人胰岛素原DNA疫苗预防非肥胖糖尿病鼠胰岛β细胞凋亡的机制探讨

Mechanisms of human proinsulin DNA vaccine preventing islet beta-cell apoptosis in NOD mice

目的探讨人胰岛素原DNA疫苗预防非肥胖糖尿病(NOD)小鼠胰岛β细胞凋亡的机制。方法(1)4周龄NOD雌鼠随机分为磷酸盐缓冲液(PBS)、质粒载体PcDNA3.1(PcDNA)、胰岛素原(PIns)3组,由胫前肌分别注射PBS、质粒PcDNA3.1、人胰岛素原DNA疫苗50μg,1周后重复1次。(2)各组取12周龄未发病的NOD鼠10只,分离胰腺,HE染色观察胰岛炎;TUNEL+SABC法检测胰岛β细胞凋亡;取脾制成细胞悬液培养72 h,ELISA法测定血清白介素4(IL-4)和γ干扰素(IFN-γ)水平;MTT法检测脾细胞增殖反应。结果(1)12周龄时PIns组正常胰岛比例高于PBS组和PcDNA组(均P<0.01),胰岛周围炎比例低于PcDNA组(P=0.02),胰岛内炎比例则低于PBS组(P<0.01)和PcDNA组(P=0.006);(2)PIns组胰岛β细胞凋亡率低于PBS组和PcD-NA组(均P<0.01);(3)PIns组血清IL-4/IFN-γ比值显著高于PBS组(P=0.014)和PcDNA组(P=0.036);(4)PIns组脾细胞对人胰岛素增殖反应的刺激指数(SI)低于PcDNA组(P=0.021)。结论人胰岛素原DNA疫苗通过上调IL-4/IFN-γ比值,使免疫平衡向Th2偏移,致NOD鼠胰岛炎减轻,胰岛β细胞凋亡减少。

Objective To investigate the mechanisms of human proinsulin DNA vaccine preventing islet β-cell apoptosis in NOD mice. Methods Female NOD mice at 4 weeks of age were randomly divided into PBS, pcDNA, and Pins groups. Mice in each group received two intramusclar injections of 0.05 ml of PBS alone, 50μg of pcDNA3.1 and 50μg of human proinsulin DNA vaccine emulsified in 0.05 ml PBS at day 1 and 8. 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 13 cells in islets were observed with double-labeling technique of TUNEL in situ combined with standard sensitive avidin-biotin complex （sABC） immunohistochemical method. MTT was used for detecting spleen cell proliferative response to human insuln. IL-4 and IFN-γ levels in sera were measured by ELISA. Results （1） At 12 weeks of age, the ratio of infiltrated islet in Pins group was higher than that in PBS group （χ^2 =27.5, P〈0. 001） and pcDNA group （χ2 =21.8, P〈0. 001）, the ratio of peri-insulitis was lower in Pins group than that in pcDNA group （χ^2 =5.4, P=0.02）, the ratio of intraislet insulitis was lower in Pins group than that in PBS group （χ^2 =20. 1, P〈0.001） and pcDNA group （χ^2 =7.5, P= 0. 006） respectively. （2） The apoptotic β cell rates in Pins group was lower than that in PBS group（χ^2 =80.8, P〈0. 001） and pcDNA group （χ^2 =62.2, P〈0. 001）. （3） The ratio of IL-4/ IFN-γ in sera was higher in Pins group than that in PBS （t=2.66, P=0.014） and pcDNA group（P=0.036） respectively. （4） The stimulative index （SI） of spleen cell to human insuln was lower in Pins group than that in pcDNA group （P=0. 021）. Conclusion Human proinsulin DNA vaccine upregulating the ratio of IL-4/ IFN-γ makes Th cells deviate to Th2, and subsequently prevents insulitis and beta-cell apoptosis in NOD mice.