小鼠胰岛的分离纯化及体外功能检测的实验研究_临床医学论文.docVIP

小鼠胰岛的分离纯化及体外功能检测的实验研究_临床医学论文 小鼠胰岛的分离纯化及体外功能检测的实验研究_临床医学论文 作者：宋科瑛　王瑞涛　卢列盛　董维平　李克【摘要】 　　目的：探讨获得高质量小鼠胰岛的分离纯化方法，评价其功能。方法：采用胆总管内胶原酶灌注膨胀消化胰腺的方法分离小鼠胰岛，不连续密度梯度离心法纯化胰岛，用双硫腙（Dithizone，DTZ）对胰岛进行特异性染色计算胰岛产量及纯度，以葡萄糖和茶碱刺激胰岛素释放检测胰岛功能。结果：胰岛的产量和活性主要与胰腺均匀膨胀和胶原酶的消化时间有关。平均每个小鼠胰腺能得到150~250个高质量胰岛，活性gt;95%，纯度gt;90%。葡萄糖及茶碱（carbachol， Cch）刺激后胰岛素释放量明显增加。结论：改良的胆总管内胶原酶灌注膨胀消化小鼠胰腺及不连续密度梯度Ficoll-400纯化胰岛的方法，可获得产量较高、纯度及功能较好的胰岛。 【关键词】 胰岛·分离·纯化·小鼠 　　Isolation， Purification and Functional Assessment of Islets in Mice 【ABSTRACT】 Objective: The technical difficulty in mouse islets isolation and purification restricts its wide use in diabetes and islet transplantation field. Here we reported and discussed a modified method with which to isolate and purify high quality islets in mice. Methods: Intraductal collagenase-P perfusion and uniform distention of the gland were applied to digest mice pancreas. A Ficoll gradient solution（25%, 23%, 20%, and 11%） was applied to make the purification. Islets were then tested for their specificity by Dithiozine（DTZ） staining. The islet function was assessed by the glucose- and carbachol-stimulated insulin secretion test. Results: The yield and viability of the islets were closely associated with good gland distention and collagenase digestion timing. We used the method discussed here, each mouse pancreas could yield 150~250 islets, with viability gt;95% and purity gt;90%. The insulin release from the in vitro islets fed with high concentration glucose and Carbachol stimuli raised to a significantly higher level. Conclusion: The above-mentioned method of mice isolation and purification might yield high quality islets for further scientific use. 【KEY WORDS】 Islets· Isolation·Purification· Mice 　　胰岛移植被认为是有望治愈胰岛素依赖性糖尿病（insulin-dependent diabetes mellitus）的新型治疗方法。小鼠品系纯正，可重复性好，可进行基因调控,自身繁殖力强，可缩短科研周期，已在国外胰岛移植和糖尿病研究领域，尤其是基因治疗领域，得到越来越广泛的应用［１-５］。但由于小鼠体积小，胰腺相关解剖结构细微，胰岛的分离纯化操作有一定的难度。本研究参考学术交流中获得的纽约西奈山医学院和美国康奈尔大学医学院的小鼠胰岛分离纯化方案，结合国内实验条件，对小鼠胰岛的分离、纯化、培养作了改进。体外功能检测证实，该方法分离纯化的小鼠胰岛可用