[连续与间断密度梯度离心法纯化人胰岛的比较]

[Comparison of continuous and discontinuous density gradient centrifugation for purification of human pancreatic islets].

作者信息

Wei Guo-hong, Sun Wei-ping, Zhang Jian, Cai De-hong, Zeng Long-yi, Chen Gui-hua, Weng Jian-ping

机构信息

Research Center of Prevention and Treatment for Diabetes Mellitus/Department of Endocrinology, First Hospital Affiliated to Sun Yat-sen University, Guangzhou 510080, China.

出版信息

Nan Fang Yi Ke Da Xue Xue Bao. 2007 Sep;27(9):1352-4.

PMID:17884776

Abstract

OBJECTIVE

To compare the effect of continuous and discontinuous density gradient centrifugation for purification of human pancreatic islets with COBE 2991 cell processor.

METHODS

Human pancreases were obtained from brain-dead donors and stored in cold UW solution. The connective tissues were removed from the pancreases, and the pancreatic ducts were perfused with a cold enzyme (Liberase). The islets were then separated by gentle mechanical dissociation and purified with discontinuous (10 pancreases) or continuous (8 pancreases) gradients of HCA-Ficoll in COBE 2991 cell processor. Samples were collected in duplicate for determination of the quantity of islets, islet equivalents (IEQ), and the purity.

RESULTS

The weights of the pancreases before and after connective tissue removal and pancreas duct perfusion, and the quantity of islets obtained (including islets quantity of different diameters and total IEQ) after dissociation were not significantly different. Continuous gradient of HCA-Ficoll, compared with discontinuous gradient, resulted in significantly greater final islet quantity (55,000 IEQ vs 206,000 IEQ, P=0.000) and islet purity (58.0%-/+8.0% vs 33.5%-/+10.3%, P=0.000) and also greater number of islets with a diameter lager than 200 microm (P<0.01).

CONCLUSION

Continuous density gradient centrifugation can be more effective than discontinuous gradient in islet purification.

摘要

目的

比较使用COBE 2991细胞处理器进行连续和不连续密度梯度离心法纯化人胰岛的效果。

方法

从脑死亡供体获取人胰腺，保存在冷的UW溶液中。去除胰腺中的结缔组织，并用冷酶（ Liberase）灌注胰管。然后通过轻柔的机械解离分离胰岛，并在COBE 2991细胞处理器中用不连续（10个胰腺）或连续（8个胰腺）的HCA-菲可密度梯度进行纯化。样本一式两份收集，用于测定胰岛数量、胰岛当量（IEQ）和纯度。

结果

去除结缔组织和灌注胰管前后胰腺的重量，以及解离后获得的胰岛数量（包括不同直径的胰岛数量和总IEQ）无显著差异。与不连续梯度相比，连续的HCA-菲可密度梯度导致最终胰岛数量显著增加（55,000 IEQ对206,000 IEQ，P = 0.000），胰岛纯度也更高（58.0%±8.0%对33.5%±10.3%，P = 0.000），并且直径大于200微米的胰岛数量也更多（P < 0.01）。