体外培养人胰岛素瘤细胞的特性

目的分离培养人胰岛素瘤细胞,并研究它的功能特性。方法原代分离人胰岛素瘤细胞,经多次筛选培养进行纯化。通过荧光显微镜观察细胞特有的自发荧光;RT-PCR方法检测胰岛素mRNA的表达和胰岛细胞自身抗原IA-2、IAR和ICAp69的表达;应用MTT方法检测抗人IA-2抗体对胰岛素瘤细胞的杀伤。结果分离的人胰岛素瘤细胞经传40代,在悬浮培养条件下可自发聚集成胰岛样结构,并有自发荧光。该细胞表达胰岛素mRNA并合成胰岛素,但明显低于大鼠胰岛素瘤细胞INS-1。该细胞也表达人胰岛细胞自身抗原IA-2、IAR和ICAp69,并且用抗人IA-2抗体与之孵育后可出现抗体剂量依赖性的细胞杀伤。结论此分离培养的人胰岛素瘤细胞表达人胰岛素和人胰岛细胞自身抗原,因此可以作为研究人Ⅰ型糖尿病发病机制的细胞模型。

Characteristics of cultured human insulinoma cells in vitro

Objective To isolate human insulinoma cells,and investigate its functional characteristics.MethodsPrimary human insulinoma cells were isolated by using collagenase type P,and then purified by repeatedly selective cell culture.The cellular autofluorescence was examined by fluorescent microscope.Expression of insulin and islet cell autoantigens such as IA-2,IAR and ICA were checked by RT-PCR and Western blot.The cytotoxicity induced by anti-human IA-2 antibody was analyzed by MTT assay.Results Human insulinoma cells may form islet-like structure under the condition of suspension culture and show autofluorescence under fluorescent microscope.The human insulinoma cells expressed lower level of insulin mRNA and protein as compared with a rat insulinoma cell line,INS-1.The insulinoma cells also expressed autoantigens of islet cells such as IA-2,IAR and ICAp69.A dose dependent cytotoxicity was found when the insulinoma cells were incubated with anti IA-2 antibody.ConclusionHuman beta cells derived from insulinoma could be purified by selective cell culture.Since the insulinoma cells expressed insulin and islet cell autoantigens,it can be used as a cell model for investigating the mechanisms of type I diabetes.