辛伐他汀对不同病理条件刺激的瘢痕疙瘩成纤维细胞功能的影响

Different effects of simvastatin on keloid fibroblasts under hypoxia and TGF-β1 treatment

目的探讨辛伐他汀在不同病理因素刺激下对瘢痕疙瘩成纤维细胞增殖、凋亡及病变相关蛋白表达的影响。方法采用组织块贴壁法培养瘢痕疙瘩成纤维细胞。CCK-8检测常氧和低氧条件下不同浓度辛伐他汀（0．01、0．1、1、10、50、100、500μmol／L）作用瘢痕疙瘩成纤维细胞24、48h后，对其增殖的影响。流式细胞技术检测常氧、低氧以及10ng/ml TGF—β、作用下10μmol／L辛伐他汀干预24、48h对瘢痕疙瘩成纤维细胞凋亡的影响，并用Western Blot检测相同干预条件下细胞I型胶原、结缔组织生长因子（connective tissue growth factor，CTGF）和TIMP-1的合成情况。结果①在常氧和低氧条件下，辛伐他汀作用瘢痕疙瘩成纤维细胞24h或48h，能明显抑制细胞增殖，随着药物浓度增加抑制作用逐渐增大，而且在低氧条件下对细胞增殖的抑制作用比常氧条件下更强。②10μmol／L辛伐他汀作用24h或48h，并不能明最影响常氧和10ng／ml TGF—β1作用下的瘢痕疙瘩成纤维细胞的凋亡，对低氧条件下的细胞则有屁著的促进凋亡的作用（24h和48h凋亡率分别增加155．6％和478．8％，P〈0．05）。③10μmol／L辛伐他汀能显著减少低氧或10ng／ml TGF-β1作用下的瘢痕疙瘩成纤维细胞I型胶原和CTGF的合成（P〈0．05），对常氧条件的I型胶原和CTGF的表达无明显影响，10μmol／L辛伐他汀还能明显增强低氧条件下的TIMP-1的表达（P〈0．05），对常氧和TGF-β1作用下的TIMP-1的表达虽然也有促进作用，但作用并不显著。结论辛伐他汀对瘢痕疙瘩成纤维细胞增殖、凋亡及纤维化相关蛋白表达的作用有剂量依赖性并受条件影响有所不同，在低氧条件下它对瘢痕疙瘩成纤维细胞的作用更强。

Objective To explore the effect of simvastatin on the proliferation, apoptosis and protein expressions of keloid fibroblasts under normoxia, hypoxia or TGF-β1 treatment. Methods Keloid fibroblasts （KFs） were isolated by explants culture method. KFs were treated with different concentrations of simvastatin under normoxia or hypoxia （2% 02 ） for 24 h and 48 h. The effects of simvastatin on cell proliferation were detected by CCK-8. Flow cytometer was used to detect the apoptosis of KFs treated with 10 μmol/L simvastatin for 24 h or 48 h under normoxia, hypoxia or 10 ng/ml TGF-β1 treatment. Then the expressions of keloid-related proteins were analyzed by Western Blot. Results It showed that simvastatin could inhibit the proliferation of KFs in a concentration- and time-dependent manner with the concentration range of 10 - 500 μmol/L for 24 h and 0.1 - 500 μmol/L for 48 h. This inhibitory effect could be significantly enhanced when cells were incubated under hypoxia for 48h with 10 -500 μmol/L simvastatin. 10μmol/L simvastatin could not influence the apoptosis of KFs under normoxia or TGF-β1 treatment, neither incubated for 24 h nor 48 h. When incubated under hypoxia, 10 μmol/L simvastatin could significantly induce the apoptosis of KFs, with the rate of 155.6% for 24 h and 478.8% for 48 h, compared with no-drug control. There are no significant influences on the expression of type I collagen, CTGF or TIMP-1 when KFs were treated with 10 μmol/L simvastatin under normoxia for 48 h. When incubated with 10 ng/ml TGF-β1 together with 10 μmol/L simvastatin for 48 h, the expression of CTGF was significantly inhibited. KFs treated with 10 μmol/L simvastatin under hypoxia for 48 h showed a significant decrease of type I collagen and CTGF, and a significant increase of TIMP-1. Conclusions Simvastatin has different effects on the proliferation, apoptosis and protein expressions of KFs in a dosedependent manner under different conditions. The effects are enhanced under hypoxia.