中华肾脏病杂志
中華腎髒病雜誌
중화신장병잡지
2010年
6期
442-447
,共6页
刘春艳%梅长林%袁莉%张懿%付莉莉%蔡厚安
劉春豔%梅長林%袁莉%張懿%付莉莉%蔡厚安
류춘염%매장림%원리%장의%부리리%채후안
多囊肾,常染色体显性%细胞周期%细胞凋亡%罗格列酮%哺乳类动物的雷帕霉素靶蛋白%p70S6K
多囊腎,常染色體顯性%細胞週期%細胞凋亡%囉格列酮%哺乳類動物的雷帕黴素靶蛋白%p70S6K
다낭신,상염색체현성%세포주기%세포조망%라격렬동%포유류동물적뢰파매소파단백%p70S6K
Polycystic kidney,autosomal dominant%Cell cycle%Apoptosis%Rosiglitazone%Mammalian target of rapamycin%p70S6K
目的 探讨过氧化物酶体增殖物激活受体γ(PPARγ)激动剂罗格列酮对常染色体显性遗传性多囊肾病(ADPKD)囊肿衬里上皮细胞增殖的抑制作用及其机制.方法 MTT法检测罗格列酮对ADPKD囊肿衬里上皮细胞系(WT9-12)细胞增殖的作用;流式细胞术检测罗格列酮对WT9-12细胞周期及凋亡的影响;Western印迹法检测罗格列酮对哺乳类动物的雷帕霉素靶蛋白(mTOR)-p70核糖体S6激酶(p70S6K)信号通路的影响.给予PPARγ特异性抑制剂GW9662及PPARγsiRNA瞬时转染WT9-12细胞,检测罗格列酮对细胞增殖及对mTOR信号通路的作用是否为PPARy依赖性的.结果 罗格列酮抑制WT9-12细胞增殖,此作用在0~200 μmol/L范围内呈剂量依赖性及时间依赖性,作用72 h的50%抑制率浓度为100μmol/L.细胞周期分析显示,罗格列酮(50、100 μmol/L)作用后,G0/G1期细胞较对照组显著增多(65.43%、64.02%比49.65%,P<0.05).50、100 μmol/L罗格列酮对细胞凋亡影响不大,高浓度(200 μmol/L)罗格列酮可使凋亡细胞达6%(正常对照组为4%).罗格列酮可呈时间及剂量依赖性下调WT9-12细胞p70S6K磷酸化表达,而对mTOR及其另一个下游4E结合蛋白1(4E-BP1)磷酸化水平无明显影响.加入GW9662及转染PPARγsiRNA阻断PPARγ表达后,可部分阻断罗格列酮对p70S6K磷酸化的影响(P<0.01).结论 罗格列酮可抑制ADPKD囊肿衬里上皮细胞增殖和阻滞细胞周期,可不依赖于mTOR途径直接下调p70S6K磷酸化表达.罗格列酮对ADPKD囊肿衬里上皮细胞p70S6K磷酸化的影响是PPARγ依赖性的.
目的 探討過氧化物酶體增殖物激活受體γ(PPARγ)激動劑囉格列酮對常染色體顯性遺傳性多囊腎病(ADPKD)囊腫襯裏上皮細胞增殖的抑製作用及其機製.方法 MTT法檢測囉格列酮對ADPKD囊腫襯裏上皮細胞繫(WT9-12)細胞增殖的作用;流式細胞術檢測囉格列酮對WT9-12細胞週期及凋亡的影響;Western印跡法檢測囉格列酮對哺乳類動物的雷帕黴素靶蛋白(mTOR)-p70覈糖體S6激酶(p70S6K)信號通路的影響.給予PPARγ特異性抑製劑GW9662及PPARγsiRNA瞬時轉染WT9-12細胞,檢測囉格列酮對細胞增殖及對mTOR信號通路的作用是否為PPARy依賴性的.結果 囉格列酮抑製WT9-12細胞增殖,此作用在0~200 μmol/L範圍內呈劑量依賴性及時間依賴性,作用72 h的50%抑製率濃度為100μmol/L.細胞週期分析顯示,囉格列酮(50、100 μmol/L)作用後,G0/G1期細胞較對照組顯著增多(65.43%、64.02%比49.65%,P<0.05).50、100 μmol/L囉格列酮對細胞凋亡影響不大,高濃度(200 μmol/L)囉格列酮可使凋亡細胞達6%(正常對照組為4%).囉格列酮可呈時間及劑量依賴性下調WT9-12細胞p70S6K燐痠化錶達,而對mTOR及其另一箇下遊4E結閤蛋白1(4E-BP1)燐痠化水平無明顯影響.加入GW9662及轉染PPARγsiRNA阻斷PPARγ錶達後,可部分阻斷囉格列酮對p70S6K燐痠化的影響(P<0.01).結論 囉格列酮可抑製ADPKD囊腫襯裏上皮細胞增殖和阻滯細胞週期,可不依賴于mTOR途徑直接下調p70S6K燐痠化錶達.囉格列酮對ADPKD囊腫襯裏上皮細胞p70S6K燐痠化的影響是PPARγ依賴性的.
목적 탐토과양화물매체증식물격활수체γ(PPARγ)격동제라격렬동대상염색체현성유전성다낭신병(ADPKD)낭종츤리상피세포증식적억제작용급기궤제.방법 MTT법검측라격렬동대ADPKD낭종츤리상피세포계(WT9-12)세포증식적작용;류식세포술검측라격렬동대WT9-12세포주기급조망적영향;Western인적법검측라격렬동대포유류동물적뢰파매소파단백(mTOR)-p70핵당체S6격매(p70S6K)신호통로적영향.급여PPARγ특이성억제제GW9662급PPARγsiRNA순시전염WT9-12세포,검측라격렬동대세포증식급대mTOR신호통로적작용시부위PPARy의뢰성적.결과 라격렬동억제WT9-12세포증식,차작용재0~200 μmol/L범위내정제량의뢰성급시간의뢰성,작용72 h적50%억제솔농도위100μmol/L.세포주기분석현시,라격렬동(50、100 μmol/L)작용후,G0/G1기세포교대조조현저증다(65.43%、64.02%비49.65%,P<0.05).50、100 μmol/L라격렬동대세포조망영향불대,고농도(200 μmol/L)라격렬동가사조망세포체6%(정상대조조위4%).라격렬동가정시간급제량의뢰성하조WT9-12세포p70S6K린산화표체,이대mTOR급기령일개하유4E결합단백1(4E-BP1)린산화수평무명현영향.가입GW9662급전염PPARγsiRNA조단PPARγ표체후,가부분조단라격렬동대p70S6K린산화적영향(P<0.01).결론 라격렬동가억제ADPKD낭종츤리상피세포증식화조체세포주기,가불의뢰우mTOR도경직접하조p70S6K린산화표체.라격렬동대ADPKD낭종츤리상피세포p70S6K린산화적영향시PPARγ의뢰성적.
Objective To investigate the antiproliferative effect of rosiglitazone, a thiazolidinedione (TZD) on autosomal dominant polycystic kidney disease (ADPKD) cystic lining epithelial cells and to explore the underlying molecular mechanism. Methods ADPKD cysticlining immortalized epithelial (WT9-12) cells were stimulated by rosiglitazone with different concentrations. After treatment, MTT method was performed to detect the level of proliferation; flow cytometry was used to determine the cell cycle distribution and the apoptosis rate. Western blotting was used to detect the protein expressions of mTOR, p70S6K, 4E-Bp1, PPARγ PPARγ siRNA was transfected into WT9-12 cells to knock down the expression of PPARγ Results Treatment of WT9-12 cells with rosiglitazone resulted in a dose-dependent and time-dependent strong inhibition of cell proliferation, an accumulation of cells in the G0/G1 phase (rosiglitazone 50 μmol/L 65.43%,rosiglitazone 100 μmol/L 64.02%, control 49.65% ) and 6% apoptosis at high concentration (rosiglitazone 200 μmol/L). Rosiglitazone reduced the phosphorylation of p70S6K in a dosedependent and time-dependent manner. The levels of phosphorylated mTOR and 4E-Bp1, the latter being a downstream substrate of mTOR related mRNA translation initiation, were not changed by rosiglitazone. Cells were pre-incubated with GW9662, a PPARγ antagonist, before the treatment with rosiglitazone, the inhibition of p70S6 kinase phosphorylation by rosiglitazone was partially prevented by GW9662 (P<0.01). Then PPARγ siRNA was transfected into WT9-12 cells, in contrast to untransfected control or cells transfected with an irrelevant siRNA, rosiglitazone did not cause an obvious inhibition of p70S6 kinase phosphorylation in PPARγ knock-down.Conclusion Rosiglitazone inhibits the proliferation of ADPKD cystic lining epithelial cells, and down-regulates p70S6 kinase phosphorylation through mTOR-independent and PPARγ-dependent signal pathway.