基于“脾胃-肠道菌群”轴研究健脾化瘀润肠法对胃癌化疗患者炎症免疫分子及胃肠道微生态平衡的影响

Traditional Chinese Medicine (TCM) believes that gastric cancer was often caused by the weakness of spleen and stomach and stagnation of liver qi. The combination chemotherapy after surgery will further aggravates the deficiency of spleen and stomach, qi-stagnation, blood stasis and lack of source for growth and development. Intestinal flora, as the main embodiment of the spleen and stomach “acquired foundation”, may also be one of the important reasons for the efficacy of TCM. This project proposes to analyze the difference and correlation of intestine micro-ecology for both the health people and patients with gastric cancer in the high incidence area and the patients with gastric cancer before and after chemotherapy, based on the theory of the connection between the inside and the outside of spleen-stomach, and the connection between the spleen-stomach and the liver-intestine. According to the inclusion criteria, we propose to randomly assign these patients into groups, with Guiqi-atractylodes soup medicine which is the typical method of invigorating spleen, removing blood stasis and tonifying intestine to intervene the chemotherapy patients, then to compare their traditional Chinese and western medicine symptom point, Karnofsky point, clinical biochemistry data, blood immune cells and serum IL-6 and IL-10. Take the stool samples from the bacterial smear, extract DNA with PCR, denaturing gradient gel electrophoresis (DGGE) and high throughput sequencing technology to study the PCR-DGGE fingerprints and composition characteristics of intestinal flora; To analyze the relevance between the micro-ecology and other index, such as inflammatory immune response, traditional Chinese and western medicine syndrome elements and biochemistry of the patients with gastric cancer, and the regulation with Guiqi-atractylodes soup medicine. It will provide modern biological basis for “spleen-stomach, intestine flora” axis theory and its clinic application.

中医学认为胃癌多为脾胃虚弱、肝气郁结所致，术后联合化疗可进一步加剧脾胃虚损、运化失司、气滞血瘀、生化无源。肠道菌群是脾胃“后天之本”主要体现者，亦可能是中药发挥疗效重要机制之一。本课题基于脾胃表里、脾胃-肝肠相通理论为指导，分析胃癌高发同一生活区健康人与患者、化疗前后肠道微生态的差异与预后相关性；并按纳入标准随机分组后，以健脾化瘀益肠法代表方归芪白术汤干预化疗患者，比较各组患者中西医症状积分、Karnofsky评分、临床生化、血液免疫细胞、血清IL-6、IL-10 等促/抑炎症免疫因子的表达；取粪便标本细菌涂片，并提取DNA利用PCR、变性梯度凝胶电泳（DGGE）及高通量测序技术等研究肠道菌群PCR-DGGE指纹图谱、组成特点；分析胃癌患者胃肠道微生态变化与炎症免疫反应、中西医证候要素、生化等指标的相关性及归芪白术汤的调控作用，为“脾胃-肠道菌群”轴理论及其临床推广应用提供现代生物学基础。

摘要：目的：通过研究武威地区胃癌患者肠道微生态、免疫炎症分子及circRNA表达、中西医证候等要素，系统评价并揭示归芪白术方治疗胃癌的作用机制，并为“脾胃-肠道菌群”轴理论及其临床推广应用提供现代生物学基础。.方法：按照纳入排除标准收录甘肃省武威肿瘤医院原发性胃癌患者与健康体检者的病历资料及相关标本，检测两组血常规、肝功能、肾功能、心肌酶等；通过气相色谱实验，分析两组间菌群代谢产物的差异；胃癌患者随机分为西医组和中西医联合治疗组，评价及检测治疗前后胃癌患者症状积分、血清免疫因子表达、肠道细菌计数、肠道菌群分析；治疗结束后检测两组患者血常规、肝功能、肾功能、心肌酶、血清肿瘤标志分子水平；检测组织中相关蛋白（IL-17、NF-κBp65、p-NF-κBp65、IKK-β、IL-6）及circRNA的表达；验证目标差异circRNA在血清样本中的表达水平及线性亲本基因的表达水平。并制备归芪白术方含药血清，检测其对胃癌细胞的增殖情况。.结果与结论：与健康对照组比较，胃癌组生活质量降低，免疫因子表达异常，肠道菌群失调，归芪白术方辅助治疗后能提高患者生活质量，降低患者中医、西医症状积分，提高胃癌术后化疗患者WBC、RBC、HGB、Neu、Lym、PLT水平，减轻胃癌化疗患者不适症状，改善免疫因子表达，保护造血功能；降低血清肿瘤标志分子CEA、CA199、CA724水平。.与健康对照组相比，胃癌患者肠道菌群相对丰度降低。肠道菌群代谢产物短链脂肪酸中，总酸、乙酸、丙酸、丁酸、异戊酸含量低于健康对照组。归芪白术方辅助治疗可能通过促进益生菌，抑制致病菌起到对胃癌的防治作用。并可通过影响胃癌患者免疫细胞及免疫因子发挥调节炎症免疫的作用。胃癌组与健康对照组，以及胃癌组治疗前后血清circRNA存在明显的表达谱差异，其差异表达可能与胃癌的发生发展以及药物的干预相关； has_circ_0000437在胃癌的临床诊断中具有一定的诊断价值。归芪白术方含药血清对人胃癌细胞MKN-45具有抑制增殖的作用。