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目的 :建立成年杂种猪胰岛分离、纯化和包埋的方法 ,并对微囊胰岛的体外分泌功能及体内移植降糖功能进行研究。方法 :采用经胰管灌注胶原酶 ,葡聚糖间断密度梯度离心 ,分离和纯化猪胰岛 ,用海藻酸钠 聚赖氨酸 海藻酸钠 (APA)包埋猪胰岛。用双硫腙染色鉴别胰岛 ,用荧光染色计数胰岛存活率。对微囊胰岛进行体外培养 ,用不同浓度葡萄糖刺激微囊胰岛考察其功能 ,并进行大鼠糖尿病模型移植。结果 :每个猪胰腺可得到的胰岛数量平均为 98,5 5 0± 13,80 4个 ,纯化后胰岛纯度达 80 %以上 ,平均胰岛回收率 43.9%。体外试验显示微囊胰岛对葡萄糖刺激发生应答 ,分泌胰岛素。微囊胰岛在 16天的培养期间可持续分泌胰岛素 ,囊内胰岛细胞生长良好 ,微囊无溶解和破碎。培养 2天的微囊胰岛对高糖刺激有明显反应 ,其胰岛素释放量分别为低糖的 1.49倍 (静态 )和 2 .45倍 (灌流 ) ,P <0 .0 5。糖尿病大鼠体内移植微囊杂种猪胰岛 5 ,0 0 0～ 6 ,0 0 0 ,可明显降低动物模型血糖 ,并持续 7～ 11d。结论 :APA微囊膜及微囊化杂种猪胰岛在体外功能良好。短期培养及体内移植降糖功能确切 ,将此法制备的微囊胰岛用于治疗I型糖尿病有良好的应用前景。
OBJECTIVE: To establish a method for the isolation, purification and embedding of islet of adult pigs, and to study the in vitro secretion of islets and the hypoglycemic function in vivo. Methods: Porcine pancreatic islets were isolated and purified by perfusion of collagenase and dextran by density gradient centrifugation through pancreatic duct. Porcine islets were embedded with sodium alginate polylysine and sodium alginate (APA). Dithizone staining was used to identify islets, and the islet survival was counted by fluorescent staining. Microcyclonal islets were cultured in vitro, with different concentrations of glucose stimulation microcapsules inspected its function, and diabetic rat model. RESULTS: The number of islets available per pancreas was 98,550 ± 13,804. The purified islets reached more than 80% purity and the average islet recovery rate was 43.9%. In vitro experiments showed that microcapsulated islets respond to glucose stimulation and secrete insulin. Microcapsulated islets continue to secrete insulin during 16 days of culture, and the islets within the capsule grow well, with no dissolution and fragmentation of the microcapsules. The microcapsule islets cultured for 2 days showed obvious response to high glucose stimulation. The insulin release amount was 1.49 times (static) and 2.45 times (perfusion) respectively, P <0. 05. Diabetic rats transplanted in vivo microvascular hybrid pig islets 500, 0 ~ 6, 0 0 0, can significantly reduce animal models of blood glucose, and continued for 7 ~ 11d. Conclusion: APA microcapsules and microencapsulated porcine islets function well in vitro. Short-term culture and in vivo glucose-lowering function of the exact graft, prepared by this method microcapsulated pancreatic islets for the treatment of type I diabetes has a good prospect.