一种简单经济提取小鼠大量肾小球的实验方法

A simple and economical method for extracting large number of mouse glomeruli

摘要:

目的 为减轻实验者用磁珠提取肾小球的经济负担，对传统的心脏灌注肾脏提取方法进行改良。方法 结扎远端腹主动脉及下腔静脉，结扎肠系膜上动脉和腹腔动脉，以减少磁珠分流。将磁珠工作液经静脉留置针灌注肾脏。加入相应体积胶原酶A，通过100?μm筛网2次，加入蛋白裂解液，离心裂解吸出含蛋白上清进行肾小球蛋白的纯化、定量。结果 与传统的心脏灌注肾脏提取方法比较，改良胸主动脉灌注肾脏提取肾小球方法磁珠用量仅为原来的1/35～1/40。在获取相同的蛋白情况下，提取肾小球的最佳浓度为4×106磁珠/ml，磁珠用量为20?ml。通过该方法灌注分离的肾小球完整，纯度为（95.7±2.3）%。结论 改良的胸主动脉灌注肾脏是一种简单经济实用磁珠提取肾小球的实验方法，可以快速获取大量完整的肾小球，蛋白质甚至RNA，为肾小球本身的研究提供新的技术支持。

Abstract:

Objective To reduce the economic burden of extracting glomeruli by magnetic beads through improving the traditional method of extracting kidney by heart perfusion. Methods The distal abdominal aorta, inferior vena cava, the superior mesenteric artery and celiac artery were ligated to reduce the magnetic bead shunt. The working fluid of magnetic beads was perfused into the kidneys through intravenous indwelling needles. The corresponding volume of collagenase A was added, and the protein lysate was added twice through 100?μm sieve. The protein supernatant was extracted by centrifugation for purification and quantification of glomeruli. Result Compared with the traditional method of kidney extraction by heart perfusion, the improved method of kidney extraction by thoracic aorta perfusion was only 1/40 to 1/35 of the original amount of magnetic beads, when the same protein and glomeruli were obtained. The optimum concentration of glomeruli was 20 ml (4?×?107 beads/ml) volume/mice. The glomeruli of mice were isolated by renal perfusion with a purity of (95.7?±?2.3) %. Conclusion The improved method of extracting glomeruli from thoracic aorta and kidney is a simple, economical and practical experimental method for extracting glomeruli by magnetic beads. It can quickly and easily obtain a large number of complete glomeruli, proteins and even RNA, and provides new technical support for the study of glomeruli itself.