嘌呤霉素损伤小鼠肾小球足细胞系中podocin表达与分布的影响及其意义

doi:10.3877/cma.j.issn.1673-5250.2008.05.103

目的

Podocin是构成肾小球足细胞裂孔隔膜(slit diaphragm，SD)完整性的重要分子，其表达异常与大量蛋白尿的形成有关。本研究采用体外培养肾小球足细胞系，探讨足细胞损伤过程中，podocin mRNA表达和分布变化与足细胞损伤的关系。

方法

将体外培养的足细胞采用随机数字表法设立为实验组与对照组。实验组采用嘌呤霉素(puromycin aminonucleoside，PAN) 刺激(剂量为50 μg/mL), 分别于8 h，24 h和48 h观察细胞形态、提取总RNA和细胞免疫组化观察podocin的分布。对照组用含10% FBS的RPMI 1640培养液培养。细胞图像应用Image J图像处理软件，计算实验组与对照组在上述各时间点200倍镜下随机选择的30个细胞的相对面积，半定量RT－PCR和间接免疫荧光法检测podocin mRNA表达和分布变化。

结果

对照组呈星形伸出树枝状突起，相邻细胞间形成相互连接。实验组细胞体缩小，足突回缩、消失，细胞间失去相互连接。上述各时间点两组间podocin mRNA表达比较，差异无显著意义(P>0.05)。Podocin在对照组呈细丝状均匀分布于细胞核膜、细胞质及细胞膜；在实验组主要沿细胞核膜呈点线样分布，刺激后8 h和24 h细胞质有少许分布，刺激48 h后出现细胞膜、细胞质分布缺失。

结论

Podocin分布异常与足细胞损伤密切相关，损伤早期即有变化；podocin分布异常是足细胞损伤过程中的重要分子效应。

关键词: 足细胞系, podocin, 嘌呤霉素

Objective

To explore the podocin mRNA expression and distribution in a time series scale in the couse of podocyte injury in vitro.

Methods

Mouse podocyte cell line (MPC5) were regenerated at 33℃ for proliferation in the presence of γ-inferon, and then subcultured at 37℃ without γ-inferon for differentiation about 8~10 days, which would be subsequently subject to puromycin aminonucleoside (PAN) treatment. The podocyte morphology was observed by the phase-contrast microscope. The podocin mRNA expression level and distribution were examined by indirect immunocytofluorescence, semi-quantitative RT-PCR at 8 h, 24 h, and 48 h, respectively.

Results

①The well-developed podocyte arborization was formed after the in vitro induction, the PAN treatment led to the podocyte foot processe retraction and effacement together with the MPC5 shrinkage and the loss of cell contact. ② Podocin was evenly distributed in a filamentous pattern with the small dots across the well-differentiated MPC5, which was shifted to an uneven, discontinuous coarse puncta limited in the nuclei immediately after PAN treatment. ③ Semi-quantitative RT-PCR found that mRNA level had no significant changes after PAN treatment (P>0.05); however, immunocytochemistry suggested podocin disappeared in the cellular membrane and cytoplasm significantly (P<0.05).

Conclusion

Because of the intimate concomitance, the changed distribution pattern of podocin was the prominent effect of the podocyte injury, which might be related to the disease mechanism.

Key words: podocyte cell line, podocin, puromycin aminonucleoside(PAN)

图1　总RNA鉴定的电泳图

图2a　PAN刺激前、后各时间点细胞形态的变化(倒置相差显微镜×200)

图2b　倒置显微镜下PAN刺激前、后各时间点足细胞面积的变化

图3　两组各时间点podocin mRNA表达的电泳条带

图4　两组各时间点podocin mRNA相对定量值(podocin/GAPDH)的变化

图5a　PAN刺激前、后各时间点podocin在足细胞的分布(荧光显微镜×400)

图5b　荧光显微镜下PAN刺激前、后各时间点podocin在足细胞胞膜、胞浆的分布定量分析

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Distribution and Expression of Podocin in Puromycin Aminonucleoside Injured Mouse Podocyte Cell Line

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Distribution and Expression of Podocin in Puromycin Aminonucleoside Injured Mouse Podocyte Cell Line