0.06)升高(均P＜0.05).實時定量RT-PCR顯示,在急性光損傷組CatB的mRNA錶達在照射後24 h(0.149±0.009)、48 h(0.173±0.009)、72 h(0.185±0.158)也較對照組(分彆為0.089±0.015、0.091±0.010、0.111±0.017)上調(均P＜0.05).10、15、20、25 J/cm2 UVA照射HDF後48 h,CatB的蛋白灰度值分彆為0.99±0.07、1.49±0.14、1.89±0.08、2.07±0.06,均較對照組(0.60±0.05)升高(均P＜0.05),CatB的mRNA錶達也較對照組升高.結論 UVA緻急性光損傷的皮膚成纖維細胞中CatB蛋白及mRNA錶達均上調.
목적 탐토조직단백매B(CatB)재급성광손상인피부성섬유세포(HDF)중적표체변화급의의.방법 체외배양원대HDF,선취제4～8대세포진행실험.실험설장파자외선(UVA)조사조화불조사적대조조.CCK8법분별검측5、10、15、20화25 J/cm2 UVA조사후HDF적증식솔.용10 J/cm2 UVA단차조사치HDF급성광손상,Western인적급실시정량역전록(RT)-PCR분별검측급성광손상조HDF급대조조HDF조사후24、48、72 h CatB단백급mRNA표체;령외분별용10、15、20、25 J/cm2 UVA조사HDF,Western인적급실시정량RT-PCR분별검측급성광손상조급대조조HDF조사후48 h CatB단백급기인표체.수거채용SPSS 13.0통계연건행방차분석,량조간비교채용최소현저차이법(LSD).결과 UVA조사도치HDF증식솔하강;당UVA제량≤10 J/cm2시,세포존활솔균보지재85％이상,각조광조분별여대조조24、48、72 h시비교,균P＜0.05.Western인적결과현시,급성광손상조(10 J/cm2 UVA조사)CatB단백회도치재조사후24 h(0.76±0.14)、48 h(1.34±0.38)、72 h(0.82±0.09)균교대조조(분별위0.35±0.01、0.45±0.12、0.61±0.06)승고(균P＜0.05).실시정량RT-PCR현시,재급성광손상조CatB적mRNA표체재조사후24 h(0.149±0.009)、48 h(0.173±0.009)、72 h(0.185±0.158)야교대조조(분별위0.089±0.015、0.091±0.010、0.111±0.017)상조(균P＜0.05).10、15、20、25 J/cm2 UVA조사HDF후48 h,CatB적단백회도치분별위0.99±0.07、1.49±0.14、1.89±0.08、2.07±0.06,균교대조조(0.60±0.05)승고(균P＜0.05),CatB적mRNA표체야교대조조승고.결론 UVA치급성광손상적피부성섬유세포중CatB단백급mRNA표체균상조.
Objective To investigate the changes in cathepsin B (CatB) expression in acutely photodamaged human dermal fibroblasts (HDFs) and their significance.Methods HDFs were isolated from the foreskin of children,and subjected to primary culture and subculture.The fourth-to eighth-passage HDFs were used in the following experiment.HDFs were divided into two groups to receive irradiation with different doses of ultraviolet A (UVA) for different durations (acutely photodamaged group) or remain unirradiated (control group).Cell counting kit-8 (CCK8) assay was conducted to evaluate the proliferative activity of HDFs after irradiation with UVA at 5,10,15,20 and 25 J/cm2 respectively.Western blot and quantitative real-time reverse transcription PCR were performed to measure the protein and mRNA expressions of CatB respectively in HDFs at 24,48 and 72 hours after exposure to UVA at 10 J/cm2,and at 48 hours after exposure to UVA at 10,15,20 and 25 J/cm2.Statistical analysis was carried out by analysis of variance and least significant difference (LSD) test using the SPSS 13.0 software.Results UVA radiation induced a decrease in the proliferative activity of HDFs.When the dose of UVA was ≤ 10 J/cm2,the survival rate of HDFs maintained higher than 85％,and significant differences were observed in cell survival rate between unirradiated and irradiated HDFs at 24,48 and 72 hours (all P ＜ 0.05).Western blot showed that the gray value of CatB protein in the acutely photodamaged group irradiated with 10 J/cm2 UVA was significantly higher than that in the control group at 24 hours (0.76 ± 0.14 vs.0.35 ± 0.01,P ＜ 0.05),48 hours (1.34 ± 0.38 vs.0.45 ± 0.12,P＜ 0.05) and 72 hours (0.82 ± 0.09 vs.0.61 ± 0.06,P＜ 0.05).Increased mRNA expressions of CatB were also observed in the acutely photodamaged group compared with the control group at 24 hours (0.149 ± 0.009 vs.0.089 ± 0.015,P ＜ 0.05),48 hous (0.173 ± 0.009 vs.0.091 ± 0.010,P ＜ 0.05) and 72 hours (0.185 ± 0.158 vs.0.111 ± 0.017,P ＜ 0.05) after UVA radiation at 10 J/cm2.The gray value of CatB protein was 0.99 ± 0.07,1.49 ± 0.14,1.89 ± 0.08,2.07 ± 0.06 in HDFs at 48 hours after exposure to UVA of 10,15,20 and 25 J/cm2,respectively,significantly higher than that in the control group (0.60 ± 0.05,all P ＜ 0.05).Similarly,the mRNA expression of CatB was up-regulated in HDFs at 48 hours after UVA radiation at 10,15,20 and 25 J/cm2 compared with the unirradiated HDFs.Conclusion The protein and mRNA expressions of CatB are up-regulated in acutely photodamaged HDFs induced by UVA radiation.
