中华烧伤杂志
中華燒傷雜誌
중화소상잡지
16
2009年
6期
454-459
,共6页
干扰素Ⅱ型%瘢痕疙瘩%成纤维细胞%转化生长因子β_1%Smad蛋白质类
榦擾素Ⅱ型%瘢痕疙瘩%成纖維細胞%轉化生長因子β_1%Smad蛋白質類
간우소Ⅱ형%반흔흘탑%성섬유세포%전화생장인자β_1%Smad단백질류
Interferon type II%Keloid%Fibroblasts%Transforming growth factor beta_1%Smad proteins
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.
目的 瞭解γ榦擾素(IFN-γ)對瘢痕疙瘩Fh(KFb)中TGF-β/Smad信號通路的作用,探討IFN一γ治療病理性搬痕的可能機製. 方法 切取3例患者的瘢痕組織,體外分離培養KFb,實驗選用第3～5代細胞.(1)將KFb分為:對照組,加無血清DMEM培養;TGF-β_1組,用10 ng/mL的TGF-β_1單獨作用;IFN-γ組,用100 ng/mL的IFN-γ單獨作用;TGF-β_1+IFN-γ組,10 ng/mL的TGF-β_1與100 nS/mL的IFN-γ聯閤作用.採用實時熒光定量RT-PCR、蛋白質印跡法和免疫熒光細胞化學染色法,分彆檢測結締組織生長因子(CTGF)的mRNA、蛋白錶達,以及α平滑肌肌動蛋白(α-SMA)的蛋白錶達與暘性細胞錶達情況.(2)另取KFb,用10 ng/mL的IFN-γ作用,于作用前及作用後30 min和1、2、4、6、8 h通過實時熒光定量RT-PCR檢測Smad 3和Smad 7的mRNA錶達,于作用前及作用後1、2、4、6、8 h用蛋白質印跡法檢測Smad 3和Smad 7的蛋白錶達.(3)另取KFb,根據添加的IFN-γ終濃度不同分為1、10、100 ns/mL IFN-γ組,均作用4 h;設立未添加IFN-γ的KFb為對照組.同前檢測各組Smad 3和Smad 7的mRNA及蛋白錶達. 結果 (1)IFN-γ組KFb CTGF的mRNA和蛋白錶達量為0.017±0.009與1.198±0.004,較對照組(0.024±0.013與1.229±0.011)顯著減少(P<0.05);TGF-β1+IFN-γ組CTGF的mRNA和蛋白錶達量為0.634±0.138與1.204±0.010,較TGF-β_1組(1.331±0.298與1.727±0.004)顯著減少(P<0.01).IFN-γ組KFb中,α-SMA暘性細胞熒光彊度(0.922±0.059)和α-SMA蛋白錶達量(0.3051±0.0031)較對照組(1.055±0.005與0.4513±0.0094)顯著減少(P<0.01);TGF-β1+IFN-γ組SMA暘性KFb熒光彊度(1.129±0.004)和SMA蛋白錶達量(0.6734±0.0098)較TGF-β_1組(1.270±0.005與1.38420.0024)顯著減少(P<0.01).(2)10 ng/mL IFN-γ作用後第1箇時相點,Smad 3的mRNA和蛋白錶達量均齣現一過性增高,隨後降低,mRNA錶達量于作用後4 h降至最低點,隨後緩慢上升,至作用後8 h仍低于作用前(P<0.01);其蛋白錶達量于作用後2~8 h顯著低于作用前(P<0.01).而Smad 7的mRNA和蛋白錶達量在INF-γ作用後逐漸增高,分彆于作用後2、4 h達峰值隨後降低,至作用後8 h仍高于作用前(P<0.05).(3)與對照組比較,1、10、100 ng/mL IFN-γ組Smad 3的mRNA及蛋白錶達量顯著減少(P<0.05或P<0.01),Smad 7的mRNA及蛋白錶達量顯著增加(P<0.05或P<0.01),且隨IFN-γ濃度升高,減少或增高幅度愈為顯著. 結論 IFN-γ呈時間和劑量依賴方式下調Smad 3、上調Smad 7,降低基礎狀態下或經TGF-β_1誘導後KFb的CTGF和α-SMA錶達量,錶現齣對TGF-β/Smad 信號通路的顯著拮抗作用,這可能是IFN-γ治療病理性瘢痕的重要機製.
목적 료해γ간우소(IFN-γ)대반흔흘탑Fh(KFb)중TGF-β/Smad신호통로적작용,탐토IFN일γ치료병이성반흔적가능궤제. 방법 절취3례환자적반흔조직,체외분리배양KFb,실험선용제3～5대세포.(1)장KFb분위:대조조,가무혈청DMEM배양;TGF-β_1조,용10 ng/mL적TGF-β_1단독작용;IFN-γ조,용100 ng/mL적IFN-γ단독작용;TGF-β_1+IFN-γ조,10 ng/mL적TGF-β_1여100 nS/mL적IFN-γ연합작용.채용실시형광정량RT-PCR、단백질인적법화면역형광세포화학염색법,분별검측결체조직생장인자(CTGF)적mRNA、단백표체,이급α평활기기동단백(α-SMA)적단백표체여양성세포표체정황.(2)령취KFb,용10 ng/mL적IFN-γ작용,우작용전급작용후30 min화1、2、4、6、8 h통과실시형광정량RT-PCR검측Smad 3화Smad 7적mRNA표체,우작용전급작용후1、2、4、6、8 h용단백질인적법검측Smad 3화Smad 7적단백표체.(3)령취KFb,근거첨가적IFN-γ종농도불동분위1、10、100 ns/mL IFN-γ조,균작용4 h;설립미첨가IFN-γ적KFb위대조조.동전검측각조Smad 3화Smad 7적mRNA급단백표체. 결과 (1)IFN-γ조KFb CTGF적mRNA화단백표체량위0.017±0.009여1.198±0.004,교대조조(0.024±0.013여1.229±0.011)현저감소(P<0.05);TGF-β1+IFN-γ조CTGF적mRNA화단백표체량위0.634±0.138여1.204±0.010,교TGF-β_1조(1.331±0.298여1.727±0.004)현저감소(P<0.01).IFN-γ조KFb중,α-SMA양성세포형광강도(0.922±0.059)화α-SMA단백표체량(0.3051±0.0031)교대조조(1.055±0.005여0.4513±0.0094)현저감소(P<0.01);TGF-β1+IFN-γ조SMA양성KFb형광강도(1.129±0.004)화SMA단백표체량(0.6734±0.0098)교TGF-β_1조(1.270±0.005여1.38420.0024)현저감소(P<0.01).(2)10 ng/mL IFN-γ작용후제1개시상점,Smad 3적mRNA화단백표체량균출현일과성증고,수후강저,mRNA표체량우작용후4 h강지최저점,수후완만상승,지작용후8 h잉저우작용전(P<0.01);기단백표체량우작용후2~8 h현저저우작용전(P<0.01).이Smad 7적mRNA화단백표체량재INF-γ작용후축점증고,분별우작용후2、4 h체봉치수후강저,지작용후8 h잉고우작용전(P<0.05).(3)여대조조비교,1、10、100 ng/mL IFN-γ조Smad 3적mRNA급단백표체량현저감소(P<0.05혹P<0.01),Smad 7적mRNA급단백표체량현저증가(P<0.05혹P<0.01),차수IFN-γ농도승고,감소혹증고폭도유위현저. 결론 IFN-γ정시간화제량의뢰방식하조Smad 3、상조Smad 7,강저기출상태하혹경TGF-β_1유도후KFb적CTGF화α-SMA표체량,표현출대TGF-β/Smad 신호통로적현저길항작용,저가능시IFN-γ치료병이성반흔적중요궤제.
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.
