长期体外培养大鼠胰岛的动态研究

Continuous observation on rat islets undergoing long-term culture in vitro

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 rat islets in secretion, gene expression and immunofluorehistochemostaining undergoing long term culture in vitro. Methods Collagenase infusion and dextran density gradient centrifugation were used to obtain rat islets; during its culture, glucose stimulated insulin secretion （GSIS） was performed on the 1st, 3rd, 5th, 7th, 10th, 15th, 20th day. Real time PCR and immunofluorescence were also performed to detect the gene expression of glucagon, insulin, PDX-1 and Glut-2. Results During islets culture, GSIS decreased sharply from the third day without reduction of the above four key gene expressions by real time PCR or by immunofluorescence. Conclusion The important function of islets-GSIS decreases during continuous islets culture in vitro without affecting expressions of the above mentioned key genes, which suggests the complicated mechanism of islets dysfunction. （Shanghai Med J, 2006, 29： 692-694）