es (6.25% vs 73.68%, [ WT9.BX P＜0.01) and delayed its onset as compared with FIA mice at ag e of 20 weeks. The serum C-peptide levels were significantly higher in GAD group th an that in control group (P＜0.05), the lymphocytic inflamma tion of pancreatic islets showed a switch from severe insulitis in control mice to peri-insulitis in GAD-treated mice with the insulitis score decreased marke dly (P＜0.01) in the GAD group. A strong proliferative respo nse of CD+8 subsets in splenocytes occured in GAD group, and the imbalance o f CD4/CD8 ratio in control mice was corrected (P＜0.01). T he 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 di fference between these two groups. Conclusion G ad prevents the onset of type 1 diabetes and reduces the severity of insulitis i n female NOD mice. Its mechanisms seem
to be related to the improvement of CD+ 8 T cell deficiency. 【Key words】 Glutamate decarboxylase; Mice, inbred NOD; Diabetes mellitus, insulin-dependent; T-lymphocyte subsets
非肥胖糖尿病(NOD)小鼠是一种自发性自身免疫性糖 尿病(DM)动物模型〔1〕。近几年国内外已有人应用谷氨酸脱羧酶(GAD)预防1型D M的尝试，表明重组人GAD65、GAD67或其活性肽段对NOD小鼠的1型DM有一定的预防或延缓作 用〔2-6〕。本研究用猪脑GAD早期免疫NOD雌性小鼠，了解其是否具有预防胰岛炎和 糖尿病发生的作用，探讨其可能的作用机制。
材料和方法
一、材料
1.动物：为NOD/Lt种小鼠。种鼠购自北京中科院实验动物研究所。在标准环境条件下饲养， 繁殖出第一代，取雌性小鼠用于实验。
2.试剂：GAD由本实验室从猪脑提纯〔7〕，为含GAD65和GAD67的混合物，用时稀 释至1 μg/μl；不完全弗氏佐剂(FIA)为Sigma公司产品。其余试剂均为AR规格。
二、方法
1.小鼠的分组与处理：4周龄时，选体重＞10g的雌性小鼠编号后分两组用药：(1)FIA对照组 (19只)：生理盐水50 μl与FIA 50 μl混匀后一次性腹腔注射(ip)；(2)GAD免疫组(32只)： GAD 50 μg与FIA 50 μl混匀后i.p.；两组皆于鼠龄8周时环磷酰胺250 mg/kg i.p.一次， 以加速糖尿病；所有小鼠从4周龄开始，每周测定体重、血糖，确诊糖尿病2周后或鼠龄20周 时处死小鼠；死前用摘眼球法取全血，4℃离心分离血清置-20℃保存以备指标测定；小鼠 死后立即取出胰腺，作如下处理：(1)置苦 味酸-多聚甲醛溶液固定24 h，洗涤数次后80%乙醇保存，备做组织病理检查；(2)用OCT包埋，干冰-丙酮法制成组织冻块，置-70℃备做免疫组化；( 3)1.5%戊二醛固定后送电镜观察；同时取胸腺和脾脏用铝箔包裹后置-70℃备用。
2.指标测定：用拜耳公司的快速血糖仪测定尾静脉血血糖(
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