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目的优化大鼠胰岛分离纯化条件,为糖尿病胰岛受损的发病机制研究及胰岛再生的应用提供必要材料。方法胆总管原位灌注胶原酶V消化胰腺并分离胰岛后,比较经典不连续密度梯度Ficoll离心法和Histopaque1077密度离心法纯化胰岛的效果,其间观察胰腺灌注液体积及消化时间不同对大鼠胰岛分离纯化的影响,并对纯化后的胰岛进行数量、纯度和功能的体外检测。结果 Histopaque 1077密度离心法比经典不连续密度梯度Ficoll离心法可得到更多优质的胰岛(P<0.01)。两种方法获得的胰岛在高糖刺激下释放胰岛素的能力差异无统计学意义(P>0.05)。胰腺灌注液之体积及消化时间对胰岛提取有十分重要的影响。结论 Histopaque 1077密度离心法简单易行且效率较高,有望成为纯化大鼠胰岛的首选方法,便于临床和基础研究工作的开展。
OBJECTIVE: To optimize the conditions of islet isolation and purification in rats and provide the necessary materials for studying the pathogenesis of islet impaired diabetes and the application of islet regeneration. Methods The pancreatic islets were digested by collagenase V in situ and isolated from pancreatic islets. The effects of classical discontinuous density gradient Ficoll centrifugation and Histopaque 1077 density centrifugation on pancreatic islets were compared. The pancreatic islets were isolated from the pancreatic islets by volume and digestion time Purification, and in vitro detection of purified islets for quantity, purity and function. Results Histopaque 1077 density centrifugation method can obtain more high-quality islets than the classical discontinuous density gradient Ficoll centrifugation method (P <0.01). There was no significant difference in the ability of islets to release insulin under high glucose stimulation (P> 0.05). Pancreatic perfusion volume and digestion time on the islet extraction has a very important impact. Conclusion The Histopaque 1077 density centrifugation method is simple and efficient, and is expected to be the first choice for the purification of pancreatic islets in the pancreas. It facilitates the development of clinical and basic research.