中华皮肤科杂志 ›› 2014, Vol. 47 ›› Issue (11): 776-779.

• 论著 • 上一篇    下一篇

组织蛋白酶B在急性光损伤皮肤成纤维细胞中的表达及意义

侯巍1,许庆芳2,刘晨3,郑跃2,赖维4   

  1. 1. 新疆医科大学第一附属医院
    2. 中山大学附属第三医院皮肤科
    3. 深圳市人民医院
    4. 广州中山大学附属第三医院皮肤科
  • 收稿日期:2013-12-13 修回日期:2014-06-21 出版日期:2014-11-15 发布日期:2014-11-01
  • 通讯作者: 赖维 E-mail:drlaiwei@163.com
  • 基金资助:
    国家自然科学基金;广东省自然科学基金

Expression of cathepsin B in acutely photodamaged fibroblasts and its significance

Wei HOU1,qingfang XUChen LIU3,Zheng Yue4,Wei Lai   

  • Received:2013-12-13 Revised:2014-06-21 Online:2014-11-15 Published:2014-11-01
  • Contact: Wei Lai E-mail:drlaiwei@163.com

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摘要： 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SPSS13.0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。

关键词: 成纤维细胞, 紫外线, 辐射损伤，实验性, 组织蛋白酶B

Abstract: Hou Wei *, Xu Qingfang, Liu Chen, Zheng Yue, Lai Wei. *Department of Dermatology, First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, China Corresponding author: Lai Wei, Email: drlaiwei@163.com 【Abstract】 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.

Key words: Fibroblasts, Ultraviolet rays, Radiation injuries, experimental, Cathepsin B