人谷氨酸脱羧酶65 DNA疫苗预防非肥胖糖尿病小鼠糖尿病的机制探讨

目的　探讨人谷氨酸脱羧酶 6 5 (GAD6 5 )DNA疫苗预防非肥胖糖尿病 (NOD)小鼠糖尿病的作用机制。方法　 (1) 6 2只 4周龄NOD雌鼠分为PBS(2 1只 )、PcDNA(2 0只 )、GAD6 5 (2 1只 ) 3组 ,由胫前肌分别注射PBS、质粒PcDNA3 1、人GAD6 5DNA疫苗 5 0 μg ,1周后重复 1次。观察 30周龄的累积糖尿病发病率。 (2 )各组取 12周龄未发病NOD鼠 (n =10 )胰腺HE染色观察胰岛炎 ;并用末端脱氧核糖核酸缺口标记法 (TUNEL)加SABC法检测胰岛 β细胞凋亡 ;ELISA法测定血清、脾细胞培养上清干扰素γ(IFN γ)和白细胞介素 4 (IL 4 )水平 ;RT PCR半定量检测脾脏IL 4、IFN γ和核因子NF ATc、NF ATpmRNA表达水平。结果　 (1) 30周龄时 ,PBS、PcDNA、GAD6 5组发病率分别为 95 2 %、80 0 %、6 1 9%。GAD6 5组发病率低于PBS组 (P =0 0 0 8)。 (2 ) 12周龄时GAD6 5组胰岛炎积分 (0 99± 0 71)和胰岛 β细胞凋亡率 (0 75 % )均低于PBS组 (2 16± 0 78,P =0 0 0 1;8 97% ,P=0 0 14 )和PcDNA组 (1 72± 0 5 9,P =0 0 2 7;2 6 5 % ,P =0 0 2 3)。GAD6 5组脾脏NF ATc、IL 4mRNA相对吸光度值及血清IL 4水平分别为 1 93± 0 34、0 70± 0 16、36 pg/ml± 8pg/ml,显著高于PBS组 (0 79± 0 15、0 4 9± 0 11、19pg/ml

Mechanisms of human glutamic acid decarboxylase 65 DNA vaccine preventing diabetes in non-obese diabetic mice

OBJECTIVE: To investigate the mechanisms of human GAD65 DNA vaccine preventing insulitis and diabetes in NOD mice. METHODS: Female NOD mice at 4 weeks of age were randomly divided into PBS (n = 21), pcDNA (n = 20), and hGAD65 (n = 21) groups. Mice in each group received two intramuscular injections of 0.05 ml PBS alone, 50 microg pcDNA3.1 and 50 microg DNA vaccine emulsified in 0.05 ml PBS 7 days apart respectively. The accumulative diabetes incidence was followed-up to 30 weeks of age in each group of NOD mice. Pancreas was removed from NOD mice at 12 weeks of age in each group (n = 10) to score insulitis severity by routine H-E staining. The apoptotic beta cells in islets were observed with double-labeling technique of TUNEL in situ combined standard sensitive avdin-biotin complex (sABC) immunohistochemical method. Their spleens were for cell culture and total RNA extraction. Spleen IL-4, IFN-gamma, NF-ATc and NF-ATp mRNA levels were tested by RT-PCR. IL-4 and IFN-gamma levels in sera and supernatants of spleen cells were measured by ELISA. RESULTS: (1) At 30 weeks of age, the diabetes incidence was 95.2%, 80.0% and 61.9% in PBS, pcDNA and hGAD65 group respectively. The diabetes incidence in the PBS group was higher than that in hGAD65 group (P = 0.008). (2) At 12 weeks of age, the insulitis scores in hGAD65 group was lower than that in PBS group (P = 0.001) and pcDNA group (P = 0.027) respectively. (3) The apoptotic beta cell rates in hGAD65 group was lower than that in PBS group (P = 0.014) and pcDNA group (P = 0.023). (4) IL-4 levels in sera, spleen IL-4 and NF-ATc mRNA level in hGAD65 group were higher than those in PBS group (all P < 0.05) and pcDNA group (all P < 0.05) respectively, NF-ATp mRNA level in hGAD65 group was lower than that in PBS group (P < 0.05). CONCLUSION: Human GAD65 DNA vaccine via downregulating NF-ATp and upregulating NF-ATc and IL-4, makes Th cells deviate to Th2, and sequently prevents insulitis, beta-cell apoptosis and diabetes onset in NOD mice.