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大鼠肾小球分离和离体培养方法的建立

周莉, 陆利民^*

复旦大学上海医学院生理与病理生理学系，上海，200032

摘要

本文旨在建立大鼠肾小球分离和离体培养的实验方法。应用差异过筛法分离技术分离大鼠肾小球，用IV型胶原酶消化法去除Bowman囊，用相差显微镜观察肾小球纯度和形态结构，台盼蓝拒染法检测肾小球的活性，双标免疫荧光技术鉴定肾小球固有细胞的标志蛋白nephrin和α-SMA的表达分布，荧光显微镜和酶标仪检测Ang II对离体培养肾小球ROS水平的影响，real-time PCR方法观察离体培养的肾小球中TGF-β1 mRNA和collagen IV mRNA的水平。结果显示，离体培养的肾小球结构完整，无包囊，毛细血管袢结构清晰；离体培养48 h后，肾小球内细胞仍具有活性；激光共聚焦显微镜下可见肾小球内足细胞和间质细胞的标志蛋白nephrin和α-SMA表达清晰；给予离体培养的肾小球Ang II刺激后，肾小球ROS的产生明显增加，TGF-β1和collagen IV mRNA水平升高。以上结果提示，大鼠肾小球的分离及离体培养技术建立成功，该分离方法获得的肾小球可以进行离体培养及药物作用观察，为基础和临床研究提供实验技术和研究手段。

关键词： 大鼠肾小球; 肾小球分离; 离体培养

分类号：R334+.1

[Isolation and culture of renal glomeruli from rats.] [Article in Chinese]

ZHOU Li, LU Li-Min^*

Department of Physiology and Pathophysiology, Shanghai Medical College of Fudan University, Shanghai 200032, China

Abstract

The aim of the study was to establish a method for isolation and culture of rat renal glomeruli. The renal glomeruli of Sprague Dawley (SD) rats were isolated by a sieving method, and Bowman’s capsule was removed by digesting the glomeruli in 0.5% type IV collagenase. The inverted phase contrast microscope was used to observe the structure of isolated renal glomeruli, and trypan blue staining was used to identify the activity of cells in glomeruli. Expressions of nephrin and α-smooth muscle actin (α-SMA) were observed by double-labeling immunofluorencence. Effects of Ang II on reactive oxygen species (ROS) generation were detected by dihydroethidium (DHE) staining. The levels of transforming growth factor-beta 1 (TGF-β1) and collagen IV mRNA were measured by real-time PCR. The results showed that the renal glomeruli with high purity and intact capillary structure were isolated by the modified protocol. The cells in the isolated glomeruli remained alive after 48 h of culture in DMEM. Confocal microscopy observations showed that nephrin and α-SMA were highly expressed in the isolated glomeruli. Treatment of the isolated renal glomeruli with Ang II enhanced ROS production. Furthermore, Ang II increased the mRNA levels of TGF-β1 and collagen IV. In conclusion, we have established a modified method that can isolate glomeruli from rat kidney, and the isolated glomeruli can be used for further observation in cultured condition. The protocol will provide a useful method for preclinical research on kidney diseases.

Key words: rat glomerulus; isolated glomeruli; primary culture

收稿日期：2015-08-10　　录用日期：2015-09-29

通讯作者：陆利民　　E-mail: lulimin@shmu.edu.cn

引用本文：

周莉, 陆利民. 大鼠肾小球分离和离体培养方法的建立[J]. 生理学报 2015; 67 (6): 629-635.

ZHOU Li, LU Li-Min. [Isolation and culture of renal glomeruli from rats.] [Article in Chinese]. Acta Physiol Sin 2015; 67 (6): 629-635 (in Chinese with English abstract).