联合注射自身胰岛细胞瘤相关抗原2基因疫苗预防NOD小鼠自身免疫性糖尿病

Vaccination with autoantigen IA-2 to prevent NOD mouse from developing autoimmune diabetes

目的 探讨自身抗原基因疫苗胰岛素瘤相关蛋白-2（pIA-2）以及分子免疫佐剂白细胞介素-4/单核细胞趋化因子-1（pIL-4/MCP-1）对糖尿病前期NOD小鼠发生自身免疫性糖尿病的预防作用.方法 利用分子克隆技术构建pIA-2自身抗原基因疫苗,将4～5周龄NOD小鼠分为pIA-2治疗组（n=5）、pIL-4/MCP-1治疗组（n=5）、联合治疗组（n=5）及对照组（n=5）,分别注射pIA-2、pIL-4/MCP-1、pIA-2联合pIL-4/MCP-1、增强型绿色荧光蛋白（pEGFP）,免疫结束后观察24周,比较各组糖尿病发病情况和胰岛炎的程度,并用RT-PCR方法和免疫组化法检测质粒在注射部位和胰岛的表达.两样本间均数的比较用t检验,率的比较用x2检验进行统计.结果 单用pIA2或pIL-4/MCP-1治疗的NOD小鼠发病与对照组相比无显著改善;联合应用该两种质粒的NOD小鼠在观察期内未发病.RT-PCR检测结果显示免疫结束后2周,注射部位肌细胞中均有胰岛细胞瘤相关抗原2（IA-2）及白细胞介素-4（IL-4）表达.免疫组化法发现,免疫结束后2周,pIA-2注射组小鼠胰岛的IA-2表达量增加;IL-4在胰岛上可见少量表达.免疫结束后观察24周,检测各组的胰岛炎发生率,对照组、pIA2组、pIL-4/MCP-1组、pIA2＋pIL-4/MCP-1组中无胰岛炎的发生率分别为4.2%（1/24）、5%（1/20）、0（0.0）、62.5%（15/24）,外周性胰岛炎发生率分别为12.5%（3/24）、10%（2/20）、5%（1/20）、29%（7/24）,中心性胰岛炎发生率（＜50%、＞50%）分别为[50%（12/24）,33.3%（8/24）]、[40%（8/20）,45%（9/20）]、[45%（9/20）,50%（10/20）]、[4.2%（1/24）,4.2%（1/24）].和对照组相比,联合治疗组中不发生胰岛炎的比率显著升高（x^2=18.38,P＜0.01）,发生中心性胰岛炎的比率显著降低（x^2=11.68,P＜0.001）,其余各组和对照组相比胰岛炎的发生比率无显著差异.以免疫组化法检测NOD小鼠胰岛内IA-2、IL-4的表达,显示免疫注射组IA-2、IL-4表达较对照组增强.结论 联合应用pIA-2及pIL-4/MCP-1基因疫苗提前免疫胰岛炎前期的NOD小鼠可抑制和延缓糖尿病的发生.

Objective To explore DNA vaccination encoding IA-2 combined immune adjuvant pIL4/MCP-1 to prevent the preclinical diabetic NOD mice from developing autoimmune diabetes. Methods The plasmid pEGFP/IA-2 was recombined with gene cloning technology. Four groups 4-5wks age NOD mice were injected with pIA-2,pIL-4/MCP-1,pIA-2 and pIL-4/MCP-1,pEGFP respectively for three times and monitored glucose and insulitis level during 24 weeks period. The expression of pIA-2 was also detected with RT-PCR and immunohistochemological methods. Results Vaccination of 4-5 week age female NOD mice with the ⅠA2-plasmid DNA or Ⅱ4/MCP-1 alone could not prevent the dia^tes and delay onset of disease when compared to the controls. None of NOD mouse developed diabetes at 31 to 32 weeks age when combined vaccination with these two plasmids DNA. RT-PCR results showed IA-2 and IL-4 mRNA in the injected muscle. IA-2 expression in islet of pIA-2 treated mice was more than that of control group. The incidence rates of non-insulitis islets for control group,pIA2,pIL-4/MCP-1,pIA2 ＋ pIL-4/MCP-1 were 4. 2% （1/24）,5% （1/20）,0（0.0） ,62.5% （15/24） ,the same incidence rates of peripheral non-insulitis islets were 12.5% （ 3/24 ）,10% （ 2/20 ）,5% （ 1/20 ）,29% （ 7/24 ）,the incidence rates of centrality inflammation of pancreatic （ 〈 50%,〉 50% ） islet were （ 50% （ 12/24 ）,33.3% （ 8/24 ） ）,（ 40% （ 8/20 ）,45% （9/20））,（45% （9/20） ,50% （10/20） ）,（4. 2% （1/24） ,4.2% （1/24） ）. Central insulitis in combined vaccination group was much less than that in control group （ x^2 = 11.68,P 〈 0. 001 ） while the number of non-insulitis islets in combined vaccination group were much higher than that in control group （ x^2 = 18.38,P 〈 0. 01 ）. The immunohistochemistry showed that the expression of IA-2 and IL-4 in pancreatic islets in vaccination group were much higher than those in control group. Conclusion Covaccination with DNA of IA2 and IL-4/MCP-1 is quite effective in abrogating diabetes in predinical diabetic NOD mice.