艰难梭菌毒素B的适配体筛选及其抗感染机制研究

Clostridium diffcile (CD) has peak infection rate, high toxicity and high resistance to most of antibiotics, and its drug-resistance rate and recurrence rate is increasing. Therefore, a novel treatment was urgently in demand. The secreted toxic protein B (TcdB) of CD is proved to be the major virulence factor, which enters host cells through receptor-mediated pattern and elicits pathogenic effect. We used SELEX technology to screen for oligonucleotide aptamers that specifically bind to surface lipids of Mycobacterium tuberculosis and Bacillus Calmette-Guerin, and these aptamers were successfully used as anti-TB drugs and immunologic adjuvants for BCG. The aim of this project was to select the oligonucleotide aptamers that specifically bind to TcdB receptor binding domains (RBD) of CD. The aptamer will inhibit the binding of TcdB to its receptor FZD, thereby reduce or even restore the suppression of TcdB-induced Wnt classical signaling pathways. Subsequently, the aptamer were proved by in vitro study that relieve the cells damage caused by toxins and observed the therapeutic effect of the aptamer on an animal model of infection. This study will elucidate a new mechanism of CD pathogenicity and further establish the basis for new anti-CD drug development.

艰难梭菌（Clostridium diffcile，CD）感染率高、毒性强，由于对大多数抗生素具有高抗性，近年来其导致疾病的耐药率和复发率也持续上升，为此亟需新型治疗手段。CD分泌的毒素蛋白B（TcdB）被证明是CD致病的主要原因，且通过受体介导进入宿主细胞内部致病。申请人前期运用SELEX技术筛选出可特异性结合结核杆菌和卡介苗表面脂糖的寡核苷酸适配体，分别成功作为抗结核治疗的药物和提高卡介苗免疫原性的佐剂。本项目拟采用该技术以TcdB受体结合区（RBD）为靶标，筛选出与之特异性结合的寡核苷酸适配体，阻断TcdB竞争性结合其受体卷曲蛋白（FZD），进而减轻甚至恢复TcdB引起的Wnt经典信号通路抑制，随后通过体外细胞实验验证适配体缓解毒素引起的细胞损伤，并在动物感染模型上观察适配体的治疗效果，为阐明CD致病新机制并进一步研发新型抗CD药物奠定基础。

艰难梭菌（Clostridium diffcile，CD）感染率高、毒性强，其导致疾病的耐药率和复发率也持续上升，为此亟需新型治疗手段。CD分泌的毒素蛋白B（TcdB）被证明是CD致病的主要原因，且通过受体介导进入宿主细胞内部致病。本项目以TcdB受体结合区（RBD）为靶标，筛选出与之特异性结合的寡核苷酸适配体RA10，阻断TcdB竞争性结合其受体卷曲蛋白（FZD），进而逆转TcdB引起的Wnt经典信号通路抑制，降低TcdB对正常肠上皮细胞的损伤；另一方面，适配体RA10还能阻断TcdB进入巨噬细胞后与白介素增强子结合因子（ILF3）的结合，显著抑制TcdB介导巨噬细胞分泌IL-1β和IL-6等炎症因子，从而进一步缓解TcdB对肠上皮细胞的损伤，并在动物模型上显示出较好的治疗效果。本研究为CD感染的治疗提供了新的靶点和研究思路，并为进一步研发新型抗CD药物奠定了基础。