基于条件重编程细胞的过敏性结膜炎模型及TLR7的激活对过敏晚期反应的免疫调节

The “late-phase reaction” of allergic conjunctivitis leads to destruction of conjunctival epithelium barrier and keratopathy which induces sight-threatening. So, we need to study the mechanism of conjunctival epithelial cell in response to anaphylaxis to control the allergic reaction and maintain the integrity of conjunctival epithelium. But there is still no suitable in vitro model for allergic conjunctivitis. We co- operated with the inventor of conditional reprogramming (CR) technology, and established CR- human conjunctival epithelial cells, which can proliferate rapidly and stable passages in vitro with conjunctival epithelium specificity and developmental potential. In our early study, we found that activation of TLR7 by imiquimod treatment impairs the secretion of proinflammatory cytokines, but the mechanism is still unclear. In this study, we will analyses and identify the differential TLR7-binding proteins by using Mass spectrometry based on CR-conjunctival epithelial cell model, and establish the knockout mice to study the function of specific protein in vivo. We will identify the related proteins and signaling pathways activated by TLR7 in the late reaction of allergic conjunctivitis, and elucidate the molecular mechanism of TLR7 involved in the late reaction of allergic conjunctivitis, and reveal the theoretical basis of TLR7 as a new target for the treatment of allergic conjunctivitis.

过敏性结膜炎“晚期反应”导致结膜上皮细胞受到破坏，严重时合并角膜病变会使视力严重下降。如何控制过敏反应以维持结膜上皮完整性，则需要对上皮细胞应答过敏反应的机制进行深入研究，但是目前仍缺乏合适的过敏性结膜炎体外研究模型。前期我们与条件重编程(CR)技术的发明人合作，建立了CR-人结膜上皮细胞，该细胞可在体外快速增殖、稳定传代，并具有结膜上皮细胞的组织特异性和分化潜能。前期研究发现TLR7的激活可以抑制CR-人结膜上皮细胞的过敏反应，但具体调控机制尚未阐明。本研究将通过建立的CR-人结膜上皮细胞过敏性结膜炎模型，利用质谱技术对TLR7激活前后结合的差异蛋白进行分析鉴定；通过基因敲除小鼠，对差异蛋白的调控功能进行体内研究，从而鉴定过敏性结膜炎晚期反应时TLR7激活的相关蛋白和信号通路，并阐明TLR7参与调控过敏性结膜炎“晚期反应”的分子机制，揭示TLR7作为抗过敏性结膜炎治疗新靶点的理论依据。

过敏性结膜炎“晚期反应”导致结膜上皮细胞受到破坏，严重时合并角膜病变会使视力严重下降。如何控制过敏反应并且维持结膜上皮完整性，则需要对上皮细胞应答过敏反应的机制进行深入研究。我们建立了CR-人结膜上皮细胞，该细胞可在体外快速增殖、稳定传代，并具有结膜上皮细胞的组织特异性和分化潜能。利用过敏反应的主要炎症因子IL-1β刺激CR-人结膜上皮细胞，可以分泌IL-6、IL-8等一系列前炎症因子，建立了一个新的研究过敏性结膜炎的体外细胞模型，申请专利并获得授权：人正常结膜上皮细胞及其用途，专利号：ZL 2018 1 0258760.0。并且已经发表了2篇论文，在国际学术会议上进行了交流。.利用这一新的细胞模型，我们发现IL-1β刺激CR-人结膜上皮细胞后，细胞分泌的IL-6和IL-8随刺激时间延长而增加，通过检索文献发现，目前没有IL-1β是否能诱导细胞分泌IL-6的相关研究。为了进一步确定IL-1β的作用，我们使用IL-1β受体的拮抗剂、IL-1β的单抗分别切断IL-1β信号的传递，进而希望发现新的IL-1β信号通路并明确这条通路的调控在治疗过敏性结膜炎方面的应用前景，相关研究成果已经在投稿中。