Liu Jia-qi, Hu Da-hai, Zhang Zhan-feng, Guan Hao, She Tao, Zhang Jun, Bai Xiao-zhi
Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.
Zhonghua Shao Shang Za Zhi. 2009 Dec;25(6):454-9.
To elucidate the effects of interferon-gamma (IFN-gamma) on the transforming growth factor beta (TGF-beta)/Smad pathway in keloid-derived fibroblasts (KFb), and to investigate the underlying mechanism in the treatment of pathologic scar with IFN-gamma.
Keloid tissue of 3 patients were obtained, and then KFb were separated and cultured in vitro. KFb from passages 3 to 5 were used for the study. (1) KFb were divided into control group (incubated with serum-free DMEM), TGF-beta(1) group (treated with 10 ng/mL TGF-beta(1)), IFN-gamma group (treated with 100 ng/mL IFN-gamma), and TGF-beta(1)+IFN-gamma group (incubated with 10 ng/mL TGF-beta(1) combined with 100 ng/mL IFN-gamma). The expression level of mRNA and protein of connective tissue growth factor (CTGF), alpha smooth muscle actin (alpha-SMA) protein and expression of alpha-SMA positive KFb were detected by real-time fluorescent quantitation RT-PCR (FQ-RT-PCR), Western blot and immunofluorescence cytochemical staining. (2) Another sample of KFb was obtained and treated with 10 ng/mL IFN-gamma. The expression level of Smad 3 and Smad 7 protein was detected by Western blot before and 1, 2, 4, 6, 8 h post stimulation (PSH). The expression level of Smad 3 and Smad 7 mRNA was assessed by FQ-RT-PCR before stimulation and 30 mins post stimulation and at PSH, 1, 2, 4, 6, 8. (3) Another sample of KFb was obtained and divided into 1, 10 and 100 ng/mL IFN-gamma groups based on the concentration of IFN-gamma, treated for 4 hours; KFb without IFN-gamma treatment was set up as control group. The expression levels of the protein and mRNA of Smad 3 and Smad 7 were measured by FQ-RT-PCR and Western blot.
(1) The level of mRNA and protein of CTGF in IFN-gamma group (0.017 +/- 0.009 and 1.198 +/- 0.004) was respectively lower than that in control group (0.024 +/- 0.013 and 1.229 +/- 0.011, P < 0.05). The level of mRNA and protein of CTGF in TGF-beta(1)+IFN-gamma group (0.634 +/- 0.138 and 1.204 +/- 0.010) was respectively lower than that in TGF-beta(1) group (1.331 +/- 0.298 and 1.727 +/- 0.004, P < 0.01). The fluorescence intensity of alpha-SMA positive KFb (0.922 +/- 0.059) and the expression level of alpha-SMA protein (0.3051 +/- 0.0031) in IFN-gamma group decreased significantly than those in control group (1.055 +/- 0.005 and 0.4513 +/- 0.0094, P < 0.01). The fluorescence intensity of alpha-SMA positive KFb (1.129 +/- 0.004) and the expression level of alpha-SMA protein (0.6734 +/- 0.0098) in TGF-beta(1)+IFN-gamma group decreased significantly than those in TGF-beta(1) group (1.270 +/- 0.005 and 1.3842 +/- 0.0024, P < 0.01). (2) The expression level of Smad 3 mRNA and protein at the first time point after IFN-gamma treatment increased temporarily then decreased gradually, and mRNA expression level reached the nadir at PSH 4, it rose gradually later, though it was still lower at PSH 8 than that before treatment (P < 0.01); protein expression level at PSH 8 was significantly lower than that before treatment (P < 0.01). The expression level of Smad 7 mRNA and protein increased gradually to the maximum at PSH 2 and 4 respectively, then decreased but was still higher at PSH 8 than that before treatment (P < 0.05). (3) Compared with those in control group, the expression levels of Smad 3 mRNA and protein in 1, 10 and 100 ng/mL IFN-gamma group were significantly lower, the expression levels of Smad 7 mRNA and protein were significantly higher (P < 0.05 or P < 0.01). The higher concentration of IFN-gamma, the more significant differences were observed.
IFN-gamma can down-regulate the expression of Smad 3 while up-regulate the expression of Smad 7 in a time- and dose-dependent manner, and reduce the expression level of CTGF and alpha-SMA in the basic state or induced by TGF-beta(1), which shows a significant inhibitory effect on the TGF-beta/Smad signal pathway. This may be an important mechanism in the treatment of pathologic scar by IFN-gamma.
阐明γ干扰素（IFN-γ）对瘢痕疙瘩来源的成纤维细胞（KFb）中转化生长因子β（TGF-β）/Smad信号通路的影响，并探讨IFN-γ治疗病理性瘢痕的潜在机制。
获取3例患者的瘢痕疙瘩组织，分离并体外培养KFb。采用第3至5代的KFb进行研究。（1）将KFb分为对照组（用无血清DMEM培养）、TGF-β(1)组（用10 ng/mL TGF-β(1)处理）、IFN-γ组（用100 ng/mL IFN-γ处理）和TGF-β(1)+IFN-γ组（用10 ng/mL TGF-β(1)与100 ng/mL IFN-γ共同培养）。通过实时荧光定量RT-PCR（FQ-RT-PCR）、蛋白质免疫印迹法和免疫荧光细胞化学染色检测结缔组织生长因子（CTGF）、α平滑肌肌动蛋白（α-SMA）的mRNA和蛋白质表达水平以及α-SMA阳性KFb的表达情况。（2）获取另一批KFb，用10 ng/mL IFN-γ处理。在刺激前及刺激后1、2、4、6、8小时（PSH），采用蛋白质免疫印迹法检测Smad 3和Smad 7蛋白的表达水平；在刺激前、刺激后30分钟及PSH 1、2、4、6、8时，采用FQ-RT-PCR评估Smad 3和Smad 7 mRNA的表达水平。（3）获取另一批KFb，根据IFN-γ浓度分为1、10和100 ng/mL IFN-γ组，处理4小时；将未用IFN-γ处理的KFb设为对照组。采用FQ-RT-PCR和蛋白质免疫印迹法检测Smad 3和Smad 7的蛋白质和mRNA表达水平。
（1）IFN-γ组CTGF的mRNA和蛋白质水平（0.017±0.009和1.198±0.004）分别低于对照组（0.024±0.013和1.229±0.011，P<0.05）。TGF-β(1)+IFN-γ组CTGF的mRNA和蛋白质水平（0.634±0.138和1.204±0.010）分别低于TGF-β(1)组（1.331±0.298和1.727±0.004，P<0.01）。IFN-γ组α-SMA阳性KFb的荧光强度（0.922±0.059）和α-SMA蛋白表达水平（0.3051±0.0031）较对照组（1.055±0.005和0.4513±0.0094）显著降低（P<0.01）。TGF-β(1)+IFN-γ组α-SMA阳性KFb的荧光强度（1.129±0.004）和α-SMA蛋白表达水平（0.6734±0.0098）较TGF-β(1)组（1.270±0.005和1.3842±0.0024）显著降低（P<0.01）。（2）IFN-γ处理后第1个时间点，Smad 3 mRNA和蛋白质表达水平先暂时升高然后逐渐降低，mRNA表达水平在PSH 4时降至最低点，随后逐渐升高，尽管在PSH 8时仍低于处理前水平（P<0.01）；PSH 8时蛋白质表达水平显著低于处理前（P<0.01）。Smad 7 mRNA和蛋白质表达水平分别在PSH 2和4时逐渐升高至最大值，然后降低，但在PSH 8时仍高于处理前（P<0.05）。（3）与对照组相比，1、10和100 ng/mL IFN-γ组Smad 3 mRNA和蛋白质表达水平显著降低，Smad 7 mRNA和蛋白质表达水平显著升高（P<0.05或P<0.01）。IFN-γ浓度越高，差异越显著。
IFN-γ可呈时间和剂量依赖性下调Smad 3表达，上调Smad 7表达，并降低基础状态下或TGF-β(1)诱导状态下CTGF和α-SMA的表达水平，对TGF-β/Smad信号通路具有显著抑制作用。这可能是IFN-γ治疗病理性瘢痕的重要机制。
