Fritschy W M, van Suylichem P T, Wolters G H, van Schilfgaarde R
Department of Surgery, University of Groningen, The Netherlands.
Diabetes Res. 1992 Feb;19(2):91-5.
Rat pancreatic islet yields obtained with dextran gradient purification were compared after suspending the digest into either the top or the bottom layer of the gradient. A 5-layer discontinuous gradient was used, which consisted of 16 ml 31% dextran as bottom layer, overlayered with 25%, 23%, 20% and 11% dextran (4 ml each). When the digest of 1 rat pancreas was suspended into the top layer of the gradient, the total number of islets obtained from the 11-20, 20-23 and 23-25% interfaces was 862 +/- 38, 240 +/- 39 and 54 +/- 5, respectively. From this gradient, also 1409 +/- 81 islets were retrieved from the bottom layer (i.e., exocrine pellet). In contrast, when the pancreas digest was suspended into the bottom layer of the gradient, 1964 +/- 63, 435 +/- 42, and 177 +/- 34 islets were obtained from the successive interfaces, and only 50 +/- 20 islets from the exocrine pellet. The total islet volume obtained from the two uppermost interfaces was 3.46 +/- 0.31 microliters after top-loading, and 4.93 +/- 0.16 microliters after bottom-loading (n = 7, p < 0.01). When the islets retrieved from one bottom loaded gradient were transplanted into either 1 (n = 6) or 2 (n = 9) diabetic recipients, glucose levels normalized in all instances. We therefore conclude that a bottom-loaded dextran gradient separates islets from exocrine tissue effectively, resulting in significantly higher islet yields than obtained with a top-loaded dextran gradient.
将消化物悬浮于葡聚糖梯度的顶层或底层后，比较了通过葡聚糖梯度纯化获得的大鼠胰岛产量。使用了一个5层不连续梯度，其底层由16毫升31%的葡聚糖组成，上面依次覆盖25%、23%、20%和11%的葡聚糖（每层4毫升）。当将1只大鼠胰腺的消化物悬浮于梯度的顶层时，从11 - 20%、20 - 23%和23 - 25%界面获得的胰岛总数分别为862±38、240±39和54±5。从这个梯度的底层（即外分泌沉淀）还回收了1409±81个胰岛。相比之下，当胰腺消化物悬浮于梯度的底层时，从连续界面获得的胰岛分别为1964±63、435±42和177±34个，而从外分泌沉淀中仅获得50±20个胰岛。顶层加载后，从最上面两层界面获得的胰岛总体积为3.46±0.31微升，底层加载后为4.93±0.16微升（n = 7，p < 0.01）。当将从一个底层加载梯度中回收的胰岛移植到1只（n = 6）或2只（n = 9）糖尿病受体中时，所有情况下血糖水平均恢复正常。因此，我们得出结论，底层加载的葡聚糖梯度能有效地将胰岛与外分泌组织分离，从而使胰岛产量显著高于顶层加载的葡聚糖梯度。
