晚期糖基化终末产物对UVA照射皮肤成纤维细胞组织蛋白酶D表达及其活性的影响

目的：研究晚期糖基化终末产物（AGE）对长波紫外线（UVA）照射皮肤成纤维细胞组织蛋白酶D （CatD）表达和活性的影响。方法原代培养来自儿童包皮成纤维细胞。CCK?8法筛选对成纤维细胞无细胞毒性的AGE?牛血清白蛋白（BSA）浓度。分别以50、100、200 mg/L AGE?BSA孵育细胞24 h,以未处理细胞作为对照组,采用RT?PCR、Western印迹及荧光法检测AGE?BSA对细胞CatD表达和活性影响。将部分成纤维细胞分为6组,即对照组（正常皮肤成纤维细胞,不接受任何处理）、AGE?BSA组、BSA组、UVA组、UVA?AGE?BSA组、UVA?BSA组。AGE?BSA组加入最大无细胞毒性浓度AGE?BSA孵育24 h,BSA组加入相同浓度BSA孵育24 h,后3组除分别接受上述处理外再接受10 J/cm2 UVA照射。处理结束后,收集细胞mRNA和蛋白,采用RT?PCR、Western印迹及荧光法检测细胞CatD表达和活性。结果50、100、200 mg/L AGE?BSA对皮肤成纤维细胞增殖活性无显著影响。50 mg/L组、100 mg/L组、200 mg/L组细胞CatD mRNA水平分别为0.267±0.007、0.348±0.007、0.418±0.006,CatD蛋白水平分别为1.403±0.181、2.233±0.090、2.477±0.111,CatD活性分别为1.760±0.080、2.330±0.060、2.890±0.080,较相应对照组CatD mRNA（0.161±0.006）、CatD蛋白（0.903±0.200）以及CatD活性水平（1.100±0.090）均显著升高,均P<0.05。AGE?BSA呈剂量依赖性地刺激细胞CatD表达和活性。对照组、UVA组和UVA?AGE?BSA组细胞中CatD mRNA表达水平分别为0.155±0.005、0.480±0.005、0.394±0.008,蛋白表达水平分别为0.920±0.235、2.583±0.199、2.070±0.125,CatD活性分别为1.110±0.040、2.970±0.110、2.560±0.060;UVA组CatD mRNA水平、蛋白表达水平、酶活性均明显高于对照组（P<0.05）,但UVA?AGE?BSA组3种指标水平均显著低于UVA组（P<0.05）。结论 AGE可升高未接受UVA照射的皮肤成纤维细胞CatD表达和活性,但AGE却抑制UVA照射上调的CatD表达和酶活性。

Effects of advanced glycation end products on the expressions and activity of cathepsin D in ultraviolet A- irradiated human dermal fibroblasts

Objective To investigate the effects of advanced glycation end products(AGE)on the expressions and activity of cathepsin D(CatD)in ultraviolet A(UVA)?irradiated human dermal fibroblasts. Methods Human dermal fibroblasts were isolated and harvested from the circumcised foreskin of children, and subjected to a primary culture. CCK?8 assay was performed to screen non?cytotoxic concentrations of AGE?bovine serum albumin (BSA). Some fibroblasts were incubated with 50, 100 and 300 mg/L AGE?BSA separately for 24 hours, with untreated cells as the control group. Then, reverse transcription(RT)?PCR, Western?blot analysis and a fluorimetric assay were performed to measure the mRNA and protein expressions as well as activity of CatD, respectively. Some fibroblasts were classified into six groups: control group receiving no treatment, AGE?BSA group and BSA group treated with the highest non?cytotoxic concentration of AGE?BSA and the same concentration of BSA respectively for 24 hours, UVA group irradiated by 10 J/cm2 UVA, UVA?AGE?BSA group and UVA?BSA group treated with AGE?BSA and BSA at the above non?cytotoxic concentration respectively for 24 hours both before and after UVA radiation at 10 J/cm2. After the treatments, RT?PCR, Western?blot analysis and a fluorimetric assay were conducted to detect mRNA and protein expressions and activity of CatD respectively. Results AGE?BSA of 50- 200 mg/L exhibited no obvious influence on cellular proliferation of fibroblasts. The fibroblasts incubated with AGE?BSA of 50, 100 and 200 mg/L showed a significant increase in the mRNA expression(0.267 ± 0.007, 0.348 ± 0.007, and 0.418 ± 0.006 respectively), protein expression (1.403 ± 0.181, 2.233 ± 0.090 and 2.477 ± 0.111 respectively), and activity(1.760 ± 0.080, 2.330 ± 0.060 and 2.890 ± 0.080 respectively)of CatD compared with the control group(mRNA:0.161 ± 0.006;protein:0.903 ± 0.200;activity:1.100 ± 0.090, all P < 0.05). AGE?BSA increased CatD expressions and activity in a dose?dependent manner. The mRNA and protein expressions as well as activity of CatD were significantly higher in the UVA group than in the control group (mRNA expression: 0.480 ± 0.005 vs. 0.155 ± 0.005; protein expression: 2.583 ± 0.199 vs. 0.920 ± 0.235;activity:2.970 ± 0.110 vs. 1.110 ± 0.040, all P<0.05), but significantly lower in the UVA?AGE?BSA group than in the UVA group(mRNA expression:0.394 ± 0.008 vs. 0.480 ± 0.005;protein expression:2.070 ± 0.125 vs. 2.583 ± 0.199;activity: 2.560 ± 0.060 vs. 2.970 ± 0.110, all P < 0.05). Conclusion AGEs could increase CatD expressions and activity in human dermal fibroblasts not receiving UVA irradiation, but inhibit their increase in UVA?induced human dermal fibroblasts.