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目的探讨分离纯化大鼠胰岛素分泌细胞的方法,并评价细胞的生物学特性。方法选用3～4周龄健康成年SD大鼠,常规外科手术显露SD大鼠胰腺和胆总管,在胆总管内插入4.5～5号头皮针后固定,逆行注入预冷的0.5mg/ml的胶原酶V溶液8～10ml,使胰腺膨胀后迅速取出胰腺放入6mlHanks液中38℃消化10min,用含10%胎牛血清的Hanks液终止消化,60目筛网过滤后用Ficoll400非连续梯度离心纯化胰岛。纯化后的细胞用DTZ染色和胰岛素释放试验来分析胰岛素的分泌情况。结果 DTZ染色后β细胞胞浆着色,为均一的猩红色。胰岛细胞分布于Ficoll400浓度为23%～20%和20%～11%的界面之间,纯度高达90%,并且细胞的胰岛素分泌情况良好。结论胶原酶V消化,Ficoll400纯化是一种简单高效的胰岛素分泌细胞分离纯化的方法,可以获取数量多,活性和纯度好的胰岛素分泌细胞。
Objective To investigate the method of isolation and purification of rat insulin-secreting cells and to evaluate the biological characteristics of the cells. Methods Healthy adult SD rats of 3 to 4 weeks old were selected. Pancreatic and common bile ducts of SD rats were exposed by conventional surgical operation. Scalp acupuncture of No. 4.5-5 was inserted in the common bile duct and fixed in retrograde injection of precooled 0.5mg / ml collagen Enzyme V solution 8 ~ 10ml, the pancreas quickly removed after the pancreas into 6ml Hanks liquid at 38 ℃ digestion 10min, with 10% fetal bovine serum Hanks liquid to stop digestion, 60 mesh sieve Ficoll400 non-continuous gradient centrifugation after purification Islet. Purified cells were analyzed for insulin secretion by DTZ staining and insulin release assay. Results After DTZ staining, the cytoplasm of β-cell was stained to be uniform scarlet. Islet cells are distributed between the Ficoll400 concentrations of 23% to 20% and 20% to 11% of the interface with a purity of up to 90% and a good insulin secretion by the cells. Conclusion Collagenase V digestion and Ficoll400 purification are simple and efficient methods for the isolation and purification of insulin secreting cells, which can obtain a large number of insulin secreting cells with good activity and purity.