原代大鼠胰岛细胞分离纯化及单细胞培养初探

A PRELIMINARY STUDY OF PRIMARY RAT ISLET CELLS SEPARATION,PURIFICATION AND UNICELLULAR CULTURE

目的:探讨获得高质量、数量多的原代大鼠胰岛细胞的有效可靠的方法。方法:以Ⅴ型胶原酶顺行灌注大鼠胆总管消化胰腺,以Histopaque 1077等密度区带离心法分离胰岛和胰腺腺泡组织。以双硫腙染色及免疫组化法分别鉴定提取物,胰岛素释放试验鉴定胰岛的活性。结果:经Histopaque 1077等密度区带离心法分离纯化,每只大鼠平均获取(251±26)枚胰岛。双硫腙染色后完整胰岛呈猩红色细胞团。胰岛单细胞培养及胰岛素释放试验起始2.8 mmol/L低糖培养胰岛素分泌量为(23.53±5.946)pmol/L,16.7mmol/L高糖刺激后胰岛素分泌量为(32.61±4.085)pmol/L,最后恢复2.8mmol/L低糖培养胰岛素分泌量为(27.81±3.616)pmol/L,3者之间比较差异均有统计学意义(P<0.05),提示胰岛细胞活性良好。结论:用Histopaque 1077等密度区带离心法分离纯化原代大鼠胰岛细胞兼具有数量多、活性好、纯度高的优点,可继续用于研究。

Objective：This study aimed to provide an effective and reliable method to obtain high quality and quantity of the primary rat islet cells.Methods：Subjects underwent retrograde perfusion of the common bile duct with type V collagenases to digest the pancreas.Histopaque 1077 isopycnic zone centrifugation was used to separate the pancreas and the pancreatic acinar tissue,and the extracts were identified by dithizone（DTZ）staining and an immunohistochemical method,respectively.The activity of the islets was subsequently determined by an insulin release test.Results：After purification by this method,an average of（251±26）islet pieces were obtained from each subject rat.The intact islets were stained scarlet with DTZ.Single-islet cell cultures and insulin release tests showed that the insulin secretion of low glucose group（2.8 mmol/L）is（23.53±5.946）pmol/L,after high glucose（16.7mmol/L）stimulation the insulin secretion is（32.61±4.085）pmol/L,finally back in low glucose（2.8 mmol/L）cultivation,the insulin secretion is（27.81±3.616）pmol/L,the statistical results show that there are significant differences between three groups,P〈 0.05.Above all shows the islet cells were active.Conclusion：This method has three advantages which is quantity,active and high purity.After purification,the islets are suitable for study.