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目的:发明一种快速易行的肾小球分离技术,为肾脏病学研究服务。方法:小鼠麻醉后,经体循环灌注四氧化三铁溶液,使肾小球内的毛细血管中充满四氧化三铁溶液。然后取出肾脏切碎,用collagenase A消化,细胞器滤过,磁铁吸附并收集含铁的肾小球。光镜、透射和扫描电镜观察肾小球的正常形态和结构。常规方法提取RNA和蛋白质,并进行qRT-PCR、micro RNA(miRNA)基因芯片和Western blot等定量检测。结果:该方法可从新生小鼠和成体小鼠的肾脏中高质量快速提纯不同发育阶段(发育早期、中期或晚期)及成熟的肾小球,分离后的肾小球光镜形态结构以及超微结构均保存完好。常规方法提取的RNA和蛋白质可用于qRT-PCR、Western blot检测以及miRNA基因芯片等分子生物学的定性和定量分析研究。结论:该研究所述的肾小球分离技术费用低廉,简便易行,为基础与临床探索肾小球对各种肾脏疾病的基因调控作用和发育分子生物学研究提供了重要的方法和技术手段。
OBJECTIVE: To develop a rapid and easy glomerular separation technique for nephrology research. Methods: After the mice were anesthetized, the ferroferric oxide solution was infused through the systemic circulation so that the capillaries in the glomeruli were filled with ferroferric oxide solution. The kidneys are then removed, shredded with collagenase A, filtered through organelles, and the magnet is adsorbed and the iron-containing glomeruli are collected. Light microscopy, transmission and scanning electron microscopy to observe the normal glomerular morphology and structure. RNA and protein were extracted by routine methods and quantified by qRT-PCR, microRNA (miRNA) microarray and Western blot. Results: The method could rapidly purify the glomeruli of different developmental stages (early stage, middle stage or late stage) and mature glomeruli from the kidneys of newborn mice and adult mice. The morphological structure of the glomerular mirror after the separation and ultrastructure Structure are well preserved. RNA and protein extracted by routine methods can be used for qualitative and quantitative analysis of molecular biology such as qRT-PCR, Western blot and miRNA microarray. CONCLUSIONS: The glomerular separation technique described in this study is inexpensive, simple and easy to perform, and provides an important method and technique for the study of gene regulation and development of glomeruli for various kidney diseases both in basic and clinical settings .