大鼠胰岛的分离和纯化

Comparison of different digestion techniques and density gradient purification procedures to prepare viable pancreatic islets from SD rats

目的：改进分离与纯化方法，以获得较高纯度和活力的大鼠胰岛。方法：用改良的酶法分离、Ｄｅｘｔｒａｎ梯度法纯化大鼠胰岛。用ＤＴＺ法判定胰岛纯度；用ＡＯ／ＰＩ法判定胰腺存活率；用大鼠胰岛移植于ＳＴＺ糖尿病小鼠法判定其功能。结果：雌、雄性ＳＤ和Ｗｉｓｔａｒ大鼠间胰岛形态无明显差异。Ⅴ型和Ⅺ型胶原酶对大鼠胰岛分离效果相似；分离以０．１％胶原酶分次消化、作用３０ｍｉｎ为宜，细胞存活率达９０％。纯化用２２％—１６％—１４％—９％梯度所获得胰岛的纯度＞９０％。胰岛移植于糖尿病小鼠可使其血糖从移植前的（２６．７０±２．５３）ｍｍｏｌ／Ｌ降低至（１３．９８±３．９２）ｍｍｏｌ／Ｌ，维持２～３ｄ。结论：所改进的制备方法可获得较高纯度和活力的大鼠胰岛。

Objective:Improving collagenase digestion methods and purification procedures to prepare pancreatic islets from rats for transplantation studies.Methods:Isolation of rat islets were performed with the basic technique described by Lim and with our modification. Islet were purified by Dextran density gradient. For identification, islet samples were stained with DTZ. Islet viabikity was determined with fluorescence (AO/PI). In vivo the rat islet function was assessed by transplantation of Kunmin mice with streptozocininduced diabetes.Results:No great quantitative differences could be detected in islet shape, number and size between SD and Wistar rats by histologic analysis. To analyze the influence of the type, consistency and reaction time of the enzyme on islet viability and yields suggests that 0.1% collagenase 30 mins was the best and that between Collagenase Ⅴ and Ⅺ gave similar results. The islets were recovered between the 9% and 14% Dextran layers, and between the 14% and 16%Dextran layers. Purity ranged 85%—95% with 22%—16%—14%—9% gradient. The islets prepared restored normoglycemia with 23 days from implantation in the diabetes mice.Conclusion:Tese results show using the digestion methods and purification procedure that we have developed could provided islets from rats for transplantation studies.