内皮抑素预处理对人皮肤成纤维细胞纤维化的作用和机制

摘要

目的 探讨内皮抑素预处理对人皮肤Fb纤维化的作用和相关机制. 方法 将人皮肤Fb体外常规培养后,取第3～5代细胞进行实验.按照随机数字表法将细胞分为空白对照组、内皮抑素组、血小板源性生长因子BB(PDGF-BB)组、内皮抑素+PDGF-BB组、TGF-β1组、内皮抑素+TGF-β1组,每组3孔.空白对照组细胞仅用DMEM培养液培养24 h;内皮抑素组细胞用含5μg/mL内皮抑素的DMEM培养液培养24 h;PDGF-BB组和TGF-β1组细胞分别用含200 ng/mL PDGF-BB、10 ng/mL TGF-β1的DMEM培养液培养24 h;内皮抑素+PDGF-BB组细胞用含5μg/mL内皮抑素的DMEM培养液预处理48 h后,再用含200ng/mL PDGF-BB的DMEM培养液培养24 h;内皮抑素+TGF-β1组细胞用含5μg/mL内皮抑素的DMEM培养液预处理48 h后,再用含10 ng/mL TGF-β1的DMEM培养液培养24 h.每组收集3孔细胞的培养上清液,ELISA法检测Ⅰ型胶原含量;每组收集3孔细胞,采用蛋白质印迹法检测细胞α平滑肌肌动蛋白(α-SMA)、PDGF受体β(PDGFRβ)、磷酸化PDGFRβ(p-PDGFRβ)、磷酸化细胞外信号调节激酶1/2(p-ERK1/2)的蛋白表达.对数据行单因素方差分析、SNK检验. 结果 (1)与空白对照组的(5.05 ±0.29) pg/mL比较,内皮抑素组细胞培养上清液中Ⅰ型胶原含量为(4.72±0.37) pg/mL,二者相近(P＞0.05);PDGF-BB组和TGF-β1组细胞培养上清液中Ⅰ型胶原含量分别为(8.60±0.57)、(9.20±0.64) pg/mL,均显著升高(p值均小于0.05).内皮抑素+PDGF-BB组细胞培养上清液中Ⅰ型胶原含量为(5.32 ±0.17)pg/mL,低于PDGF-BB组(P＜0.05);内皮抑素+TGF-β1组细胞培养上清液中Ⅰ型胶原含量为(5.41±0.20) pg/mL,低于TGF-β1组(P＜0.05).(2)与空白对照组细胞α-SMA、PDGFRβ、p-PDGFRβ、p-ERK1/2蛋白表达量比较,内皮抑素组无明显差异(P值均大于0.05),PDGF-BB组、TGF-β1组均明显增加(P值均小于0 01).内皮抑素+ PDGF-BB组、内皮抑素+TGF-β1组细胞α-SMA、PDGFRβ、p-PDGFRβ、p-ERK1/2蛋白表达量均分别低于PDGF-BB组、TGF-β1组(P值均小于0.05). 结论 内皮抑素预处理可抑制人Fb纤维化和向肌Fb转化,这可能与内皮抑素下调p-PDGFRβ、PDGFRβ和p-ERK蛋白表达有关.%Objective To explore the effects of endostatin pretreatment on fibrosis of human skin fibroblasts and the mechanisms.Methods Human skin fibroblasts were routinely cultured in vitro,and then the cells of passage 3 to 5 were used in the following experiments.The ceils were divided into blank control,endostatin,platelet-derived growth factor-BB (PDGF-BB),endostatin + PDGF-BB,transforming growth factor-β1 (TGF-β1),and endostatin + TGF-β1 groups according to the random number table,with 3 wells in each group.Cells in blank control group were cultured with DMEM medium for 24 h.Cells in endostatin group were cultured with DMEM medium containing 5 μg/mL endostatin for 24 h.Cells in PDGF-BB group and TGF-β1 group were cultured with DMEM medium containing 200 ng/mL PDGF-BB and 10 ng/mL TGF-β1 for 24 h,respectively.Cells in endostatin + PDGF-BB group were pretreated with DMEM medium containing 5 μg/mL endostatin for 48 h and then cultured with DMEM medium containing 200 ng/mL PDGF-BB for 24 h.Cells in endostatin + TGF-β1 group were pretreated with DMEM medium containing 5 μg/mL endostatin for 48 h and then cultured with DMEM medium containing 10 ng/mL TGF-β1 for 24 h.The content of type Ⅰ collagen in the cell culture supernatant of three wells in each group was determined by enzyme-linked immunosorbent assay.The protein expression levels of α-smooth muscle actin (α-SMA),PDGF receptor β (PDGFRβ),phosphorylated PDGFRβ (p-PDGFRβ),and phosphorylated extracellular signal-regulated protein kinases 1/2 (p-ERK1/2) of three wells in each group were detected by Western blotting.Data were processed with one-way analysis of variance and SNK test.Results (1)Compared with (5.05 ± 0.29) pg/mL in blank control group,content of type Ⅰ collagen in the cell culture supernatant of endostatin group [(4.72 ± 0.37) pg/mL] was close to it (P ＞ 0.05),content of type Ⅰ collagen in the cell culture supernatant of PDGF-BB group and TGF-β1 group [(8.60 ± 0.57) and (9.20 ±0.64) pg/mL,respectively] was higher (with P values below 0.05).Content of type Ⅰ collagen in the cell culture supernatant of endostatin + PDGF-BB group [(5.32 ± 0.17) pg/mL] was lower than that of PDGF-BB group (P ＜ 0.05),and content of type Ⅰ collagen in the cell culture supernatant of endostatin + TGF-β1 group [(5.41 ±0.20) pg/mL] was lower than that of TGF-β1 group (P ＜0.05).(2) Compared with those in blank control group,protein expression levels of α-SMA,PDGFRβ,p-PDGFRβ,and pERK1/2 of cells in endostatin group showed no obvious differences (with P values above 0.05),while those in PDGF-BB and TGF-β1 group were significantly higher (with P values below 0.01).Protein expression levels of α-SMA,PDGFRβ,p-PDGFRβ,and p-ERK1/2 of cells in endostatin + PDGF-BB group and endostatin + TGF-β1 group were significantly lower than those in PDGF-BB group and TGF-β1 group,respectively (with P values below 0.05).Conclusions Pretreatment of endostatin can inhibit the fibrosis of hu man skin fibroblast and its transformation into myofibroblast,which may be related to the down-regulation of protein expression of p-PDGFRβ,PDGFRβ,and p-ERK.