尾加压素Ⅱ对皮肤成纤维细胞表达Ⅰ型胶原蛋白的影响

Effects of urotensin Ⅱ on the expression of type Ⅰ collagen in human skin fibroblasts

目的 探讨血管活性物质尾加压素Ⅱ对人皮肤成纤维细胞表达Ⅰ型胶原蛋白及其迁移的影响和细胞内信号转导机制.方法 在离体培养的人皮肤成纤维细胞上,用酶联免疫吸附试验和Transwell迁移试验,观察尾加压素Ⅱ对细胞的促分泌和迁移作用.加入不同的细胞内信号转导阻断剂,观察其对尾加压素Ⅱ效应的影响.结果 尾加压素Ⅱ以浓度和时间依赖方式促进成纤维细胞分泌Ⅰ型胶原蛋白.尾加压素Ⅱ浓度为10^-10、10^-9、10^-8、10^-7、10^-6 mol/L的实验组Ⅰ型胶原蛋白分泌量分别比对照组依次增加21.2％（P＞ 0.05）、52.2％（P＜0.05）、84.4％（P＜ 0.05）、83.6％（P＜ 0.05）和77.1％（P＜ 0.05）.从4h开始,Ⅰ型胶原蛋白浓度明显增加,其分泌量较对照组增加23.2％（P＞ 0.05）、12h时增加69.5％（P＜ 0.05）和24 h时增加84.1％（P＜ 0.05）.与对照组相比,10^-8 mol/L尾加压素Ⅱ组可明显促进细胞迁移（P＜0.05）.尾加压素Ⅱ的效应能被丝裂原活化的蛋白激酶阻断剂PD98059、钙通道阻断剂尼卡地平和钙调素激酶阻断剂环孢素A所阻断,其中对尾加压素Ⅱ促成纤维细胞分泌Ⅰ型胶原蛋白的抑制率依次为18.2％、15.9％、19.7％,对迁移的抑制率为38.3％（P＜ 0.05）、20.7％（P＜0.05）和81.4％（P＜ 0.05）.结论 尾加压素Ⅱ可促进人皮肤成纤维细胞Ⅰ型胶原蛋白的分泌和成纤维细胞的迁移,其促进效应可能通过Ca2＋、钙调素激酶以及丝裂原活化的蛋白激酶通路来介导.

Objective To evaluate the effect of a vasoactive substance urotensin Ⅱ on the expression of type Ⅰ collagen and migration of human skin fibroblasts,and to explore the underlying mechanisms of signal transduction.Methods Fibroblasts were isolated from human foreskin tissues and subjected to primary culture.After a series of subculture,fibroblasts were classified into several groups to be treated with different concentrations （10^-10 to 10^-6 mol/L） of urotensin lⅡ for 24 hours,urotensin Ⅱ of 10^-s mol/L for different durations （0,4,12,24 hours）,or pretreated with PD98059 （a mitogen-activated protein kinase kinase inhibitor）,nicardipine （a calcium channel blocker） and ciclosporin （a calcineurin inhibitor） of 10^-5 mol/L respectively for 30 minutes followed by treatment with urotensin Ⅱ of 10^-8 mol/L for 24 hours.The cells receiving no treatment served as the control.Subsequently,enzyme-linked immunosorbent assay was performed to determine the level of urotensin Ⅱ in the supernatant of fibroblasts,and Transwell assay to estimate the migration activity of fibroblasts.Statistical analysis was carried out by t test and analysis of variance.Results Urotensin Ⅱ promoted the expression of type Ⅰ collagen in a time-and concentrationdependent manner.The level of type Ⅰ collagen was increased by 21.2% （P 〉 0.05）,52.2% （P 〈 0.05）,84.4% （P 〈0.05）,83.6% （P 〈 0.05） and 77.1% （P 〈 0.05） in the supernatant of fibroblasts treated with 10^-10,10^-9,10^-8,10^-7 and 10^-6 mol/L of urotensin Ⅱ for 24 hours respectively,by 23.2% （P 〉 0.05）,69.5% （P 〈 0.05） and 84.1% （P 〈0.05） in the supernatant of fibroblasts treated with urotensin Ⅱ of 10^-8 mol/L for 4,12 and 24 hours respectively,compared with the untreated control fibroblasts.The migration activity was markedly enhanced in fibroblasts treated with urotensin Ⅱ of 10^-8 mol/L for 24 hours compared with the control fibroblasts （P 〈 0.05）.PD98059,nicardipine and cyclosporin A inhibited the secretion of type Ⅰ collagen by 18.2%,15.9% and 19.7% respectively,and suppressed the migration of fibroblasts by 38.3% （P 〈 0.05）,20.7% （P 〈 0.05） and 81.4% （P 〈 0.05） respectively in the groups receiving pretreatment compared with those treated with urotensin Ⅱ alone.Conclusions Urotensin Ⅱ can promote the secretion of type Ⅰ collagen by and migration of fibroblasts,which may be realized through the Ca2＋,calmodulin kinase,and mitogen-activated protein kinase pathways.