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γ干扰素对瘢痕疙瘩成纤维细胞转化生长因子β/Smad信号通路的作用

Effects of interferon-gamma on the transforming growth factor beta/Smad pathway in keloid-derived fibroblasts

摘要:

目的 了解γ干扰素(IFN-γ)对瘢痕疙瘩Fh(KFb)中TGF-β/Smad信号通路的作用,探讨IFN一γ治疗病理性搬痕的可能机制. 方法 切取3例患者的瘢痕组织,体外分离培养KFb,实验选用第3~5代细胞.(1)将KFb分为:对照组,加无血清DMEM培养;TGF-β_1组,用10 ng/mL的TGF-β_1单独作用;IFN-γ组,用100 ng/mL的IFN-γ单独作用;TGF-β_1+IFN-γ组,10 ng/mL的TGF-β_1与100 nS/mL的IFN-γ联合作用.采用实时荧光定量RT-PCR、蛋白质印迹法和免疫荧光细胞化学染色法,分别检测结缔组织生长因子(CTGF)的mRNA、蛋白表达,以及α平滑肌肌动蛋白(α-SMA)的蛋白表达与阳性细胞表达情况.(2)另取KFb,用10 ng/mL的IFN-γ作用,于作用前及作用后30 min和1、2、4、6、8 h通过实时荧光定量RT-PCR检测Smad 3和Smad 7的mRNA表达,于作用前及作用后1、2、4、6、8 h用蛋白质印迹法检测Smad 3和Smad 7的蛋白表达.(3)另取KFb,根据添加的IFN-γ终浓度不同分为1、10、100 ns/mL IFN-γ组,均作用4 h;设立未添加IFN-γ的KFb为对照组.同前检测各组Smad 3和Smad 7的mRNA及蛋白表达. 结果 (1)IFN-γ组KFb CTGF的mRNA和蛋白表达量为0.017±0.009与1.198±0.004,较对照组(0.024±0.013与1.229±0.011)显著减少(P<0.05);TGF-β1+IFN-γ组CTGF的mRNA和蛋白表达量为0.634±0.138与1.204±0.010,较TGF-β_1组(1.331±0.298与1.727±0.004)显著减少(P<0.01).IFN-γ组KFb中,α-SMA阳性细胞荧光强度(0.922±0.059)和α-SMA蛋白表达量(0.3051±0.0031)较对照组(1.055±0.005与0.4513±0.0094)显著减少(P<0.01);TGF-β1+IFN-γ组SMA阳性KFb荧光强度(1.129±0.004)和SMA蛋白表达量(0.6734±0.0098)较TGF-β_1组(1.270±0.005与1.38420.0024)显著减少(P<0.01).(2)10 ng/mL IFN-γ作用后第1个时相点,Smad 3的mRNA和蛋白表达量均出现一过性增高,随后降低,mRNA表达量于作用后4 h降至最低点,随后缓慢上升,至作用后8 h仍低于作用前(P<0.01);其蛋白表达量于作用后2~8 h显著低于作用前(P<0.01).而Smad 7的mRNA和蛋白表达量在INF-γ作用后逐渐增高,分别于作用后2、4 h达峰值随后降低,至作用后8 h仍高于作用前(P<0.05).(3)与对照组比较,1、10、100 ng/mL IFN-γ组Smad 3的mRNA及蛋白表达量显著减少(P<0.05或P<0.01),Smad 7的mRNA及蛋白表达量显著增加(P<0.05或P<0.01),且随IFN-γ浓度升高,减少或增高幅度愈为显著. 结论 IFN-γ呈时间和剂量依赖方式下调Smad 3、上调Smad 7,降低基础状态下或经TGF-β_1诱导后KFb的CTGF和α-SMA表达量,表现出对TGF-β/Smad 信号通路的显著拮抗作用,这可能是IFN-γ治疗病理性瘢痕的重要机制.

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abstracts:

Objective To elucidate the effcts of interferon-gamma(IFN-γ)on the transforming growth factor beta(TGF-β)/Smad pathway in keloid-derived fibroblasts(KFb),and to investigate the un-derlying mechanism in the treatment of pathologic scar with IFN-γ. Methods 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-β_1 group(treated with 10 ng/mL-TGF-β_1),IFN-γ group(treated with 100 ng/mL IFN-γ),and TGF-β_1+IFN-γ group(incu-bated with 10 rig/mL TGF-β_1 combined with 100 ng/mL IFN-γ).The expression level of mRNA and protein of connective tissue growth factor(CTGF),α smooth muscle aetin(α-SMA)protein and expression of ot-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-γ.The expression level of Smad 3 and Smad 7 protein was detected bv Western blot be-fore 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-γ groups based off the con-centration of IFN-γ, treated for 4 hours;KFb without IFN-γ treatment was set up as centrel group.The ex-pression levels of the protein and mRNA of Smad 3 and Smad 7 were measured by FO-RT-PCR and Western blot. Results (1)The level of mRNA and protein of CTGF in IFN-γ group(0.01 7±0.009 and 1.198±0.004) was respectively lower than that in centrel group (0.024 4-0.013 and 1.229±0.011,P< 0.05).The level of mRNA and protein of CTGF in TGF-β_1+IFN-γ group(0.634±0.138 and 1.204±0.010)was respectively lower than that in TGF-β_1 group(1.331±0.298 and 1.727±0.004,P<0.01).The fluorescence intensity of(α-SMA positive KFb(0.922±0.059)and the expression level of α-SMA pro-tein (0.3051±0.0031) in IFN-γ group decreased significantly than those in control group (1.055±0.005 and 0.4513±0.0094,P<0.01).The fluorescence intensity of α-SMA positive KFb (1.129±0.004) and the expression level of α-SMA protein (0.6734±0.0098) in TGF-β_1+IFN-γ group decreased significantly than those in TGF-β_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-γ treatment increased temporarily then de-creased gradually,and mRNA expression level reached the nadir at PSH 4,it rose gradually later,though it was stillJower at PSH 8 than that before treatment(P<0.01):protein expression level at PSH 8 was sig-nificantly 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-γ 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 concen-tration of IFN-γ,the more significant differences were observed. Conclusions IFN-γ 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 α-SMA in the basic state or induced by TGF-β_1,which shows a significant inhibitory effect on the TGF-β/Smad signal pathway.This may be an important mechanism in the treatment of pathologic scar by IFN-γ.

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