尿紧张素Ⅱ诱导人皮肤成纤维细胞增殖和α平滑肌肌动蛋白表达的机制研究  

Mechanisms underlying the urotensin Ⅱ-induced proliferation of and α-smooth muscle actin in human dermal fibroblasts

作　　者：罗丽敏 李军[2] 刘劲松 朱红 王郧莲 刘菡 

机构地区：[1]湖北医药学院附属东风医院皮肤科,湖北十堰442000 [2]湖北医药学院附属太和医院心内科

出　　处：《中华皮肤科杂志》2017年第12期894-898,共5页Chinese Journal of Dermatology

基　　金：十堰市科学技术研究与开发项目（15Y54）;十堰市太和医院科研专项计划（2014PY03）

摘　　要：目的 探讨尿紧张素Ⅱ（UⅡ）对体外培养人正常皮肤成纤维细胞（NF）增殖和α平滑肌肌动蛋白（α？SMA）表达的生物学功能影响及调控机制。方法 原代培养NF，采用反转录PCR和Western印迹法分别检测UⅡ及其受体mRNA和蛋白表达水平。采用CCK8法观察不同浓度（10？10、10？9、10？8、10？7、10？6 mol/L）UⅡ作用0、6、12、24、48 h对NF增殖的影响，筛选出UⅡ最佳作用浓度为10？8 mol/L，刺激时间为24 h。将NF分为5组：对照组（不含UII）、UⅡ组、UⅡ ＋ 尼卡地平（钙通道阻断剂，10？5 mol/L）组、UⅡ ＋ PD98059［丝裂原活化的蛋白激酶（MAPK）抑制剂，10？5 mol/L］组和UⅡ ＋ 环孢素［钙蛋白激酶（CaM PK）阻断剂，10？5 mol/L］组，UⅡ浓度均为10？8 mol/L，刺激NF 24 h后，采用CCK8法检测各组NF的增殖活性，以实时定量荧光PCR和Western印迹法分别检测各组α？SMA mRNA与蛋白的相对表达水平。结果 NF表达UⅡ受体，不表达UⅡ。对照组、UⅡ组、UⅡ＋ 尼卡地平组、UⅡ＋ PD98059组和UⅡ＋ 环孢素组NF细胞增殖活性（A450值）分别为1.036 ± 0.046、1.405 ± 0.158、1.121 ± 0.109 、1.192 ± 0.089和1.141 ± 0.056，组间差异有统计学意义（F = 9.587，P 〈 0.01），UⅡ组高于对照组、UⅡ＋ 尼卡地平组、UⅡ＋ PD98059组、UⅡ＋ 环孢素组（q值分别为8.263、6.355、4.774、5.912，均P 〈 0.05）。5组间NF细胞α？SMA mRNA与蛋白的相对表达量差异亦均有统计学意义（F值分别为6.351、7.045，均P 〈 0.01），且UⅡ组α？SMA mRNA及蛋白表达量均高于另4组（均P 〈 0.05）。结论 UⅡ可能通过钙通道、MAPK和CaM PK通路诱导NF增殖和α？SMA的表达。Objective To evaluate the effects of urotensin Ⅱ on cell proliferation of and α-smooth muscle actin （α-SMA） in normal human dermal fibroblasts （NFs）, and to explore their regulatory mechanisms. Methods NFs were isolated from foreskin tissues and subjected to primary culture in vitro. Reverse transcription PCR and Western blot analysis were performed to measure the mRNA and protein of urotensin Ⅱ and its receptor, respectively. Cell counting kit-8 （CCK-8） assay was conducted to estimate the proliferation of NFs, which were treated with urotensin Ⅱ at different concentrations of 0, 10-10, 10-9, 10-8, 10-7 and 10-6 mol/L for 0, 6, 12, 24 and 48 hours separately, and then the optimal concentration and duration of urotensin Ⅱ exposure were selected to be 10-8 mol/L and 24 hours respectively. Some cultured NFs were divided into 5 groups： control group receiving no treatment, UⅡ group treated with 10-8 mol/L urotensin Ⅱ, UⅡ＋ nicardipine group treated with 10-8 mol/L urotensin Ⅱ and the calcium channel blocker nicardipine at the concentration of 10-5 mol/L, UⅡ＋ PD98059 group treated with 10-8 mol/L urotensin Ⅱ and the mitogen activated protein kinase（MAPK）inhibitor PD98059 at the concen-tration of 10-5 mol/L, and UⅡ＋ cyclosporine group treated with 10-8 mol/L urotensin Ⅱ and the calcium-dependent protein kinase （CaM PK） inhibitor cyclosporine at the concentration of 10-5 mol/L. After 24-hour treatment, CCK-8 assay was conducted to evaluate the proliferation of NFs in the above groups, real-time fluorescence-based quantitative PCR and Western blot analysis were performed to determine the mRNA and protein of α-SMA respectively. Results Urotensin Ⅱ receptor was expressed in NFs, but urotensin Ⅱ was not. The proliferative activity of NFs significantly differed among the control group, UⅡ group, UⅡ＋ nicardipine group, UⅡ＋ PD98059 group and UⅡ＋ cyclosporine group （the mean absorbance value at 405 nm： 1.036 ± 0.046, 1.405 ± 0.158, 1.121 ± 0.109, 1.1

关 键 词：尿紧张素类 成纤维细胞 细胞增殖 肌动蛋白类 瘢痕 尿紧张素类受体 

分 类 号：R751[医药卫生—皮肤病学与性病学]