耐具核梭杆菌拮抗的双歧杆菌及其重组活载体防治结直肠癌的实验研究

Intestinal flora plays an important role in the occurrence and treatment of tumor. The dysbiosis of the human intestinal microbiota is linked to colorectal cancer (CRC). The increase number of the tumor-associated bacteria such as Fusobacterium nucleatum (F. nucleatum), and the decrease number of probiotic such as bifidobacteria was the important factor to induce the tumorigenesis of CRC. Although it is found that supplementation of probiotics has a specific inhibitory effect on animal intestinal tumors, it is difficult to achieve the desired effect in human clinical trials. Our previous study found that secretion of bacteriocin-like substance (BLS) of F. nucleatum which have stronger antagonistic capability to G + bacillus, inhibit the growth of probiotics including bifidobacteria, Lactobacillus, and so on. The DNA copy of F. nucleatum and bifidobacteria showed a negative correlation in the intestinal tract of CRC patients. Our findings provide new clues for the use of probiotics intervention strategies for human CRC. In the present project, we will screen drug resistance bifidobacteria through induction of BLS resistant to bifidobacteria, which means the bifidobacteria have the resistance to F. nucleatum. We will reconstruct the balance of microbiota of intestinal and use of the strategy to explore effective in the prevention and indoleamine 2,3-dioxygenase inhibitor therapy of CRC; In addition, we will further prepare the apoptosis peptide, which were expressed by recombinant Bb bacteria and targeted to solid tumor by hypoxia, to explore targeted CRC treatment of live vector.

肠道菌群在肿瘤的发生及治疗中均发挥重要作用。现已证实结直肠癌（CRC）患者肠道菌群失调：具核梭杆菌（Fn）等促癌细菌数量增高，双歧杆菌（Bb）等益生菌数量降低是CRC发生发展的重要因素；且Bb菌能提高免疫检测点抑制剂的抗癌疗法效果。尽管在实验动物研究中发现补充益生菌对肠道肿瘤具有明确抑制作用，但令人困惑的是在人体临床试验中却很难达到预想效果。本项目前期研究发现：Fn分泌的细菌素类似物（BLS）对G+杆菌具拮抗作用，抑制Bb菌、乳杆菌等益生菌的生长，致使CRC患者肠道内Fn与Bb的数量呈现反向相关。此发现为利用益生菌干预人类CRC策略提供了新的线索。本项目拟通过诱导法筛选BLS耐药株（即耐受Fn菌拮抗的Bb株），重构肠道益生菌和Fn之间平衡，并利用该策略探讨其在CRC的预防和IDO抑制剂免疫治疗中的有效性；并利用Bb对实体瘤的低氧靶向性，构建表达穿膜凋亡肽的重组菌，探索抗肿瘤活载体治疗策略。

结直肠癌（CRC)患者肠道菌群失调，表现为具核梭杆菌（F. nucleatum, Fn）等促癌细菌数量显著增高，双歧杆菌（Bifidobacterium, Bb）等益生菌数量显著降低现象。补充耐受Fn菌的益生菌，重构肠道微生态平衡将有助于CRC的预防和治疗。本项目筛选出耐具核梭杆菌的双歧杆菌和乳酸杆菌，并基于粪便培养组学分离出多株新的益生菌；通过qPCR定量分析CRC患者粪便中Fn菌和益生菌的数量，阐明了Fn菌对益生菌双歧杆菌等的杀伤作用，筛选出用于结直肠早期筛查的粪便微生物标志物（Fn/Bb +Fn/Fp)；克隆表达Fn菌新的毒力因子NAP（FN1079）蛋白，证实了Fn1079通过CXCL3/CXCR2轴具有强的促肿瘤转移作用；此外，通过多组学手段解析了Fn菌感染结肠癌细胞后分泌的外泌体携带特异性miR-1246/92b-3p/27a-3p和特异性蛋白CXCL16/IL8/Rho促结直肠癌转移的新机制；开展粪便培养组学筛选本土人群益生菌，建立了本土益生菌库，分出两株新菌种，已发掘具商业化价值的乳酸菌和双歧杆菌菌株多株，其中一株婴儿型本土耐酸的副干酪乳杆菌已完成商业转化。以上研究为结直肠癌的早期筛查、清除具核梭杆菌、补充耐受具核梭杆菌的益生菌调节肠道微生态平衡有积极的预防和治疗作用。