中华肾脏病杂志
中華腎髒病雜誌
중화신장병잡지
2013年
5期
370-374
,共5页
刘念%崔英春%贾冶%远航%罗萍%苗里宁
劉唸%崔英春%賈冶%遠航%囉萍%苗裏寧
류념%최영춘%가야%원항%라평%묘리저
转化生长因子β1%纤溶酶原激活物抑制物1%乙酰化作用%启动子%Smad3
轉化生長因子β1%纖溶酶原激活物抑製物1%乙酰化作用%啟動子%Smad3
전화생장인자β1%섬용매원격활물억제물1%을선화작용%계동자%Smad3
Transforming growth factor beta1%Plasminogen activator inhibitor 1%Acetylation%Promoter%Smad3
目的 探讨转化生长因子β1(TGF-β1)对1型纤溶酶原激活物抑制物(PAI-1)基因启动子及转录区域内组蛋白尾部乙酰化修饰的影响.方法 采用染色质共免疫沉淀与实时定量PCR技术,观察高糖及TGF-β1对PAI-1基因启动子及转录区组蛋白H3赖氨酸残基9位乙酰化(H3K9Ac)修饰的影响；并应用共免疫沉淀方法探讨转录因子CREB结合蛋白(CBP)与Smad3、Sp1蛋白之间的相互作用.结果 在PAI-1基因启动子的4个区域内,TGF-β1(10 μg/L)刺激显著增加了P1、P2、P3区域的H3K9Ac修饰(均P＜0.05),而在距离转录起始点较远的P4区域,H3K9Ac修饰水平没有明显改变；而在PAI-1基因的转录区,TGF-β1刺激显著增加了T1区域的H3K9Ac修饰(P＜0.05),而T2区没有明显改变.高糖刺激引起系膜细胞内PAI-1基因的表达水平及其启动子P1区域H3K9Ac修饰显著增加(P＜0.05),应用TGF-β1中和性抗体预处理显著抑制了高糖引起的PAI-1基因启动子区H3K9Ac修饰(P＜0.01).TGF-β1刺激能够显著诱导Smad3、CBP蛋白结合在P1、P2、P3区域(均P＜0.05)；同时,TGF-β1刺激能够诱导CBP、Sp1与Smad3蛋白的结合,进而引起H3K9Ac修饰.结论 TGF-β1能够诱导PAI-1基因启动子与转录起始区发生H3K9Ac修饰,促进Smad3与Sp1及CBP蛋白相互结合,促进PAI-1基因的转录表达.
目的 探討轉化生長因子β1(TGF-β1)對1型纖溶酶原激活物抑製物(PAI-1)基因啟動子及轉錄區域內組蛋白尾部乙酰化脩飾的影響.方法 採用染色質共免疫沉澱與實時定量PCR技術,觀察高糖及TGF-β1對PAI-1基因啟動子及轉錄區組蛋白H3賴氨痠殘基9位乙酰化(H3K9Ac)脩飾的影響；併應用共免疫沉澱方法探討轉錄因子CREB結閤蛋白(CBP)與Smad3、Sp1蛋白之間的相互作用.結果 在PAI-1基因啟動子的4箇區域內,TGF-β1(10 μg/L)刺激顯著增加瞭P1、P2、P3區域的H3K9Ac脩飾(均P＜0.05),而在距離轉錄起始點較遠的P4區域,H3K9Ac脩飾水平沒有明顯改變；而在PAI-1基因的轉錄區,TGF-β1刺激顯著增加瞭T1區域的H3K9Ac脩飾(P＜0.05),而T2區沒有明顯改變.高糖刺激引起繫膜細胞內PAI-1基因的錶達水平及其啟動子P1區域H3K9Ac脩飾顯著增加(P＜0.05),應用TGF-β1中和性抗體預處理顯著抑製瞭高糖引起的PAI-1基因啟動子區H3K9Ac脩飾(P＜0.01).TGF-β1刺激能夠顯著誘導Smad3、CBP蛋白結閤在P1、P2、P3區域(均P＜0.05)；同時,TGF-β1刺激能夠誘導CBP、Sp1與Smad3蛋白的結閤,進而引起H3K9Ac脩飾.結論 TGF-β1能夠誘導PAI-1基因啟動子與轉錄起始區髮生H3K9Ac脩飾,促進Smad3與Sp1及CBP蛋白相互結閤,促進PAI-1基因的轉錄錶達.
목적 탐토전화생장인자β1(TGF-β1)대1형섬용매원격활물억제물(PAI-1)기인계동자급전록구역내조단백미부을선화수식적영향.방법 채용염색질공면역침정여실시정량PCR기술,관찰고당급TGF-β1대PAI-1기인계동자급전록구조단백H3뢰안산잔기9위을선화(H3K9Ac)수식적영향；병응용공면역침정방법탐토전록인자CREB결합단백(CBP)여Smad3、Sp1단백지간적상호작용.결과 재PAI-1기인계동자적4개구역내,TGF-β1(10 μg/L)자격현저증가료P1、P2、P3구역적H3K9Ac수식(균P＜0.05),이재거리전록기시점교원적P4구역,H3K9Ac수식수평몰유명현개변；이재PAI-1기인적전록구,TGF-β1자격현저증가료T1구역적H3K9Ac수식(P＜0.05),이T2구몰유명현개변.고당자격인기계막세포내PAI-1기인적표체수평급기계동자P1구역H3K9Ac수식현저증가(P＜0.05),응용TGF-β1중화성항체예처리현저억제료고당인기적PAI-1기인계동자구H3K9Ac수식(P＜0.01).TGF-β1자격능구현저유도Smad3、CBP단백결합재P1、P2、P3구역(균P＜0.05)；동시,TGF-β1자격능구유도CBP、Sp1여Smad3단백적결합,진이인기H3K9Ac수식.결론 TGF-β1능구유도PAI-1기인계동자여전록기시구발생H3K9Ac수식,촉진Smad3여Sp1급CBP단백상호결합,촉진PAI-1기인적전록표체.
Objective To explore the effect of transforming growth factor β1 (TGF-β1) on epigenetic histone lysine acetylation in the plasminogen activator inhibitor 1 (PAI-1) promoter and transcribe regions in glomerular mesangial cells (GMCs).Methods Chromatin immunoprecipitation assay and real-time quantitative PCR were used to detect Histone3K9 acetylation (H3K9Ac) in the PAI-1 promoter and transcribe regions induced by TGF-β1 and high glucose.Immunoprecipitation was also used to see the cooperation of Smad3,CBP and Sp1 proteins.Results In the four target regions of PAI-1 promoter,TGF-β1 treatment enhanced H3K9Ac at P1,P2 and P3 in GMCs (P ＜ 0.05),but no change was seen in the P4 region which was far from the transcription starting site.TGF-β1 obviously induced H3K9Ac in the T1 transcribe region of PAI-1 instead of T2 (P ＜ 0.05).High glucose increased PAI-1 mRNA expression and H3K9Ac around P1 promoter region (P＜ 0.05).TGF-β1 neutralizing antibody abrogated high glucose-induced H3K9Ac at PAI-1 promoter (P ＜ 0.01).TGF-β1 treatment could recruit Smad3 and CBP protein binding to the PAI-1 promoter regions (P1,P2,P3),and induce their cooperation in GMCs,which were responsing to TGF-β1 associated H3K9Ac.Conclusion TGF-β1 can induce H3K9Ac in the promoter and transcribe regions of PAI-1,promote Smad3 recruition and cooperation with Sp1 and CBP,which are associated with PAI-1 gene's regulation in GMCs.
