嘌呤霉素致足细胞损伤细胞模型的建立

Establishment of a podocyte cell injury model induced by puromycin aminonucleoside

目的:建立小鼠足细胞损伤的细胞模型,为进一步从细胞、分子水平研究足细胞的生物学作用,以及足细胞特别是裂孔隔膜(slit diaphragm)分子在蛋白尿发生、发展中的分子机制提供稳定、可靠的足细胞损伤模型。方法:应用不同浓度的嘌呤霉素(15mg/L、45mg/L和75mg/L)作用于足细胞,通过流式细胞仪检测作用24h和48h后足细胞的凋亡情况,以确定嘌呤霉素的作用浓度和时间,建立足细胞损伤的细胞模型;并以噻唑蓝(MTT)检测足细胞损伤后的活力,应用免疫荧光染色观察裂孔隔膜关键分子Nephrin、Podocin以及骨架蛋白F-actin在足细胞损伤中的分布来评价足细胞损伤。结果:45mg/L嘌呤霉素作用48h以及75mg/L嘌呤霉素作用24h或48h,足细胞明显凋亡。结论:嘌呤霉素可以呈时间和剂量依赖性诱导小鼠足细胞凋亡,45mg/L嘌呤霉素作用48h后诱导的足细胞损伤模型稳定、可靠,可应用于足细胞损伤的研究。

Objective：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. Methods： MPC5 were treated for 24 and 48 hours by 15, 45 and 75 mg/L PAN, respectively. The podoeyte 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-immnofluorescent staining under confocal microscope. The distribution of F-actin was revealed with direct-immnofluorescent staining under microscope. Results ： 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 ）. Conclusion： We set up an in vitro podocytc injury model treated with PAN for the first time, This reliable cell model is a good basis for further studies on podocyte injury.