CAJ | 학술논문

目的观察甲状旁腺激素(PTH)对人肾小管上皮细胞(HK-2)间充质转分化的影响,探讨Wnt信号通路在其中的作用.方法细胞被分为对照组和PTH处理组.实时定量-聚合酶链反应(RT-PCR)和Western印迹法检测α平滑肌肌动蛋白(α-SMA)及钙黏素E(E-cadherin) mRNA及蛋白的表达;10-10mol/L PTH作用细胞48 h后,采用实时定量PCR芯片技术检测Wnt通路相关基因表达.不同浓度PTH作用后和10-10 mol/L PTH作用不同时间后,用RT-PCR、Western印迹法检测Wnt4 mRNA及蛋白的表达.构建Wnt4过表达及敲除Wnt4的细胞模型,用RT-PCR、Western印迹法检测α-SMA、E-cadherin、Wnt4 mRNA及蛋白质表达.结果与PTH处理组相比,PTH+DKK1组α-SMA mRNA和蛋白表达量明显降低;E-cadherinmRNA及蛋白表达量明显升高(均P＜0.01).PTH处理组细胞有18个Wnt信号通路基因显著上调和9个基因下调;与对照组相比,PTH处理组细胞Wnt4 mRNA和蛋白表达量明显增加(均P＜0.01);与PTH处理组相比,PTH+Wnt4过表达组α-SMA mRNA和蛋白表达量明显上调,E-cadherin mRNA和蛋白表达量明显下调(均P＜0.05).与PTH处理组相比,PTH+Wnt4 siRNA组α-SMA mRNA和蛋白表达量降低,E-cadherin mRNA和蛋白相对表达量升高(均P＜0.05).结论甲状旁腺激素诱导HK-2细胞间充质转分化.其机制可能与PTH激活Wnt信号通路,尤其Wnt4信号通路有关.
목적 관찰갑상방선격소(PTH)대인신소관상피세포(HK-2)간충질전분화적영향,탐토Wnt신호통로재기중적작용.방법 세포피분위대조조화PTH처리조.실시정량-취합매련반응(RT-PCR)화Western인적법검측α평활기기동단백(α-SMA)급개점소E(E-cadherin) mRNA급단백적표체;10-10mol/L PTH작용세포48 h후,채용실시정량PCR심편기술검측Wnt통로상관기인표체.불동농도PTH작용후화10-10 mol/L PTH작용불동시간후,용RT-PCR、Western인적법검측Wnt4 mRNA급단백적표체.구건Wnt4과표체급고제Wnt4적세포모형,용RT-PCR、Western인적법검측α-SMA、E-cadherin、Wnt4 mRNA급단백질표체.결과 여PTH처리조상비,PTH+DKK1조α-SMA mRNA화단백표체량명현강저;E-cadherinmRNA급단백표체량명현승고(균P＜0.01).PTH처리조세포유18개Wnt신호통로기인현저상조화9개기인하조;여대조조상비,PTH처리조세포Wnt4 mRNA화단백표체량명현증가(균P＜0.01);여PTH처리조상비,PTH+Wnt4과표체조α-SMA mRNA화단백표체량명현상조,E-cadherin mRNA화단백표체량명현하조(균P＜0.05).여PTH처리조상비,PTH+Wnt4 siRNA조α-SMA mRNA화단백표체량강저,E-cadherin mRNA화단백상대표체량승고(균P＜0.05).결론 갑상방선격소유도HK-2세포간충질전분화.기궤제가능여PTH격활Wnt신호통로,우기Wnt4신호통로유관.
Objective To observe the effect of parathyroid hormone on transdifferentiation of human renal proximal tubular epithelial cell (HK-2),and to investigate the role of Wnt signaling pathway in this process.Methods The expression of α-SMA,E-cadherin mRNA and protein was detected by real-time PCR and Western blotting.After the induction of 10-10 mol/L PTH for 48 h,the Wnt pathway associated gene expression profiling was detected by quantitative PCR-microarray.The expression of Wnt4 mRNA and protein under various concentrations of PTH or after exposed to 10-10 mol/L PTH for different time was detected by real-time PCR and Western blotting.The overexpression and knock-down plasmids of Wnt4 were constructed and the expression of α-SMA,E-cadherin,Wnt4 mRNA and protein was detected by real-time PCR and western blotting after overexpression and knockdown of Wnt4.Results Compared with PTH group,the expression of α-SMA mRNA and protein in PTH+DKK1 group was significantly down-regulated,while E-cadherin mRNA and protein expression was significantly up-regulated (all P ＜ 0.01).PTH treatment resulted in the up-regulation of 18 genes and down-regulation of 9 genes associated with Wnt pathway.Compared with control group,the expression of Wnt4 mRNA and protein increased markedly in PTH group (all P ＜ 0.01).The expression of α-SMA mRNA and protein was significantly up-regulated and E-cadherin mRNA and protein expression was significantly down-regulated after overexpression of Wnt4 and PTH treatment (all P ＜ 0.05),while the expression of α-SMA mRNA and protein was significantly down-regulted and E -cadherin mRNA and protein expression was significantly down-regulated after knockdown of Wnt4 and PTH treatment (all P ＜ 0.05).Conclusions PTH-induced EMT in HK-2 cells is mediated by Wnt signal pathway,and Wnt4 might be a key gene during PTH-induced EMT.