SD大鼠胰岛的分离纯化及培养

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。

Isolation and purification and culture of SD rat islet cells

Objective To develop a method of isolating and purifying and culturing rat islet cells and to evaluate the biological characteristics of obtained cells. Methods 3～4 weeks old healthy adult SD rats were selected,conventional surgery was used to reveal SD rat pancreas and bile duct,4.5～5 No. Scalp needle was inserted into the common bile duct and 0.5mg/ml（total 8-10ml） of collagenase V solution was injected into the common bile duct. The pancreas was quickly removed into the 6 ml Hanks pancreatic digestive fluid（38℃） for 10 minutes and then put into Hanks solution containing 10% fetal bovine serum to terminate the digestion. Ficoll400 discontinuous gradient centrifugation was utilized to purify the islet cells after 60 mesh screen filter. The purified cells were detected by DTZ staining and insulin secretion was analyzed with HPLC method. Results DTZ staining showed the positive stained β-cells with homogeneous scarlet in cytoplasm. Insulin-secreting cells were found to be distributed in the interface between 23%～20% and 20%～11% concentrations of Ficoll400,the purity degree of the cells was more than 90% with better insulin secretion condition. Conclusion The present method is a simple and efficient insulin-secreting cell separation and purification method.