CAJ | 학술논문

目的观察新型过氧化物酶体增殖物激活受体(PPARγ)激动剂DH9对人多囊肾囊肿衬里上皮细胞(WT9-12)中Wnt-β连环素(catenin)信号通路的影响.方法予以不同浓度的DH9作用WT9-12细胞72 h,MTT法检测细胞增殖情况;RT-PCR技术检测DH9干预后β-catenin mRNA水平.予GSK3β抑制剂(SB216763)或PPARγ抑制剂GW9662预处理WT9-12细胞后,再予以60 μmol/L DH9或单给DH9干预,Western印迹分别分析β-catenin、磷酸化β-catenin、GSK3β、磷酸化GSK3β蛋白水平.结果 DH9对WT9-12细胞增殖有抑制作用,60μmol/L DH9干预72 h对细胞的增殖抑制达50％,并随着干预时间延长,抑制率增高.60μmol/L DH9可下调β-catenin的蛋白表达(P＜0.01),上调磷酸化β-catenin的蛋白表达(P＜0.01),具有剂量依赖性.而DH9对β-catenin的mRNA水平无明显改变.DH9可上调磷酸化GSK3β的蛋白表达(P＜0.01),但对GSK3β的表达无明显影响.在SB216763预处理WT9-12细胞与DH9共处理72 h后,发现可逆转DH9对β-catenin的下调作用(P＜0.01),但GW9662对DH9下凋β-catenin的作用无明显改变.结论 DH9抑制WT9-12细胞的增殖具有时间和剂量依赖性,并且这种增殖抑制作用可通过GSK3β依赖的方式下调β-catenin的蛋白表达从而抑制Wnt-β-catenin信号通路实现,与PPARγ途径无关.
목적 관찰신형과양화물매체증식물격활수체(PPARγ)격동제DH9대인다낭신낭종츤리상피세포(WT9-12)중Wnt-β련배소(catenin)신호통로적영향.방법 여이불동농도적DH9작용WT9-12세포72 h,MTT법검측세포증식정황;RT-PCR기술검측DH9간예후β-catenin mRNA수평.여GSK3β억제제(SB216763)혹PPARγ억제제GW9662예처리WT9-12세포후,재여이60 μmol/L DH9혹단급DH9간예,Western인적분별분석β-catenin、린산화β-catenin、GSK3β、린산화GSK3β단백수평.결과 DH9대WT9-12세포증식유억제작용,60μmol/L DH9간예72 h대세포적증식억제체50％,병수착간예시간연장,억제솔증고.60μmol/L DH9가하조β-catenin적단백표체(P＜0.01),상조린산화β-catenin적단백표체(P＜0.01),구유제량의뢰성.이DH9대β-catenin적mRNA수평무명현개변.DH9가상조린산화GSK3β적단백표체(P＜0.01),단대GSK3β적표체무명현영향.재SB216763예처리WT9-12세포여DH9공처리72 h후,발현가역전DH9대β-catenin적하조작용(P＜0.01),단GW9662대DH9하조β-catenin적작용무명현개변.결론 DH9억제WT9-12세포적증식구유시간화제량의뢰성,병차저충증식억제작용가통과GSK3β의뢰적방식하조β-catenin적단백표체종이억제Wnt-β-catenin신호통로실현,여PPARγ도경무관.
Objective To investigate the effects of a novel PPARγ agonist DH9 on Wntβ-catenin pathway in human polycystic kidney cystic-lining epithelial cells (WT9-12).Methods WT9-12 cells were treated with different concentrations of DH9 for 72 hours and the proliferation was assessed by MTT.WT9-12 cells were pretreated with SB216763 or GW9662 for two hours and then treated with DH9 for 72 hours.Western blotting was applied to detect the protein expression of β-catenin,phospho-β-catenin,GSK3β,phospho-GSK3β.Results DH9 could effectively inhibit the proliferation of the cells.60 μmol/L DH9 could facilitate β-catenin down-regulation (P＜0.01) and phospho-β-catenin up-regulation (P＜0.01).Inhibition of GSK3β by SB216763 could protect WT9-12 cells against DH9-facilitated β-catenin repression in a dose-dependent manner despite phosphorylating deactivation,but PPARγ inhibitor GW9662 couldn't.Conclusions DH9can effectively block the proliferation of WT9-12 cells.The effect may be mediated by facilitating the down-regulation of β-catenin via GSK3β-dependent mechanism.