不同培养时间对NIT-1细胞胰岛素分泌及相关基因表达的影响

Effects of culture time on insulin secretion and expression of related genes in NIT-1 cells

目的:研究体外生理浓度葡萄糖培养胰岛β细胞株NIT-1细胞在不同时间出现的功能变化及其可能机制。方法:观察5.6 mmol/L葡萄糖浓度的DMEM完全培养基培养的NIT-1细胞在8周培养时间内细胞的形态变化;胰岛素免疫细胞化学染色;应用放射免疫法测定8周葡萄糖刺激的胰岛素分泌水平;并以RT-PCR半定量测定各基因Insulin、Glut2、GK、PDX-1、NeuroD1、Pax4、Pax6 mRNA表达水平变化。结果:(1)传代培养过程中细胞生长良好呈多边形,不同培养时间的NIT-1细胞形态无显著差异;(2)胰岛素免疫细胞化学染色示GSIS组胞浆染色阳性,呈淡黄褐色;(3)1-8周每周测定的葡萄糖刺激的胰岛素分泌(GSIS)与第1周数据比较,显示胰岛细胞分泌功能改变差异显著(P<0.05);(4)1-8周每周测定的胰岛素基因、PDX-1基因、GK基因、Glut2基因、Neu-roD1基因和Pax6基因mRNA的表达与第1周比较,显示基因mRNA的改变差异显著(P<0.05);Pax4基因mRNA的改变无显著差异(P>0.05)。结论:生理浓度葡萄糖培养的NIT-1细胞的葡萄糖刺激的胰岛素分泌功能随体外培养时间的延长呈逐步下降趋势;这可能与维持β细胞功能有关的基因Insulin、PDX-1、GK、Glut2、NeuroD1和Pax6表达下降有关。

AIM ： To investigate the glucose stimulated insulin secretion （GSIS） in β cell cultured in vitro and the role of relevant transcriptional factors in the process. METHODS ： （ 1 ） The murine insulinoma cell line NIT - 1 was cultured in complete DMEM medium with 5.6 mmol/L glucose and maintained in routine passages for 8 weeks. Morphological changes were observed with invert microscopy. （2） Insulin expression in NIT - 1 cells was measured by immunohistochemical staining. （3） During 8 weeks, the concentrations of insulin in supernatant were determined by RIA after stimula- ted with glucose. （4） mRNA expressions of genes including insulin, PDX - 1, GK Glut2, NeuroD1, Pax4 and Pax6 were detected by RT-PCR. RESULTS： （1） NIT- 1 remained normal polygon -shape without great morphological changes during culture. （2） Immunohistochemical staining showed that insulin expression in the cytoplasm of test group was positive with light brown color. （3） Compared with the first week, insulin concentrations secreted during 8 weeks decreased progressively, significant difference among groups was observed （P 〈 0. 05 ）. （4） The expression of genes including insulin, PDX - 1, GK, Glut2, NeuroD1 and Pax6 decreased progressively with time, compared to the first week （ P 〈 0. 05 ） except Pax4 gene expression （ P 〉 0. 05 ）. CONCLUSION ： GSIS as well as expression of related genes such as insulin, PDX - 1, GK, Glut2, NeuroD1 and Pax6 decreases progressively with time in NIT - 1 cells cultured in vitro with physiological concentration of glucose, implying possible correlation between decreased gene expression and impaired function of pancreatic β cells.