西藏小型猪胰岛细胞体外分离和纯化方法

摘要

Objectives Identify the optimized techniques for the isolation and purification of pancreas islets to supply high quality islets for xenotransplantation in the treatment of diabetes. Methods Compare the quantities and effects of different mass concentration (1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml) and the same mass concentration conditions at different time points (0 min, 15 min, 30 min, 45 min, 60 min, 75 min, 90 min) on Tibet minipig pancreas islets isolation. Compare the quantities and effects of Ficoll400 density gradient centrifugation, lymphatic separation liquid centrifugal, centrifugal, and placing the 4 ways on purification of islets. With AO/PI dyeing calculation islet cell survival rate. Results The best time points of different mass concentration collagen enzyme to acquire islets were confirmed, 1 mg/ml and 2 mg/ml groups for 45 min,3 mg/ml and 4 mg/ml groups for 30 min respectively. Among the 4 best time points, 1 mg/ml group gets the most amount of islets . Each group gets islets of complete morphology at best time point respectively. Islets number by many to less is placing> Ficoll400 density gradient centrifugation> lymphatic separation liquid centrifugal>centrifugal among 4 kinds of purification ways. The AO/PI staining after testing the survival rate of pancreatic islet cells for more than 95.00%. Conclusions In the condition of cutting tissue into pieces, collagen enzyme mass concentration 1 mg/ml, 45 min of digesting time can get more islets, Ficoll400 density gradient centrifugation collects islets most effectively.%目的 确定成体西藏小型猪胰岛细胞体外分离、纯化的理想条件,为异种移植治疗糖尿病提供高质量的胰岛细胞.方法 对比不同质量浓度的胶原酶(1 mg/ml,2 mg/ml,3 mg/ml,4 mg/ml)和同一质量浓度不同时间点(0 min,15 min,30 min,45 min,60 min,75 min,90 min)分离获得胰岛细胞的数量和效果;使用Ficoll400密度梯度离心、淋巴分离液分离、离心和静置4种方法纯化胰岛细胞悬液,比较最终收集到的胰岛细胞数量和效果；用DTZ染色计算胰岛细胞的纯度；用AO/PI染色计算胰岛细胞存活率.结果 确定了不同质量浓度的胶原酶获取胰岛细胞的最佳时间,胶原酶l mg/ml和2 mg/ml组为45 min,3 mg/ml和4 mg/ml组为30 min,各组在最佳时间点获得胰岛细胞的数量最多的为1 mg/ml组,胰岛细胞形态完整；4种纯化方法中,收集的胰岛细胞数量由多到少依次为静置组＞ Ficoll400密度梯度离心组＞离心组＞淋巴分离液离心组.经AO/PI染色后检测胰岛细胞的存活率为95.00％以上.结论 在组织块剪碎消化的情况下,胶原酶质量浓度1 mg/ml、消化时间45 min的分离条件可获得数量较多的胰岛细胞,Ficoll400密度梯度离心的纯化手段收集到的胰岛细胞效果最好.