CAJ | 학술논문

目的探讨天冬氨酸组织蛋白酶(cathepsin D)及半胱氨酸组织蛋白酶(cathepsin K)在光老化成纤维细胞中的表达变化.方法培养原代人皮肤成纤维细胞,在50 mg/L的8-甲氧沙林(8-MOP)培养基中避光孵育24 h后,用80 kJ/m~2 UVA照射,体外诱导培养细胞光老化.衰老相关-β-半乳糖苷酶(SA-β-Gal)染色证明老化诱导成功.Western印迹及实时定量RT-PCR对比检测光老化成纤维细胞及正常成纤维细胞eathepsin K和cathepsin D蛋白及基因表达.结果 Western印迹结果显示,光老化组表达的cathepsin D的灰度值为3.25 4±0.33,对照组为14.18 ±2.25,t=30.61,P<0.01.两组间差异有统计学意义;光老化组表达的cathepsin K灰度值为2.39±0.66,对照组为29.38±4.62,t=12.63,P<0.01,两组差异有统计学意义.光老化组cathepsin D mRNA的△Ct值为2.79±0.17,对照组为4.54±0.34,根据2~(-△△Ct)值计算得eathepsin D mRNA在光老化组的表达下调为对照组的0.24±0.021(t=20.78,P<0.01);光老化组cathepsin K mRNA的△Ct值为-0.92±0.06,对照组为2.57±0.13,根据2~(△△Ct)值计算得cathepsin K mRNA在光老化组的表达下调为对照组的0.09±0.005(t=28.50,P<0.01).结论 cathepsin D及cathepsin K在光老化成纤维细胞中表达均下调.
목적 탐토천동안산조직단백매(cathepsin D)급반광안산조직단백매(cathepsin K)재광노화성섬유세포중적표체변화.방법 배양원대인피부성섬유세포,재50 mg/L적8-갑양사림(8-MOP)배양기중피광부육24 h후,용80 kJ/m~2 UVA조사,체외유도배양세포광노화.쇠로상관-β-반유당감매(SA-β-Gal)염색증명노화유도성공.Western인적급실시정량RT-PCR대비검측광노화성섬유세포급정상성섬유세포eathepsin K화cathepsin D단백급기인표체.결과 Western인적결과현시,광노화조표체적cathepsin D적회도치위3.25 4±0.33,대조조위14.18 ±2.25,t=30.61,P<0.01.량조간차이유통계학의의;광노화조표체적cathepsin K회도치위2.39±0.66,대조조위29.38±4.62,t=12.63,P<0.01,량조차이유통계학의의.광노화조cathepsin D mRNA적△Ct치위2.79±0.17,대조조위4.54±0.34,근거2~(-△△Ct)치계산득eathepsin D mRNA재광노화조적표체하조위대조조적0.24±0.021(t=20.78,P<0.01);광노화조cathepsin K mRNA적△Ct치위-0.92±0.06,대조조위2.57±0.13,근거2~(△△Ct)치계산득cathepsin K mRNA재광노화조적표체하조위대조조적0.09±0.005(t=28.50,P<0.01).결론 cathepsin D급cathepsin K재광노화성섬유세포중표체균하조.
Objective To investigate the expression changes of aspartic proteinase (cathepsin D) and cysteine proteinase (cathepsin K) in photoaged fibroblasts. Methods The senescence of human fibroblasts was induced via culture in the presence of 8-methoxypsralen (MOP) of 50 mg/L in darkness for 24 hours followed by irradiation with UVA of 80 kJ/m~2. Then, aged fibroblasts were confirmed by senescence-associated β-galactosidase (SA-β-gal) staining. Real-time RT-PCR and Western blot were carried out to detect the mRNA and protein expressions of cathepsin D and cathepsin K in photoaged and normal control fibroblasts, respectively. Results Western blot showed a significant difference between photoaged and control fibroblasts in the grey scale of cathepsin D and cathepsin K (3.25 ± 0.33 vs 14.18 ± 2.25, f = 30.61, P < 0.01; 2.39 ± 0.66 vs 29.38 ± 4.62, t = 12.63, P< 0.01). The △Ct values for cathepsin D and cathepsin K mRNA were 2.79 ± 0.17 and -0.92 ± 0.06, respectively, in photoaged fibroblasts, significantly lower than those in the control fibroblasts (4.54 ± 0.34, 2.57 ± 0.13, t = 20.78, 28.50, respectively, both P < 0.01). According to the value of 2~(-△△Ct), the expression of cathepsin D and cathepsin K mRNA decreased 0.24 ± 0.021 and 0.09 ± 0.005 folds, respectively, in photoaged fibroblasts compared with the control fibroblasts. Conclusion The expression of cathepsin D and cathepsin K is decreased in photoaged fibroblasts.