CAJ | 학술논문

目的以嘌呤霉素(PAN)损伤足细胞,以地塞米松(DEX)干预,观察不同时间点瞬时受体电位阳离子通道蛋白6(TRPC6)表达和分布的变化,探讨在足细胞损伤过程中,DEX对TRPC6表达与分布的影响及与足细胞损伤的关系.方法体外培养足细胞,设立对照组、PAN刺激组和DEX干预组.对照组用含0.02％二甲亚砜(DMSO)的RPMI 1640培养液培养,PAN刺激组加入PAN(50 μg/ml),DEX干预组同时加入PAN(50 μg/ml)和溶解于0.02％ DMSO的DEX(1 μmol/L),处理8h、24 h、48 h后观察细胞形态并拍摄,采用图像处理软件分析各组细胞形态及胞体面积的差别.采用流式细胞仪检测足细胞凋亡率,采用实时荧光定量PCR、Western印迹和间接免疫荧光染色检测各时间点TRPC6的mRNA、蛋白表达及分布变化.结果经PAN刺激后细胞体显著缩小(P＜0.05),足突回缩、消失,细胞间失去相互连接,刺激后48 h足细胞凋亡率显著升高(P＜0.05).PAN刺激组在刺激24 h和48 h后TRPC6 mRNA及蛋白表达量显著上升(P＜0.05),TRPC6在足细胞上的分布亦出现异常.DEX干预后细胞胞体显著大于PAN刺激组(P＜0.05),可维持正常的足突形态;干预48 h后足细胞凋亡率显著低于PAN刺激组,TRPC6 mRNA及蛋白的表达、分布均趋于正常水平.结论 DEX通过稳定重要的足细胞分子TRPC6的表达及分布来抑制足细胞损伤,从而发挥保护足细胞和抗蛋白尿的作用.
목적 이표령매소(PAN)손상족세포,이지새미송(DEX)간예,관찰불동시간점순시수체전위양리자통도단백6(TRPC6)표체화분포적변화,탐토재족세포손상과정중,DEX대TRPC6표체여분포적영향급여족세포손상적관계.방법 체외배양족세포,설립대조조、PAN자격조화DEX간예조.대조조용함0.02％이갑아풍(DMSO)적RPMI 1640배양액배양,PAN자격조가입PAN(50 μg/ml),DEX간예조동시가입PAN(50 μg/ml)화용해우0.02％ DMSO적DEX(1 μmol/L),처리8h、24 h、48 h후관찰세포형태병박섭,채용도상처리연건분석각조세포형태급포체면적적차별.채용류식세포의검측족세포조망솔,채용실시형광정량PCR、Western인적화간접면역형광염색검측각시간점TRPC6적mRNA、단백표체급분포변화.결과 경PAN자격후세포체현저축소(P＜0.05),족돌회축、소실,세포간실거상호련접,자격후48 h족세포조망솔현저승고(P＜0.05).PAN자격조재자격24 h화48 h후TRPC6 mRNA급단백표체량현저상승(P＜0.05),TRPC6재족세포상적분포역출현이상.DEX간예후세포포체현저대우PAN자격조(P＜0.05),가유지정상적족돌형태;간예48 h후족세포조망솔현저저우PAN자격조,TRPC6 mRNA급단백적표체、분포균추우정상수평.결론 DEX통과은정중요적족세포분자TRPC6적표체급분포래억제족세포손상,종이발휘보호족세포화항단백뇨적작용.
Objective To observe the changes of foot processes,expression and distribution of transient receptor potential cation channel 6 (TRPC6) in podocytes by puromycin aminonucleoside (PAN) and dexamethasone (DEX) intervention,then to investigate the function of TRPC6 in podocytes and its relation to proteinuria in kidney diseases.Methods Podocytes cultured in vitro were divided into three group:control group,PAN stimulation group and DEX intervention group.Mouse podocyte cell line (MPC5) were cultured in 0.02％ dimethyl sulfoxide (DMSO) in control group,subjected to PAN (50 μg/ml) treatment alone or with DEX (1 μmol/L) in other two groups for 8 h,24 h,48 h.The podocyte morphology was observed and took pictures by phase-contrast microscope,then the differences of morphology and areas were analyzed.The distribution,mRNA expression and protein expression of TRPC6 were detected by indirect immunocyto-fluorescence,real-time quantitative PCR and Western blotting,respectively.Results The well-developed podocyte arborization and interconnection was formed in control group,but PAN led to significant shrinkage of podocytes (P ＜ 0.05),together with podocyte foot process retraction,effacement and loss of cell contact.DEX significantly prevented the shrinkage and apoptosis of podocytes.The apoptosis rate was significantly increased after PAN stimulated 48 h (P ＜ 0.05).Real-time quantitative PCR and Western blotting found TRPC6 mRNA and protein expression were prone to increase in PAN group compared with control group (P ＜ 0.05).The distribution of TRPC6 becamed abnormal in PAN group.DEX decreased TRPC6 mRNA and protein expression at 48 h compared with PAN group (P ＜ 0.05).The abnormal distribution of TRPC6 was also alleviated by the protection of DEX.Conclusion DEX exerts a direct action to podocyte which retains the integrity of slit diaphragm against podocyte injury,and alleviates proteinuria via stabilizing mRNA,protein expression and distribution of TRPC6.