CAJ | 학술논문

目的探讨铜蓝蛋白(Ceruloplasmin,Cp)在石英诱导的人胚肺成纤维细胞(HELF)中PI3K/PTEN信号通路改变中的作用.方法建立稳定转染空载体pGenesil1.1的HELF细胞及pGenesil 1.1与shRNA PTEN质粒共转染的HELF细胞(简称PT),复苏转染p85显性失活突变体质粒的HELF细胞(简称DN-p85).实验中石英浓度为100 μg/ml,Cp浓度为10、20、30 μg/ml,Cp在石英作用1h后加入.在HELF、PT和DN-p85 3个不同细胞系中,分别设立对照组、石英染毒组、石英+不同浓度Cp组.用噻唑蓝(MTT)比色法观察分别抑制PTEN和p85后,Cp对石英诱导的HELF细胞增殖的影响.蛋白免疫印迹(Western blot)实验检测PTEN、p85、p110、AKT及AKT308位点和AKT473位点磷酸化水平、总ERK、JNK及其磷酸化水平的变化.结果抑制PTEN后,100 μgml石英与30 μg/ml Cp共同诱导的p85蛋白水平增加被明显抑制(P＜0.05),p110蛋白水平无变化(P＞0.05).抑制p85后,石英以及石英+不同浓度Cp诱导的细胞增殖加快被抑制;100 μg/ml石英与30 μg/ml Cp共同诱导的AKT308位及AKT473位磷酸化水平、总ERK、磷酸化ERK和磷酸化JNK水平的增加低于相应的对照组(P＜0.05),但是总JNK水平的变化没有统计学差异(P＞0.05).结论 Cp通过PI3K/AKT/MAPK信号通路参与石英诱导的HELF细胞增殖,PTEN可以影响PI3K调节亚单位p85蛋白水平.
목적 탐토동람단백(Ceruloplasmin,Cp)재석영유도적인배폐성섬유세포(HELF)중PI3K/PTEN신호통로개변중적작용.방법 건립은정전염공재체pGenesil1.1적HELF세포급pGenesil 1.1여shRNA PTEN질립공전염적HELF세포(간칭PT),복소전염p85현성실활돌변체질립적HELF세포(간칭DN-p85).실험중석영농도위100 μg/ml,Cp농도위10、20、30 μg/ml,Cp재석영작용1h후가입.재HELF、PT화DN-p85 3개불동세포계중,분별설립대조조、석영염독조、석영+불동농도Cp조.용새서람(MTT)비색법관찰분별억제PTEN화p85후,Cp대석영유도적HELF세포증식적영향.단백면역인적(Western blot)실험검측PTEN、p85、p110、AKT급AKT308위점화AKT473위점린산화수평、총ERK、JNK급기린산화수평적변화.결과 억제PTEN후,100 μgml석영여30 μg/ml Cp공동유도적p85단백수평증가피명현억제(P＜0.05),p110단백수평무변화(P＞0.05).억제p85후,석영이급석영+불동농도Cp유도적세포증식가쾌피억제;100 μg/ml석영여30 μg/ml Cp공동유도적AKT308위급AKT473위린산화수평、총ERK、린산화ERK화린산화JNK수평적증가저우상응적대조조(P＜0.05),단시총JNK수평적변화몰유통계학차이(P＞0.05).결론 Cp통과PI3K/AKT/MAPK신호통로삼여석영유도적HELF세포증식,PTEN가이영향PI3K조절아단위p85단백수평.
Objective To investigate the roles of ceruloplasmin (Cp) in PI3K/PTEN cell signaling pathway change in human embryonic lung fibroblasts (HELFs) induced by silica.Methods HELFs transfected with pGenesil1.1 plasmid and pGenesil1.1 with PTEN shRNA (PT) plasmid were successfully established.100 μg/ml silica and different concentrations of Cp (10、20、30 μg/ml) were used in this experiment and Cp were treated cells after exposed to silica for 1 h.Three different cell lines (including HELFs,PT and cells were transfected with p85 dominant negative mutant plasmid (DN-p85)) were divided into control groups,silica groups and silica+different concentrations of Cp groups.MTT assay was used to dctect the effects of Cp on silica-induced cell proliferation after inhibiting PTEN and p85.When supressing the expression of PTEN and p85,western blot assay was performed to detect the levels of p85,pll0,AKT308,AKT473 and ERK,JNK and their phosphorylated levels.Results After inhibition of PTEN,the high levels of p85 induced by 100 μg/ml silica with 30 μg/ml Cp were markedly decreased (P＜0.05).When suppressing p85,the increased cell proliferation was not observed.And the high levels of AKT308,AKT473,ERK and phosphorylated JNK and ERK stimulated by 100 μg/ml silica with 30 μg/ml Cp were decreased (P＜0.05).Conclusion Cp could further strengthened silica-induced cell proliferation by PI3K/AKT/MAPK cell signaling pathway,of which the level of p85 was regulated by PTEN.