重组人核心蛋白多糖固相拮抗转化生长因子β_1对瘢痕成纤维细胞的刺激作用

The antagonistic effect of recombinant human decorin on TGF-β_1 stimulation of fibroblasts in collagen lattices of scar

目的模拟人体内核心蛋白多糖和转化生长因子β_1(TGF-β_1)接触方式,观察核心蛋白多糖固相拮抗TGF-β_1刺激瘢痕成纤维细胞的效果。方法制备成纤维细胞胶原网格(FPCL)体外三维培养模型,将其分为4组。对照组：向FPCL中加入培养液；核心蛋白多糖组：向FPCL中混入终浓度2mg/L的重组人核心蛋白多糖,再加入培养液；TGF-β_1组：向FPCL中加入含5μg/L TGF-β_1的培养液；TGF-β_1+核心蛋白多糖组：向FPCL中混入终浓度2 mg/L的重组人核心蛋白多糖,然后加入含5μg/L TGF-β_1的培养液。在培养12、24、48、72、96h时观察各组FPCL的收缩情况,并用蛋白质印迹法与逆转录聚合酶链反应分别检测FPCL中瘢痕成纤维细胞Ⅰ型纤溶酶原激活物抑制因子1 (PAI-1)、α平滑肌肌动蛋白(α-SMA)的蛋白及mRNA表达水平。结果各培养时相点下,TGF-β_1组FPCL收缩比对照组明显增强,核心蛋白多糖组FPCL收缩则比对照组明显减弱。TGF-β_1组的PAI1、α-SMA的蛋白及相应mRNA表达水平(3 482±211、4 320±272；0．89±0．15、0．56±0．11)显著高于对照组(1764±147、1699±146；0．29±0．06、0．21±0．06,P＜0．01)；其余两组相应检测指标与对照组比较差异无统计学意义(P＞0．05)。结论重组人核心蛋白多糖混入胶原凝胶,可显著抑制TGF-β_1对瘢痕成纤维细胞的刺激作用,表明在体外核心蛋白多糖具有拈抗TGF-β_1的作用。提示皮肤组织损伤后,由于创面机械性缺少核心蛋白多糖,TGF-β_1活性上调,可能是瘢痕增生的一个重要因素。

Objective To mimic contact pattern between deeorin and TGF-β1 in vivo, and investigate the antagonistic effect of recombinant human decorin on TGF-β1 stimulation of hypertrophic scar fibroblasts in collagen lattices. Methods Fibroblasts populated collagen lattices （FPCL） model was adopted in the study, and they were divided into control group, decorin group [2mg/L recombinant human decorin （rh-decorin） was administered to FPCL] , TGF-β1 group （5 μg/LTGF-β1 was administered to the culture medium） , and TGF-β1 ± decorin group（ 2mg/L rh-decorin was administered to FPCL, then culture medium containing 5 μg/L TGF-β1 was added into FPCL）. Changes in PAI-1 and α-SMA protein expression in scar fibroblasts in collagen lattices were detected with Western blotting at 12 post-administration hour （PAH） , 24 PAH, 48 PAH, and 72 PAH, and expressions of PAI-1 and α-SMA mRNA were concomitantly examined by RT-PCR. Results The contraction of FPCL at each time-point in control group was obviously attenuated compared with that in decorin group, but it was significantly intensified compared with that in TGF-β1 group. The expression of PAI-1 and α-SMA mRNA and protein in TGF-β1 group （3 482 ± 211, 4 320 ± 272, 0.89 ± 0. 15, 0.56 ± 0.11 ） were markedly increased than those in control group（ 1 764 ± 147, 1 699 ± 146, 0.29 ± 0.06, 0.21 ± 0.06, P 〈 0.01 ） , while no obvious difference of them was found between control and other two groups. Conclusion Stimulation of scar fibroblasts by TGF-β1 can be suppressed when rh-decorin is blended into collagen lattices, indicating that decorin is effective in neutralizing TGF-β1 in vitro. The pathogensis of hypertrophic scar might be related to up-regulation of TGF-β1 with the lack of decorin after cutaneous injury.