非肥胖型糖尿病小鼠胸腺异位基因及MHC-Ⅱ类分子的表达

目的研究非肥胖型糖尿病(NOD)小鼠胸腺异位基因及MHC-Ⅱ类分子表达水平,探讨胸腺异位基因及MHC-Ⅱ类分子在中枢免疫耐受中的作用。方法对NOD小鼠用GA-PAP试剂盒检测血糖;尿糖试纸检测尿糖;ELISA间接法检测IAA的含量;光镜观察胰岛的组织学改变情况;MTT法检测胸腺T淋巴细胞功能;ELISA夹心法检测胸腺细胞IL-4、IFN-γ分泌水平;流式细胞术检测胸腺细胞MHC-Ⅱ类分子表达水平;RT-PCR法检测胸腺异位基因的表达水平。结果NOD小鼠血糖水平与正常BALB/c小鼠差异无显著意义,尿糖水平均为阴性;NOD小鼠自身抗体水平与正常BALB/c小鼠差异无显著意义;与对照组相比,NOD小鼠胰岛数量减少,分布稀疏,胰岛细胞数亦减少,同时有大量炎细胞浸润;NOD小鼠胸腺细胞对ConA刺激的应答水平与正常BALB/c小鼠差异无显著意义;NOD小鼠胸腺细胞IFN-γ的分泌与正常BALB/c小鼠差异无显著意义,而IL-4的分泌能力明显低于正常BALB/c小鼠;NOD小鼠胸腺内Insulin水平显著减少,而GAD67和PLP水平无显著变化;NOD小鼠MHC-Ⅱ类分子的表达水平明显下降。结论NOD小鼠在糖尿病发病前期存在着胸腺异位基因及MHC-Ⅱ类分子表达缺陷,表明胸腺异位基因及MHC-Ⅱ类分子在中枢免疫耐受中起着重要作用。

Expression of Promiscuous Gene and MHC-Ⅱ Molecules in Thymus of Mice with Nonobese Diabetic

Objective To explore the expression level of promiscuous gene and MHC-Ⅱ molecules in the thymus of mice with nonobese diabetic(NOD) and their roles in central immunotolerance.Methods Determine the blood sugar of NOD mice by GA-PAG kit,urinary sugar by test paper and IAA content by indirect ELISA.Observe the histological change of pancreatic island by light microscopy.Determine the function of thymic lymphocytes by MTT method and the secretions of IL-4 and IFN-γ in thymic lymphocytes by sandwich ELISA.Test for the expression of MHC-Ⅱ molecules by flow cytometry,and that of promiscuous gene by RT-PCR.Results The blood sugar level of NOD mice showed no significant difference from that of normal BALB/c mice,and all the determination results of their urinary surgar were negative.Compared with that of normal BALB/c mice,the autoantibody level of NOD mice showed no significant difference.However,the pancreatic islands of NOD mice decreased significantly in quantity,and were scattered in distribution.The number of pancreatic island cells also decreased significantly,accompanied with the infiltration of a large quantity of inflammatory cells.No significant difference was observed between the secretion level of IFN-γ in thymic lymphocytes of NOD and normal mice,while the secretion level of IL-4 of the former was significantly lower than that of the latter.The insulin level in thymus of NOD mice decreased significantly,while GAD67 and PLP levels showed no significant change.The expression level of MHC-Ⅱ molecules decreased significantly.Conclusion The defect in the expression of promiscuous gene and MHC-Ⅱ molecules in thymus of NOD mice at stage of prediabete indicated their important roles in central immunotolerance.