关键词: 肾小球 , 肾小球分离 , 四氧化三铁 , 肾病研究

Abstract:

Objective: To develop a method for glomerular purification that can further perform the molecular biology for nephrology research.  Medthodology: After the anesthetization, the thorax and abdomen cavity of the mouse was opened.  Iron oxide solution was microperfused into the microvascular circulation of the whole body that include kidneys. Kidneys were dissected and minced into small pieces, then digested with collagenase A in PBS. The tissue was diluted in PBS and gently pressed through a filter followed by rinsing with PBS. Glomeruli that contained iron oxide were isolated with a magnetic particle concentrator and washed with PBS. Electron microscope was used for the observation of glomerular morphology. Extracted total RNA and protein from isolated glomeruli were further employed for quantitative Real-Time polymerase Chain Reaction (qRT-PCR), miroRNA microarray and Western blotting experiments.  Results: We developed a method that allows largely and quickly to purify the glomerulus at different developing/developed stage from postnatal day 1 to adult mice. The structure and ultrastructure of glomerulus purified were entirely kept and normal under the electro-microscope. The total RNA and protein extracted from the glomerulus purified were successfully used to the molecular biological research, including qRT-PCR, Western blotting and microRNA microarray.  Conclusion: This method is simple and cheaper to purify the glomerulus, which provides a useful and powerful tool for basic science and clinic research relevant to nephrology.

Key words: glomeruli , glomerular isolation , iron oxide , kidney research