中国综合临床
中國綜閤臨床
중국종합림상
CLINICAL MEDICINE OF CHINA
2011年
10期
1049-1054
,共6页
酒精性肝病%内毒素%importinα%NF-κBp50%人工多肽cSN50
酒精性肝病%內毒素%importinα%NF-κBp50%人工多肽cSN50
주정성간병%내독소%importinα%NF-κBp50%인공다태cSN50
Alcoholic liver disease%Endotoxin%Importin α%NF-κB%cSN50
目的 研究乙醇、内毒素是否通过核转录因子NF-kappaB即IκB-NF-κB-importinα途径引起细胞炎症、凋亡和诱导下游基因TNF-α、Caspase-3的表达；cSN50作为一种穿膜多肽通过阻断NF-κB与importinα结合,从而抑制NF-κB核转位及其下游基因的表达,起到保护细胞的作用.方法 应用流式细胞仪观察HepG2经不同浓度乙醇、内毒素刺激后的细胞凋亡率；分光光度计法测Caspase-3活性；ELISA法检测细胞培养上清TNF-α；Western blot法检测NF-κB p50、importinα3、IκBα,间接免疫荧光法测p50、IκBα、importinα3的活化水平.结果 乙醇、内毒素刺激后TNF-α、Caspase-3的值量效上与空白对照组比差异均有统计学意义(P均＜0.05),细胞核内p50显著增加(P＜0.05),胞浆IκBα下降(P＜0.05),cSN50( 100 μmol/L)能部分阻断P50的核转位及其下游因子的表达(P＜0.05).结论 急性酒精肝损伤中,NF-κB的核转位在此损伤中起重要作用,cSN50能部分抑制被刺激后的HepG2细胞核中NF-κB活性及其下游基因的表达.
目的 研究乙醇、內毒素是否通過覈轉錄因子NF-kappaB即IκB-NF-κB-importinα途徑引起細胞炎癥、凋亡和誘導下遊基因TNF-α、Caspase-3的錶達；cSN50作為一種穿膜多肽通過阻斷NF-κB與importinα結閤,從而抑製NF-κB覈轉位及其下遊基因的錶達,起到保護細胞的作用.方法 應用流式細胞儀觀察HepG2經不同濃度乙醇、內毒素刺激後的細胞凋亡率；分光光度計法測Caspase-3活性；ELISA法檢測細胞培養上清TNF-α；Western blot法檢測NF-κB p50、importinα3、IκBα,間接免疫熒光法測p50、IκBα、importinα3的活化水平.結果 乙醇、內毒素刺激後TNF-α、Caspase-3的值量效上與空白對照組比差異均有統計學意義(P均＜0.05),細胞覈內p50顯著增加(P＜0.05),胞漿IκBα下降(P＜0.05),cSN50( 100 μmol/L)能部分阻斷P50的覈轉位及其下遊因子的錶達(P＜0.05).結論 急性酒精肝損傷中,NF-κB的覈轉位在此損傷中起重要作用,cSN50能部分抑製被刺激後的HepG2細胞覈中NF-κB活性及其下遊基因的錶達.
목적 연구을순、내독소시부통과핵전록인자NF-kappaB즉IκB-NF-κB-importinα도경인기세포염증、조망화유도하유기인TNF-α、Caspase-3적표체；cSN50작위일충천막다태통과조단NF-κB여importinα결합,종이억제NF-κB핵전위급기하유기인적표체,기도보호세포적작용.방법 응용류식세포의관찰HepG2경불동농도을순、내독소자격후적세포조망솔；분광광도계법측Caspase-3활성；ELISA법검측세포배양상청TNF-α；Western blot법검측NF-κB p50、importinα3、IκBα,간접면역형광법측p50、IκBα、importinα3적활화수평.결과 을순、내독소자격후TNF-α、Caspase-3적치량효상여공백대조조비차이균유통계학의의(P균＜0.05),세포핵내p50현저증가(P＜0.05),포장IκBα하강(P＜0.05),cSN50( 100 μmol/L)능부분조단P50적핵전위급기하유인자적표체(P＜0.05).결론 급성주정간손상중,NF-κB적핵전위재차손상중기중요작용,cSN50능부분억제피자격후적HepG2세포핵중NF-κB활성급기하유기인적표체.
Objective To investigate whether polypeptide cSN50,as a transmembrane peptides,can inhibit NF-κB nuclear translocation and its downstream gene expression to play a role to protect cells by blocking the combination of NF-κB with the importinα3 during the alcohol and endotoxin-induced inflammation and apoptosis.Methods Flow cytometry method was used to observe the apoptosis rate of HepG2 by different concentration of alcohol and/or endotoxin.Spectrophotometer method was used to detect Caspase-3 activity.TNF- α in cell culture supernatant was detected by ELISA assay.p50,importinα3,and IκBαunder the stimulation of alcohol and/or endotoxin at selected optimal concentration were detected by Western blot.Indirect immunofluorescence assay was used to measure the activation of p50,IκBα,and importinα3.Results The change of TNF-α and Caspase-3 in the dose-effect course,compared with control group,was significant (P ＜0.05 ).p50 was increased in the nucleus ( P ＜ 0.05 ),and IκBα was decreased in the cytoplasm ( P ＜ 0.05 ).cSN50( 100 μmol/L) partially blocked nuclear translocation of p50 and its downstream factor( P ＜ 0.05 ).Conclusion NF-κB nuclear translocation may play an important role in acute alcoholic liver injury.cSN50 can effectively inhibit NF-κB activity and its downstream gene expression in HepG2 cells.
