谷氨酸脱羧酶通过调节T细胞亚群预防NOD小鼠发生1型糖尿病

Glutamic acid decarboxylase prevents type 1 diabetes by regulating the subsets of Tlymphocytes in NOD mice

目的 了解谷氨酸脱羧酶 (GAD)对非肥胖糖尿病 (NOD)小鼠的 1型糖尿病是否具有预防作用并探讨其免疫作用机制。方法 用猪脑GAD和不完全弗氏佐剂 (FIA)混合后给 4周龄雌性NOD小鼠腹腔注射 (32例 ) ,同时单独注射等量FIA作为对照组。 8周龄时腹腔注射环磷酰胺以加速糖尿病。每周测定体重、血糖 ,糖尿病形成 2周或 2 0周龄处死小鼠后测定血清C 肽、GAD抗体(GAD Ab)和脾组织T细胞亚群 ,观察胰腺病理、免疫组化和超微结构变化。结果 2 0周龄时 ,FIA对照组的糖尿病总发病率为 73 .6 8% ,而GAD组仅为 6 .2 5 % (P <0 .0 1) ,发病时间也明显延缓 ;GAD组的胰岛炎症分数较FIA组明显降低 ,且炎症程度减轻 (P <0 .0 5 ) ;GAD组的C 肽水平也明显高于对照组 (P <0 .0 5 ) ;两组的GAD Ab阳性率无明显差别 ;FIA组的脾脏T细胞以CD+4 亚群为主 ,GAD组则CD+8亚群比例增高 ,CD4 /CD8比值显著下降 (P <0 .0 1) ;免疫组化也表明FIA组胰岛浸润淋巴细胞为CD+4 T细胞 ,而GAD组胰岛CD+4 细胞明显减少 ,且局限于胰周。结论 猪脑GAD有预防NOD小鼠胰岛炎和糖尿病发生的作用 ,其机制可能与改善小鼠缺陷的CD+8T细胞亚群有关。

Objective To observe whether glutamic acid decarboxylase (GAD) can prevent type 1 diabetes in nonobese diabetic (NOD) mice and to study its influence on the immune system. Methods One injection was performed intraperitoneally (i.p.) with GAD purified from pig brain, together with Freund incomplete adjuvents (FIA), into female NOD mice at the age of 4 weeks as treated group (n=32), other 19 mice were injected FIA 100 μl alone as control group. Diabetes was accelerated 4 weeks later by a single injection of cyclophosphamide. Blood glucose and body weight were measured weekly. At the age of 20 weeks or 2 weeks after the onset of diabetes, mice in two groups were killed and the serum levels of C peptide and antibodies against GAD were detected with radioimmunoassay and ELISA respectively. The subsets of T cells in spleen were determined by two color FACS analysis, the ultrastructure of β cells was observed, and pancreatic histopathological and immunocytochemical studies were performed using the anti CD 4 or anti CD 8 monoclonal antibodies. Results Immunization with GAD reduced the incidence of diabetes (6.25% vs 73.68%, P<0.01) and delayed its onset as compared with FIA mice at age of 20 weeks. The serum C peptide levels were significantly higher in GAD group than that in control group (P<0.05), the lymphocytic inflammation of pancreatic islets showed a switch from severe insulitis in control mice to peri insulitis in GAD treated mice with the insulitis score decreased markedly (P<0.01) in the GAD group. A strong proliferative response of CD + 8 subsets in splenocytes occured in GAD group, and the imbalance of CD 4/CD 8 ratio in control mice was corrected (P<0.01). The lymphocytes infiltrating islets showed a marked reduction of CD + 4 T cells, even the small number of CD + 4 subsets existed only outside the islets in GAD immunized mice. The prevalence of GAD antibody showed no significant difference between these two groups. Conclusion Gad prevents the onset of type 1 diabetes and reduces the severity of insulitis in female NOD mice. Its mechanisms seem to be related to the improvement of CD + 8 T cell deficiency.