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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 被引量:5

Expression of cathepsin B in acutely photodamaged fibroblasts and its significance
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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. 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.
出处 《中华皮肤科杂志》 CAS CSCD 北大核心 2014年第11期776-779,共4页 Chinese Journal of Dermatology
基金 国家自然科学基金(81171523) 广东省自然科学基金(10151008901000117)
关键词 成纤维细胞 紫外线 辐射损伤 实验性 组织蛋白酶B Fibroblasts Ultraviolet rays Radiation injuries, experimental Cathepsin B
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