组织蛋白酶D调控皮肤成纤维细胞降解晚期糖基化终末产物的研究

doi:10.3760/cma.j.issn.0412-4030.2018.09.003

摘要/Abstract

摘要： 目的探究组织蛋白酶D（CatD）对皮肤成纤维细胞降解吞入胞内的晚期糖基化终末产物（AGE）的调控作用。方法分别以CatB + CatL、CatD以及20S蛋白酶体的酶活性抑制剂CA074Me、天冬氨酸酶抑制剂（pepstatin A）、MG-132处理皮肤成纤维细胞，CCK8和荧光法检测细胞活性及蛋白酶活性。CA074Me、pepstatin A、MG-132组分别加入AGE-BSA孵育8 h，而对照组加入磷酸盐缓冲液（PBS）。孵育8 h后，置换含相应抑制剂的新鲜培养基继续培养24 h，流式细胞仪检测各时间点胞内AGE-BSA平均荧光强度。以不同浓度pepstatin A（37.5、75、150 μmol/L）或PBS处理皮肤成纤维细胞，加入AGE-BSA孵育，方法同前，ELISA检测各时间点胞内AGE-BSA平均浓度，计算降解率。慢病毒转染皮肤成纤维细胞，设空白对照组、空载病毒转染组、CatD高表达慢病毒转染组，荧光显微镜观察，RT-PCR、Western印迹法和荧光法检测各组CatD mRNA和蛋白表达及酶活性。分别加入AGE-BSA孵育，处理及检测方法同前，计算降解率。结果 1 μmol/L CA074Me组、75 μmol/L pepstatin A组及1 μmol/L MG-132组细胞增殖活性均在90%以上，与对照组（100%）差异无统计学意义（F = 1.525，P > 0.05）。去除AGE-BSA 24 h后，CA074Me + AGE-BSA组和MG-132 + AGE-BSA组胞内AGE-BSA荧光强度（275.00 ± 10.15、259.00 ± 11.14）均较其相应8 h点荧光强度（295.00 ± 6.56、285.67 ± 8.74）显著下降（配对t值分别为4.778、6.154，均P < 0.05），而pepstatin A + AGE-BSA组8 h和32 h点细胞内AGE-BSA荧光强度差异无统计学意义（均P > 0.05）。37.5、75和150 μmol/L pepstatin A组24 h内对吞入胞内AGE-BSA的降解率分别为9.64% ± 1.27%、5.62% ± 0.47%和3.21% ± 0.73%，各组间差异有统计学意义（F = 45.876，P < 0.05），且该降解率随pepstatin A浓度增加而降低（P < 0.05）。荧光显微镜下观察，空白对照组细胞未见荧光，空载病毒转染组和CatD高表达慢病毒转染组细胞荧光阳性率均在80%以上。CatD高表达慢病毒转染组CatD mRNA、蛋白表达及活性均较另2组显著上调（均P < 0.05）。CatD高表达慢病毒转染 + AGE-BSA组24 h内AGE-BSA的降解率显著高于AGE-BSA组和空载病毒转染 + AGE-BSA组（均P < 0.05）。结论 CatD可促进皮肤成纤维细胞降解吞入胞内的AGE-BSA。

关键词: 组织蛋白酶D, 成纤维细胞, 糖基化终产物, 高级, 降解

Abstract: Xu Xinya, Xu Qingfang, Zheng Yue, Li Yuying, Huang Yunfen, Gong Zijian, Lu Chun, Lai Wei Department of Dermatology, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou 510630, China Corresponding author: Xu Qingfang, Email: xqf69@163.com 【Abstract】 Objective To investigate the regulatory role of cathepsin D （CatD） in the degradation of intracellular advanced glycation end products （AGEs） endocytosed by human dermal fibroblasts （HDFs）. Methods Cultured HDFs were treated with 1 μmol/L CA074Me （an inhibitor of CatB and CatL）, 75 μmol/L pepstatin A （an inhibitor of CatD） and 1 μmol/L MG-132 （an inhibitor of 20S proteasome） separately for 4 hours, and then cell counting kit 8 （CCK8） assay and fluorometric assay were performed to determine the cellular viability and protease activity, respectively. The cells in the CA074Me group, pepstatin A group and MG-132 group were additionally treated with AGE-bovine serum albumin （BSA） for 8 hours, and the cells in the blank control group were treated with phosphate-buffered saline （PBS） alone. After 8-hour cultivation, the cells in the above groups were subsequently reincubated with fresh culture medium containing the corresponding inhibitors for 24 hours. Then, flow cytometry was performed to assess the mean fluorescence intensity of intracellular AGE-BSA at different time points. Some other HDFs were treated with 37.5, 75 and 150 μmol/L pepstatin A and PBS separately for 4 hours, and then the cells in the 4 groups were treated with 200 mg/L AGE-BSA for 8 hours, followed by the removal of AGE-BSA from the medium and the treatment with 37.5, 75 and 150 μmol/L pepstatin A and PBS respectively. Enzyme-linked immunosorbent assay （ELISA） was conducted to measure the mean concentration of intracellular AGE-BSA at different time points, and the degradation rate of AGE was calculated. Some HDFs were divided into 3 groups: blank control group receiving no treatment, NC group transfected with an empty vector, and CatD group transfected with a CatD-overexpressing lentiviral vector. Fluorescence microscopy was conducted to estimate the transfection efficiency. Reverse transcription-PCR, Western blot analysis and fluorometric assay were performed to determine the mRNA and protein , and activity of CatD respectively. Then, the cells in the above 3 groups were incubated with AGE-BSA for 8 hours, followed by the removal of AGE-BSA from the medium and the treatment with fresh culture medium. The detection methods were same as the above experiment, and the degradation rate was calculated. Results The cellular proliferative activity in the 1-μmol/L CA074Me group, 75-μmol/L pepstatin A group and 1-μmol/L MG-132 group was more than 90%, and there was no significant difference between the 3 groups and the control group （100%, F = 1.525, P > 0.05）. Twenty-four hours after the removal of AGE-BSA from the medium, the fluorescence intensities of intracellular AGE-BSA in the CA074Me + AGE-BSA group （275.00 ± 10.15） and MG-132 + AGE-BSA group （259.00 ± 11.14） significantly decreased compared with those at the 8-hour time point （295.00 ± 6.56 and 285.67 ± 8.74 respectively; paired t test, t = 4.778, 6.154 respectively, both P < 0.05）, while no significant difference was observed in the fluorescence intensities of intracellular AGE-BSA in the pepstatin A + AGE-BSA group between the 8-hour time point and 32-hour time point （P > 0.05）. The degradation rates of intracellular AGE-BSA within 24 hours in the 37.5, 75 and 150 μmol/L pepstatin A groups were 9.64% ± 1.27%, 5.62% ± 0.47% and 3.21% ± 0.73% respectively; there were significant differences among the 3 groups （F = 45.876, P < 0.05）, and the degradation rate significantly decreased along with the increase of pepstatin A concentration （P < 0.05）. Fluorescence microscopy showed no fluorescent cells in the blank control group, while the NC group and CatD group both showed a high proportion （> 80%） of fluorescent cells. The mRNA and protein as well as the activity of CatD were significantly higher in the CatD group than in the blank control group and NC group （all P < 0.05）. The CatD + AGE-BSA group showed a significantly higher degradation rate of intracellular AGE-BSA within 24 hours compared with the AGE-BSA group and NC + AGE-BSA group （both P < 0.05）. Conclusion CatD can promote the degradation of intracellular AGE-BSA endocytosed by HDFs.

Key words: Cathepsin D, Fibroblasts, Glycosylation end products, advanced, Degradation

中图分类号:

许新雅许庆芳郑跃黎钰莹黄云芬龚子鉴陆春赖维. 组织蛋白酶D调控皮肤成纤维细胞降解晚期糖基化终末产物的研究[J]. 中华皮肤科杂志, 2018, 51(9): 647-652.

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