Liu Shu Fang, Ding Jie, Fan Qing Feng, Zhang Han
Department of Pediatrics, Peking University First Hospital, Beijing, China.
Beijing Da Xue Xue Bao Yi Xue Ban. 2008 Dec 18;40(6):586-9.
To establish a podocyte cell injury model induced by puromycin aminonucleoside (PAN), an in vitro model for studying the role of podocytes, especial the slit diaphragm molecules in proteinuria at the cellular and molecular levels.
MPC5 were treated for 24 and 48 hours by 15, 45 and 75 mg/L PAN, respectively. The podocyte molecular behavior during podocyte injury was evaluated: the apoptotic podocyte cells were revealed with FITC-Annexin V and Propidium Iodide (PI) assay and the proliferative podocyte cells detected with MTT assay after PAN treatment. The distribution of Nephrin and Podocin was revealed with indirect-immunofluorescent staining under confocal microscope. The distribution of F-actin was revealed with direct-immunofluorescent staining under microscope.
The percentage of apoptotic podocyte cells was increased in a dose- and time-dependent manner after PAN treatment. In PAN-treated group, the apoptosis was obviously increased at hour 48, the PAN-45 treated group was 33.48% +/- 14.55% and PAN-75 treated group 38.01% +/- 12.13% vs the control group 6.38% +/- 0.50% (P<0.01).
We set up an in vitro podocyte injury model treated with PAN for the first time. This reliable cell model is a good basis for further studies on podocyte injury.
建立嘌呤霉素氨基核苷（PAN）诱导的足细胞损伤模型，一种在细胞和分子水平上研究足细胞作用，特别是裂孔隔膜分子在蛋白尿中作用的体外模型。
分别用15、45和75mg/L的PAN处理MPC5细胞24小时和48小时。评估足细胞损伤过程中的足细胞分子行为：用FITC-Annexin V和碘化丙啶（PI）检测法检测凋亡的足细胞，用MTT检测法检测PAN处理后增殖的足细胞。在共聚焦显微镜下用间接免疫荧光染色显示Nephrin和Podocin的分布。在显微镜下用直接免疫荧光染色显示F-肌动蛋白的分布。
PAN处理后，凋亡足细胞的百分比呈剂量和时间依赖性增加。在PAN处理组中，48小时时凋亡明显增加，PAN-45处理组为33.48%±14.55%，PAN-75处理组为38.01%±12.13%，而对照组为6.38%±0.50%（P<0.01）。
我们首次建立了用PAN处理的体外足细胞损伤模型。这个可靠的细胞模型是进一步研究足细胞损伤的良好基础。
