过氧化物酶体增殖物激活受体γ激动剂拮抗人皮肤成纤维细胞转化生长因子β1机制研究

目的 了解过氧化物酶体增殖物激活受体γ(PPARγ)激动剂拈抗TGF-β1促皮肤瘢痕化的效应机制.方法 体外培养正常成人皮肤Fb,根据培养基中所加刺激物不同分为空白对照组(无血清DMEM培养基),TGF-β1组(DMEM培养基加入含终浓度10 ng/mL TGF-β1作用48 h),曲格列酮组(DMEM培养基加入含终浓度10μmol/L曲格列酮作用2 h,再加入10 ng/mL TGF-β1作用48h),15-脱氧前列腺素J2(15d-PGJ2)组(DMEM培养基加入含终浓度10 μmol/L 15d-PGJ2作用2 h,再加入10 ng/mL TGF-β1作用48 h).应用蛋白质印迹法检测结缔组织生长因子(CTGF)的表达,实时荧光RT-PCR检测CTGF、基质金属蛋白酶1(MMP-1)及血小板源性生长因子(PDGF)的mRNA水平变化.对数据进行单因素方差分析.结果 TGF-β1组的CTGF蛋白和mRNA表达水平均高于空白对照组,曲格列酮组和15d-PGJ2组的CTGF蛋白及mRNA表达水平低于TGF-β1组.空白对照组、TGF-β1组、曲格列酮组、15d-PGJ 2组MMP-1 mRNA表达水平分别为1.281±0.195、0.193±0.051、0.417±0.043、0.485±0.027,其中TGF-β1组低于空白对照组(F=12.811,P＜0.01),曲格列酮组、15d-PGJ2组高于TGF-β1组(F=12.811,P值均小于0.01).空白对照组、TGF-β1组、曲格列酮组、15d-PGJ2组PDGF mRNA表达水平分别为0.349±0.057、1.044±0.237、0.677±0.055、0.511±0.017,其中TGF-β1组高于空白对照组(F=16.848,P＜0.01),曲格列酮组、15d-PGJ2组低于TGF-β1组(F=16.848,P值均小于0.01).结论 激活后的PPARγ对TGF-β1下游反应因子CTGF的抑制作用,可能是其拮抗TGF-β1促皮肤瘢痕化的主要机制,而对细胞因子MMP-1、PDGF的影响可能也是其机制之一.

Mechanism underlying the inhibitory effects of peroxisome proliferator-activated receptor γ agonists on transforming growth factor β1 in adult skin fibroblasts

Objective To study the mechanism underlying the inhibitory effects of peroxisome proliferator-activated receptor γ (PPARγ) agonists on transforming growth factor β 1 (TGF-β1)-induced scarring of skin. Methods Fibroblasts isolated from healthy adult skin were cultured in vitro and divided into blank control group (serum-free DMEM culture) , TGF-β1 group (with stimulation of 10 ng/mL TGF-β1 for 48 hours) , troglitazone group (with the same treatment as in TGF-β1 group after stimulation of 10 μmol/L troglitazone for 2 hours), and 15-deoxygen prostaglandin J2 (15d-PGJ2) group (with the same treatment as in TGF-β1 group after stimulation of 10 μmol/L 15d-PGJ2 for 2 hours) according to the stimulation added into DMEM. The expression of connective tissue growth factor (CTGF) was determined with Western blot.The mRNA levels of CTGF, matrix metalloproteinase-1 (MMP-1) and platelet-derived growth factor (PDGF) were determined with real-time fluorescence RT3-PCR. Data were processed with one-way analysis of variance. Results The expression of CTGF at mRNA and protein levels in skin fibroblasts were significantly increased in TGF-β group as compared with control group; while expression of CTGF at mRNA and protein levels in 15d-PGJ2 and troglitazone groups were significantly decreased as compared with that in TGF-β1 group. The mRNA level of MMP-1 in TGF-β1 group (0. 193 ±0.051 ) was obviously lower than that in blank control group ( 1.281 ±0. 195, F = 12.811, P ＜0.01 ), while the mRNA levels of MMP-1 in troglitazone group (0.417 ±0. 043) and 15d-PGJ2 group (0. 485 ±0. 027) were significantly increased as compared with that in TGF-β1 group ( F = 12.811 , P values all below 0. 01 ). The mRNA level of PDGF in IGF-β1 group ( 1. 044 ± 0. 237 ) was obviously higher than that in control group ( 0. 349 ± 0. 057, F =16. 848, P ＜0.01 ), while the levels in troglitazone group (0.677 ±0.055) and 15d-PGJ2 group (0.511 ±0. 017) were significantly decreased as compared with that in TGF-β1 group ( F = 16. 848, P values all below 0. 01 ). Conclusions The inhibitory effect of activated PPARγ on the expression of CTGF induced by TGF-β1 may be the main mechanism of its inhibitory effect on TGF-β-induced scarring on skin, and its influence on MMP-1 and PDGF may also be one of the underlying mechanisms.