CpG-A20纳米DNA载体治疗溃疡性结肠炎的作用及机制研究

Ulcerative colitis (UC) belongs to one of the inflammatory bowel disease (IBD), which is a significant public health problem with an increasing incidence. UC is characterized by inflammation in the colon associated with aberrant mucosal immune responses to constituents of the intestinal microbiota characterized by imbalances in effector versus regulatory immune cells activity. Dendritic cells play a pivotal role in initiating, regulating immune reaction. Tumor necrosis factor, alpha-induced protein 3 (A20) is a potent anti-inflammatory protein that utilizes de-ubiquitinating, E3 ligase and ubiquitin binding functions to regulate NF-κB signaling. The expression of A20 in dendritic cell preserves immune homeostasis and prevents colitis. In our previous study, A20 expressing eukaryotic expression vector was constructed with inserted CpG sequence. We will further investigate that：The optimum process conditions for the preparation of nano-DNA vector and cells absorption and metabolism in the body; The therapeutic effect of CpG-A20 expressing nano-DNA vector on the intestinal inflammation, Th cells differentiation, mucosa barrier function; Elucidating the underlying mechanism of A20 expressing nano-DNA vector regulating p53, mucosa epithelia cells and inflammatory cells involved. This study may provide important basic information to the targeted biological immunotherapy for ulcerative colitis.

作为常见的炎性肠病，溃疡性结肠炎（UC）发病机制的关键因素是免疫系统针对肠道菌群及食物抗原的异常炎症反应，目前国内外仍缺少针对UC靶向性治疗方法的研究。肠道树突状细胞（DC）处在启动、调控免疫反应的关键地位，通过表达A20可负性调控肠道免疫炎症、诱导耐受型DC（Tol-DC）的产生，有望成为UC治疗靶点。我们首次构建了插入CpG寡核苷酸序列，表达负性免疫调节分子A20的DNA载体（P-pCA），本课题拟在前期工作基础上进一步研究：P-pCA纳米DNA载体制备最佳工艺条件、细胞摄取情况、体内吸收及代谢；明确P-pCA纳米DNA载体对小鼠溃疡性结肠炎的治疗效果及对肠道局部炎症、Th细胞失衡及肠道黏膜屏障的调节作用；阐明P-pCA纳米DNA载体调控p53蛋白影响溃疡性结肠炎肠道黏膜上皮细胞及炎症细胞增殖及凋亡的分子机制。有望为炎性肠病靶向性生物治疗手段研发提供理论依据并开发出新型免疫药物。

近年来，随着人民生活水平的提高和环境的污染，IBD 发病率和患病率在我国呈明显增长趋势，严重影响患者生活和工作，给社会、经济带来巨大负担。目前国内外仍缺少针对溃疡性结肠炎靶向性DNA 载体治疗方法的研究。本研究探讨了一种表达去泛素化酶A20 的纳米DNA 载体靶向性治疗溃疡性结肠炎的作用及其机制，首先，我们构建了A20真核表达载体，并经聚乳酸-乙醇酸（PLGA）包封制备了A20纳米DNA疫苗，确定了其形态、大小、粒度分布等指标，评价了其转染进入细胞的效率。其次，构建了小鼠溃疡性结肠炎模型，并经结肠黏膜给予A20纳米DNA疫苗进行治疗，进一步评价了其对小鼠结肠炎症的抑制效果。最后，按照年度研究计划，评价了A20纳米DNA疫苗对结肠炎小鼠肠道黏膜屏障功能及相关炎症因子表达水平的影响，完成既定研究计划。研究结果显示，成功制备出了以PLGA 包封的pVAX1- A20 纳米DNA 载体，并可有效体外转染肠道上皮细胞； PLGA-pVAX1A20 纳米DNA 载体可以抑制溃疡性结肠炎小鼠结肠炎症，抑制NF-kB下游信号的激活，并诱导Treg细胞产生，降低炎性结肠黏膜通透性，抑制Th2细胞反应；肠道黏膜DC细胞可表达Foxp3，并可经葡萄球菌肠毒素刺激后上调Foxp3的表达；miRNA-17-92可抑制肠道黏膜B细胞中IL-10的表达。目前已发表课题相关SCI论文10篇以上，提交申请发明专利5项，培养硕士研究生3名，完成课题研究目标。本课题后续研究仍在进行中，通过本课题的实施，有望为炎症性肠病提供新的治疗方法。