一种抗原基因耐受型DNA疫苗治疗大鼠胶原诱导关节炎的效应和机理研究

The exploration of the treatment strategy of rheumatoid arthritis (RA) has always been the focus and difficulty in the field of autoimmune diseases. Since the pathogeny of RA is not clear, no specific radical cure is found to date. It is reported that the heterologous type Ⅱ collagen administrated orally has been found available by inducing immune tolerance, while DNA vaccine offers the new promising strategies for the treatment of RA in recent years. Our previous results indicated that the naked DNA plasmid encoding chicken type Ⅱ collagen (cloned successfully in the early stage of this project) administrated by injection has shown the comparative efficacy in the treatment of RA as the current “gold standard” methotrexate (MTX), and no obvious side effects was observed. Therefore, we speculate that the gene of chicken type Ⅱ collagen can specifically express in vivo to induce immune tolerance and regulate the overall network balance of the immune system to treat RA effectively and safely. In this project, two major strategies of oral immune tolerance and DNA vaccine will be combined to develop an antigenic gene tolerizing oral DNA vaccine with the gene of chicken type Ⅱ collagen carried by the attenuated Salmonella typhimurium SL7207. Subsequently, the biological effects of the oral DNA vaccine will be evaluated systemically on RA rat. Furthermore, it will be explored and revealed that the related mechanisms of inducing immune tolerance and regulating immune balance through gastrointestinal mucosal pathway. The results are expected to provide a new strategy for the future clinical treatment of rheumatoid arthritis.

类风湿性关节炎（RA）治疗策略的探索始终是自身免疫性疾病研究领域前沿的焦点和难点。由于其发病机制尚未完全明确，迄今尚无特异性根治疗法。文献报道口服异源Ⅱ型胶原诱导免疫耐受治疗RA初见成效，DNA疫苗又是近年来备受国际推崇用于探索治疗RA的重大策略之一。本项目组前期成功克隆编码鸡Ⅱ型胶原的DNA裸质粒注射免疫模型动物后亦显示其与目前治疗RA的“金标准”药物甲氨蝶呤疗效相当，且没有明显副作用。由此我们推测：该基因可在体内特异表达以诱导免疫耐受从而调节免疫系统整体网络平衡来有效安全地治疗RA。本项目拟将口服免疫耐受与DNA疫苗两大策略充分结合，研制以减毒鼠伤寒沙门氏菌SL7207为载体，以鸡Ⅱ型胶原基因为核心的抗原基因特异耐受型DNA疫苗，并系统评价其口服治疗大鼠关节炎的生物学效应，探讨通过胃肠黏膜途径诱导机体产生免疫耐受进而调节免疫平衡的相关机理。研究结果有望为临床RA的治疗提供新策略。

类风湿性关节炎（RA）治疗策略的探索始终是自身免疫性疾病研究领域前沿的焦点和难点。由于其发病机制尚未完全明确，迄今尚无特异性根治疗法。文献报道口服异源Ⅱ型胶原诱导免疫耐受治疗RA初见成效，DNA疫苗又是近年来备受国际推崇用于探索治疗RA的重大策略之一。本项目组前期成功克隆编码鸡Ⅱ型胶原的DNA裸质粒注射免疫模型动物后亦显示其与目前治疗RA的“金标准”药物甲氨蝶呤疗效相当，且没有明显副作用。由此我们推测：该基因可在体内特异表达以诱导免疫耐受从而调节免疫系统整体网络平衡来有效安全地治疗RA。本项目研制成功一种以减毒鼠伤寒沙门氏菌SL7207为载体，以鸡Ⅱ型胶原基因为核心的特异性耐受型DNA疫苗SL7207/pcDNA-CCOL2A1，经验证可以在回肠组织内有效表达。进一步通过体征观察、关节肿胀评价、放射影像学检查、组织病理学检查以及血清中类风湿相关抗体因子的检测等系统评价了其口服治疗大鼠RA的生物学效应，结果显示低剂量DNA疫苗预防组（108CFU/只）与高剂量DNA疫苗治疗组（1010CFU/只）可安全有效地延缓关节炎疾病进程并降低发病率，同时显著降低抗Ⅱ型胶原抗体及抗环瓜氨酸肽抗体水平。最后通过基因表达-时间分析、粘膜免疫分泌型IgA抗体测定、促炎/抑炎细胞因子高通量测定、淋巴细胞增殖试验以及外周血T淋巴细胞亚群流式分析等，探讨分析了DNA疫苗通过胃肠黏膜途径防治RA的作用机理，初步研究表明该DNA疫苗在体内能提高CD3+CD8+C28- Ts及CD4+CD25+FoxP3+ Treg细胞比例，有效抑制CD3+CD8+CD28+ Tc与Th细胞比例，明显降低Th1型促炎细胞因子与相关趋化因子水平，促进Th2与Th3型抑炎细胞因子分泌。本项目将口服免疫耐受与DNA疫苗两大策略充分结合，实现了DNA疫苗口服给药调节免疫系统整体网络平衡并诱导免疫耐受治疗RA的目标，回答了特异抑制炎性疾病进程而又不诱导普遍免疫抑制的关键科学问题。研究结果为基因疫苗类药物的临床应用提供了新参考，为临床类风湿性关节炎的防治提供了新策略。