高通量测序鉴定导致结直肠病变的肠道“核心菌群”及其辅助诊断价值

Colorectal cancer (CRC) is a serious threat to human healthy. Early diagnosis of CRC is particularly important, as the mortality is closely related to the stage of disease at time of diagnosis. Recent studies had shown the structure changes of intestinal microbial community in the samples of CRC patient, indicating the potential association of intestine microbial structure changes and developing of CRC. However, little has been done to investigate the interactions of host-hut microbiota, and whether there is “core” pathogenic gut bacteria participated in developing of CRC still remain unknown. Our previous high-throughput sequencing studies had demonstrated dynamic changes of intestinal microbial community structure at different stages of the CRC, suggesting “cross-talk” interactions between gut microbiota and host. Based on the results, we speculate that the significantly changed gut flora included the “core” pathogenic bacteria that play key roles in carcinogenesis. Next, we will increase sequencing numbers of the CRC samples to preliminarily analyze the composition of the “core” pathogenic bacteria, and then verify them by qPCR. Subsequently, the effect of the “core” pathogenic bacteria on key genes involved in carcinogenesis will be studied by the bacteria-animal model/human intestinal epithelial cell interactions. Our potential results not only can confirm the composition of “core” pathogenic bacteria, but also can give a clue for understanding the preliminary roles of intestinal flora in the carcinogenesis of colorectal cancer. Finally, the distribution rule of “core flora” in stool samples will be examined through a comparative analysis of healthy people, patients with bowel polyps, adenomas and colorectal cancer, and then the possibilities to use “core” pathogenic bacteria as auxiliary biomarkers for early diagnosis of colorectal cancer will be evaluated.

结直肠恶性病变严重威胁人类健康，其高致死率与早期诊断困难紧密相关。最新研究表明肠道菌群结构变化与结直肠癌发生发展密切相关；然而菌群与宿主的相互作用关系及其中是否存在发挥主要致病作用的“核心”细菌等问题均未阐明。本课题前期测序分析初步发现了肠道菌群构成在不同分期的癌／癌旁组织中呈现动态变化；推测肠道菌群与宿主发生“对话式”交互作用导致了肿瘤发展和菌群动态改变；在显著变化细菌中，可能包含着在癌变中发挥重要作用的“核心菌群”。本课题拟通过增加样本量，初步确定“核心菌群”组成并利用实时定量PCR进行确认；随后挑取2－4种可培养细菌感染肠道上皮细胞系，分析其对结直肠癌发生关键基因的影响，以期为初步了解“核心菌群”在结直肠肿瘤发生中的作用机制奠定基础；最后将通过比较分析健康人群、肠息肉患者、腺瘤患者和结直肠癌患者粪便样本中“核心菌群”的分布规律,探讨其作为结直肠癌早期诊断辅助指标的可能性。

结直肠恶性病变严重威胁人类健康，其高致死率与早期诊断困难紧密相关。最新研究表明肠道菌群结构变化与结直肠癌发生发展密切相关; 然而菌群与宿主的相互作用关系及其中是否存在发挥主要致病作用的“核心”细菌等问题均未阐明。本项目利用Illumina测序技术平台对结直肠癌／癌旁组织和不同病理分期结直肠癌组织中的肠道群菌构成变化进行了比较分析。我们的研究结果表明癌组织和癌旁组中菌群构成和丰度无显著变化，肠道菌群构成随着结直肠癌的发生发展呈现动态变化，并初步鉴定出在结直肠癌不同发展阶段显著差异的10个细菌菌属。之后通过比较癌前病变、癌症早期和晚期样本中菌群构成的异同进一步呈现了肠道菌群结构随着结直肠癌发生发展的动态变化趋势；并利用显著差异细菌在各个样本中的分布规律分析初步鉴定了在结直肠癌发生发展中具有重要作用的可能的关键致病菌群，主要包括拟杆菌属的脆弱拟杆菌、消化链球菌属、普氏菌属、梭杆菌属、希瓦氏菌属和埃希菌属。脆弱拟杆菌、普氏菌属、消化链球菌属、梭杆菌属呈现随着结直肠癌发生发展而递增的趋势；而变形菌门的希瓦氏菌属、志贺菌属和大肠埃希菌在癌症样本中普遍增高。进一步的体外试验分析发现上述菌属的代表菌株能够促进癌细胞系HCT116的体外迁移，暗示这些临床重要的条件致病菌也在结直肠癌的发展中发挥着一定作用，可能是结直肠癌发生发展中的“核心”致病菌群。上述菌属在各个样本中较为一致的分布变化规律暗示联合监测上述菌群在个体中的变化趋势可能为结直肠癌的早期检测提供新的生物学指标。此外，我们的结果还揭示出随着癌症发生发展一些益生菌群的消逝，最为典型的如乳酸菌。综上所述，我们的研究结果对于了解结直肠癌发生发展中肠道菌群与宿主的交互作用机制具有一定的作用，同时对于结直肠癌的预防诊断具有一定的应用价值。