靶向B7:CD28共刺激信号系统的新型DNA疫苗治疗I型糖尿病的效应与机制研究

It has been proved that type 1 diabetes mellitus(T1DM) is a organ specific important autoimmune disease characterized by infiltration of multiple inflammatory cells (mainly T cells), causing insulitis and immune destruction of the insulin-producing β cells of the panereatie islets, leading to insulin deficiency, and hyperglycemia. Current clinically available treatments for T1DM fail to cure the disease or unsatisfactorily halt disease progression, thus, T1DM remains incurable disease. To overcome these limitations, the development of therapeutic DNA vaccines and boosters may offer new promising strategies. Based on the facts that there are quite a bit of abnormal activation of both CD28+CD4+Th1(Th1) and CD28+CD8+CTL(CTL) effector cells, and quantity or/and quality declines of both CD4+CD25+Treg(Treg) and CD8+CD28-Ts(Ts) regulatory cells in T1DM, we originally put forward a new idea that the development of a novel therapeutic DNA vaccine targeted B7-1/CD28 costimulatory signal system for treating effectively T1DM. The developed novel DNA vaccine could specifically kill both of Th1 and CTL effector cells, and selectively enhance the quantity or/and quality of both Treg and Ts regulatory cells. Furthermore, we will systemically evaluated the curtive effects of the novel DNA vaccine on non-obese diabetie(NOD) mouse by a series of clinical indexes, blood glucose analysis, histopathological examination of panereatie islets and immunlogical indexes. In addition, we will also explore preliminarily the mechanisms of the novel DNA vaccine. We expect that our study will provide a new strategy for clinical treatment of T1DM. To our knowledge, far not see the same study reported.

业已证实，I型糖尿病(T1DM)主要是由T淋巴细胞介导的器官特异性重大自身免疫性疾病(IAIDs)，临床上治疗甚为棘手。近年来国际上探索采用DNA疫苗治疗IAIDs已成该领域倍加关注的热点之一。鉴于T1DM的发生与CD28+CD4+Th1(Th1)与CD28+CD8+CTL(CTL)效应细胞的异常活化尤其是与CD4+CD25+Treg(Treg)与CD8+CD28-Ts(Ts)调节性细胞的数量/功能下降紧密相关这一事实，我们在国际上原创性提出研制基于B7-1/CD28信号系统能靶向杀伤Th1/CTL效应细胞而又选择性促进Treg/Ts调节性细胞，从而达到能有效防治T1DM的新型DNA疫苗设想。通过一系列临床指标、血糖测定、胰岛组织病理、免疫学功能检测，系统观察该新型DNA疫苗防治非肥胖I型糖尿病小鼠的效果，初步探讨其作用机制，为临床T1DM的防治提供新策略。经查新证实这在国际上尚未见报道。

I型糖尿病（T1DM）是一种严重危害人类健康的多发性、难治性自身免疫性疾病。时至今日，国际上针对T1DM的主流治疗策略始终是采用胰岛素或胰岛素类似物的长期补充疗法。长期使用胰岛素常常会带来诸多的严重毒副作用。鉴于 B7:CD28/CTLA4 共刺激信号途径在介导T1DM发生发展的免疫病理生理过程中发挥重要作用这一事实，我们在国际上率先原创性地提出研制基于B7:CD28信号系统靶向杀伤高表达CD28+CD4+Th1及CD28+CD8+CTL细胞而选择性促进外周血CD4+CD25+Treg以及CD8+CD28-Ts细胞两类调节性T细胞从而达到能有效治疗T1DM的新型DNA疫苗的设想。.首先，我们分子设计并构建优化成功治疗性基因疫苗 pcDNA3.1/Zeo(+)-B7-2-PE40真核表达载体。肌肉注射一次，在2-50天内均可检测出B7-2-PE40融合基因的mRNA。尤其是对正常鼠无任何毒副作用，病理组织学检查显示，心、肝、脾、肺、肾脏器均未发生明显的病理学改变。.其次，系统证实所研创的该新型DNA疫苗能有效防治T1DM。在治疗性组，该DNA疫苗能有效治疗T1DM小鼠NOD/LTJ，使血糖降低、血胰岛素增加，其效果明显优于甲氨蝶呤阳性对照。在预防组，该疫苗能非常显著地预防T1DM小鼠NOD/LTJ的发生与发展，使T1DM小鼠NOD/LTJ的血糖含量保持在正常水平、血胰岛素含量明显增加。.第三，进一步证实该DNA疫苗在有效防治T1DM模型中，能显著降低T1DM小鼠NOD/LTJ血中自身抗体ICA和GAD浓度。其效果明显优于甲氨蝶呤阳性对照。尤其是自身抗体GAD的降低在预防组效果十分突出，而自身抗体ICA的降低在治疗组与预防组是相同的。而自身抗体IAA无显著变化。.最后，对作用机理的初步研究表明，该DNA疫苗在体内能有效靶向杀伤CD28+ T细胞，提高CD4+CD25+ Treg及CD8+ CD28-Treg细胞亚群的比例；有效纠正T1DM产生的Th1偏移,诱导Th1向Th2/Th3转化，明显降低炎性细胞因子浓度,促进抗炎细胞因子分泌。免疫病理检查进一步揭示，该DNA疫苗能有效减少胰岛炎性的严重程度，有效恢复受损胰岛细胞的数量和功能。.这是具有我国完全自主知识产权在国际上首创的第一个具有靶向性有效防治T1DM的全新型外毒素融合基因疫苗。为临床T1DM的治疗提供新策略。