论文部分内容阅读
目的观察长期体外培养大鼠胰岛在分泌功能、基因表达及免疫荧光组织化学染色法的动态变化。方法采用胶原酶灌注消化和nextran密度梯度离心分离、纯化获得大鼠胰岛,连续培养20 d,定期检测葡萄糖刺激的胰岛素分泌(GSIS);实时聚合酶链反应(RT-PCR)和免疫荧光组织化学染色法检测胰高糖素(Glu)、胰岛素、胰腺十二指肠同源盒基因(PDX)-1、葡萄糖转运蛋白(Glut)-2基因mRNA及蛋白水平的表达。结果连续培养胰岛的GSIS水平持续下降;但RT-PCR及免疫荧光显示随培养时间延长,胰岛内关键基因表达并未见明显减弱。结论长期体外培养大鼠胰岛的胰岛素分泌功能持续下降,并非由胰岛素含量减少所致,可能存在更深层次的原因。
OBJECTIVE: To observe the dynamic changes of secretory function, gene expression and immunofluorescence histochemical staining of long-term islet cultured rat islets. Methods The pancreatic islets were isolated and purified by collagenase digestion and nextran density gradient centrifugation. The pancreatic islets were cultured and cultured continuously for 20 days. Glucose-stimulated insulin secretion (GSIS) was detected periodically. Real-time polymerase chain reaction (RT-PCR) and immunofluorescence histochemistry The expressions of glucagon, insulin, PDX-1 and Glut-2 mRNA and protein in pancreatic duodenum were detected by staining. Results The GSIS level of pancreatic islets decreased continuously. However, RT-PCR and immunofluorescence showed that the expression of key genes in islets was not significantly decreased with the prolongation of culture time. Conclusion Long-term in vitro cultured rat islets insulin secretion continued decline, not by the reduction of insulin content, there may be a deeper reason.