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摘要:
目的 探讨新型胰岛分离液（IPS液）在小鼠胰岛分离中的分离效率及胰岛保护作用。 方法 将消化后的小鼠胰腺按体积平均分为两组（IPS组和UW组），分别应用IPS-Optiprep液及UW-Optiprep液进行连续梯度密度离心分离胰岛，比较两组分离液的分离纯化效率和分离后的胰岛活性。将诱导成功的糖尿病小鼠随机分为3组：实验组（n=10），接受采用IPS-Optiprep液分离纯化的胰岛移植；对照组（n=10），接受采用UW-Optiprep液分离纯化的胰岛移植；假性移植组（n=5），仅给予手术但并不进行胰岛移植。分析3组小鼠术后血糖水平以及测实验组和对照组小鼠术后21 d的腹腔葡萄糖耐量试验的血糖水平。比较两种分离液的配制成本。 结果 与UW组相比，IPS组的IPS-Optiprep液可提供更高的胰岛当量（IEQ）、胰岛纯度、回收率及胰岛完整度。胰岛形态观察可见，IPS组胰岛被膜完整，直径明显大于UW组。UW组纯化后的胰岛活性率高于IPS组[（88±5）%比（84±3）%，P < 0.01]。与UW-Optiprep液相比，IPS-Optiprep液可获得相当的在体胰岛功能。IPS-Optiprep液可显著降低胰岛分离纯化成本。 结论 新型胰岛分离液IPS-Optiprep液在胰岛分离提纯中显示出较好的分离效率，增加了胰岛的纯度、完整度与回收率，并显著降低了纯化成本，但对胰岛细胞活性的保护作用稍逊，可能与胰岛的高完整度及IPS-Optiprep液中的内毒素有关。 Abstract:Objective To investigate the isolation and protective effect of a new islet purification solution (IPS)- Optiprep solution on the islet in mouse models. Methods The digested pancreatic islets were divided into the IPS andUW groups according to the islet volume. The pancreatic islets were isolated by the continuous gradient density centrifugation using IPS-Optiprep or UW-Optiprep solutions. The purification efficiency and isolated islet activity of purification solution were compared between two groups. The diabetic mouse models were successfully induced and randomly assigned into three groups. In the experimental group (n=10), the mice received pancreatic islet transplantation using islets isolated and purified by the IPS-Optiprep solution. In the control group (n=10), the mice underwent pancreatic islet transplantation using islets isolated and purified by the UW-Optiprep solution. In the sham surgery group (n=5), the mice merely underwent surgery without pancreatic islet transplantation. Postoperative blood glucose levels were detected and compared among three groups. The blood glucose levels of intraperitoneal glucose tolerance test at postoperative 21 d were statistically compared between the experimental and control groups. The cost of the preparation of two isolation solutions was also compared. Results Compared with the UW group, the islet equivalent (IEQ), islet purity, recovery rate and islet integrity were significantly higher in the IPS group. Islet morphological observation revealed that the islet membrane was complete and the islet diameter in the IPS group was considerably larger than that in the UW group. The activity of purified islets in the UW group was significantly higher than that in the IPS group [(88±5)% vs. (84±3)%, P < 0.01]. Compared with the UW-Optiprep solution, identical in vivo islet function was obtained in the IPS-Optiprep solution. The cost of IPS-Optiprep solution was significantly less than that of the UW-Optiprep solution. Conclusions The new IPS-Optiprep solution yields higher islet isolation efficiency, purification, integrity and recovery rate and significantly reduces the purification cost compared with the UW-Optiprep solution. Nevertheless, IPS-Optiprep solution exerts a less protective effect on the activity of islet cells, which is probably correlated with the high islet integrity and the endotoxin in the IPS-Optiprep solution. -
图 1 两组分离液分离纯化后的胰岛形态（双硫腙，×100）
A图为经UW-Optiprep液纯化的胰岛；B图为经IPS-Optiprep液纯化的胰岛
Figure 1. Islets morphology after purification by isolation solution of two groups
图 2 两组分离液分离纯化后的胰岛荧光图片（FDA/PI，×100）
A图为经UW-Optiprep液纯化的胰岛；B图为经IPS-Optiprep液纯化的胰岛；红色荧光为死细胞，绿色荧光为活细胞
Figure 2. Fluorescence images of the purified islets by isolation solution of two groups
图 3 3组糖尿病小鼠术后的血糖变化
Figure 3. Changes of blood glucose in diabetic mice of 3 groups after operation
图 4 两组糖尿病小鼠胰岛移植后21 d腹腔葡萄糖耐量试验的血糖变化
Figure 4. Changes of blood glucose in diabetic mice of two groups by intraperitoneal glucose tolerance test at 21 d after islet transplantation
表 1 两组分离液的胰岛分离纯化效率的比较
Table 1. Comparison of islet isolation and purification efficiency of isolation solutionbetween two groups (x±s)
组别 纯化后IEQ 纯度（%） 完整度 回收率（%） IPS组 371±43 46±11 0.87±0.17 85±19 UW组 285±47 33±60 0.70±0.15 65±15 P值 < 0.01 < 0.01 < 0.05 < 0.05 
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