CAJ | 학술논문

目的观察嘌呤霉素(PAN)和地塞米松(DEX)对足细胞分子podocin表达和分布的影响,探讨DEX改善蛋白尿的机制.方法体外培养小鼠足细胞(MPC5),分为对照组、PAN组和DEX组.对照组用含0.02%DMSO的RPMI-1640培养液培养;PAN组加入PAN;DEX组同时加入PAN和DEX.处理后8 h、24 h和48 h,观察细胞形态并摄像,用图像处理软件分析各组细胞形态及胞体面积的差异.用RT-PCR、Western印迹和间接免疫荧光染色榆测各时间点pedocin mRNA和蛋白的表达及分布.结果正常足细胞呈星形,胞体大并有树样突起,细胞相互连接.PAN刺激8 h,足细胞胞体面积缩小,为对照组的43%;24 h为10%;48h为5.7%(P<0.01);部分足细胞足突及细胞连接消失.DEX组在8 h、24 h和48 h,足细胞胞体面积显著大于PAN组,分别为对照组的43.9%、26.2%及29.6%(P<0.05),足突形态正常.PAN组podocin mRNA表达量呈下降趋势,蛋白表达量显著降低(P<0.01),分布异常.DEX组podocin mRNA和蛋白的表达量及分布与对照组相似,48 h时mRNA和蛋白的表达量均显著高于PAN组(P<0.05).结论 DEX直接作用于足细胞,稳定pedocin mRNA和蛋白的表达量及分布,该作用可能与其能保护足细胞及改善蛋白尿有关.
목적 관찰표령매소(PAN)화지새미송(DEX)대족세포분자podocin표체화분포적영향,탐토DEX개선단백뇨적궤제.방법 체외배양소서족세포(MPC5),분위대조조、PAN조화DEX조.대조조용함0.02%DMSO적RPMI-1640배양액배양;PAN조가입PAN;DEX조동시가입PAN화DEX.처리후8 h、24 h화48 h,관찰세포형태병섭상,용도상처리연건분석각조세포형태급포체면적적차이.용RT-PCR、Western인적화간접면역형광염색유측각시간점pedocin mRNA화단백적표체급분포.결과 정상족세포정성형,포체대병유수양돌기,세포상호련접.PAN자격8 h,족세포포체면적축소,위대조조적43%;24 h위10%;48h위5.7%(P<0.01);부분족세포족돌급세포련접소실.DEX조재8 h、24 h화48 h,족세포포체면적현저대우PAN조,분별위대조조적43.9%、26.2%급29.6%(P<0.05),족돌형태정상.PAN조podocin mRNA표체량정하강추세,단백표체량현저강저(P<0.01),분포이상.DEX조podocin mRNA화단백적표체량급분포여대조조상사,48 h시mRNA화단백적표체량균현저고우PAN조(P<0.05).결론 DEX직접작용우족세포,은정pedocin mRNA화단백적표체량급분포,해작용가능여기능보호족세포급개선단백뇨유관.
Objective To observe the effects of puromycin aminonucleoside (PAN) and dexamethasone (DEX) on the expression and distribution of pedocin in vitro, and to explore the possible mechanism of DEX in improving proteinuria. Methods Mouse podecyte cells (MPCs) in control group were cultured with RPMI-1640 plus 0.02% DMSO, and were subjected to PAN treatment alone (PAN group) or PAN plus DEX (DEX group) for 8, 24,48 hours respectively. The pedocyte morphology was observed by phase-contrast microscope, and was analyzed by Image J. The distribution, mRNA and protein expression of podocin were detected by indirect immunocytofluorescence, semi-quantitative RT-PCR and Western blot, respectively. Results The well-developed arborization and interconnection of podocytes were found in control group. PAN treatment led to significant shrinkage of pedocytes with decreased distribution at 43% of control group at 8 h, 10% at 24 h and 5.7% at 48 h (P<0.01), respectively, together with podocyte foot process retraction as well as effacement and loss of cell contact. RT-PCR revealed podoein mRNA expression prone to decrease. Western blot showed podoein protein expression was significantly decreased and immunocytochemistry revealed podoein expression was disappeared in the cellular membrane after PAN treatment. DEX significantly prevented the shrinkage of podcytes, with decreased area at 43.9% of control at 8 h, 26.2% at 24 h and 29.6% at 48 h (P<0.05), respectively, and up-regulated the mRNA and protein expression of podocin at 48 h (P<0.05). The abnormal distribution of podocin was also alleviated by DEX. Conclusion DEX exerts a direct action on podocyte via stabilizing mRNA, protein expression and distribution of podocin, which may be associated with the improvement of proteinuria.