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

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

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。

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 intramusclar injections of 0 05 ml PBS alone , 50 μg pcDNA3 1 and 50 μg DNA vaccine emulsified in 0 05 ml PBS 7 days apart respectivly. The accumulative diabetes incidence was followed up to 30weeks 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 β 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 γ, NF ATc and NF ATp mRNA levels were tested by RT PCR. IL 4 and IFN γ 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 β 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.