天冬氨酸/半胱氨酸组织蛋白酶在光老化成纤维细胞中的表达变化

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/m2 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cathepsin 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 ± 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ΔCt值为2.79 ± 0.17，对照组为4.54 ± 0.34，根据2-ΔΔCt值计算得cathepsin 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ΔCt值为-0.92 ± 0.06，对照组为2.57 ± 0.13，根据2-ΔΔCt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。

Expressions of aspartic proteinase and cysteine proteinase in photoaged fibroblasts

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.