癌变生物标志物与溃疡性结肠炎脾胃气虚证的相关性研究

Ulcerative Colitis (UC) is a refractory disease, its incidence increased year by year. Ulcerative Colitis Associated Colorectal Carcinoma (CAC) is one of the complications of UC with poor prognosis. Regular surveillance of risk factors can improve the early diagnosis of CAC. Chinese medicine believes that the pathogenesis feature of UC is deficiency in origin and excess in appearance. However, deficiency of spleen qi and stomach qi is always the main pathological basis of UC. On the basis of existing research, we hypothesized that the patients with deficiency of spleen qi and stomach qi have a higher risk of developing CAC than the patients with other TCM syndromes. The project intend to start off from the CAC-related biomarkers, and then observe the morphology of the colonic mucosa, determination the gene expression and protein levels of p53, K-ras and APC in the colonic mucosa, measure the content of reactive oxygen species in the mucosa and study the activation of the PI3K/Akt signaling pathway on the levels of organs-tissue-cell. To explain the pathophysiological implications of deficiency of spleen qi and stomach qi and provide a scientific basis for the diagnosis of TCM syndromes. Meanwhile, this has important significance to assess the prognosis of patients and monitoring disease progression with TCM syndromes.

溃疡性结肠炎作为难治性疾病，发病率逐年增高。溃疡性结肠炎相关性大肠癌是该病的并发症之一，且预后不良。加强癌变危险因素的监测可以提高CAC的早期诊断率。中医认为溃疡性结肠炎病机表现为本虚标实的复杂证候，但总是以脾胃虚弱为主要的发病基础。结合已有研究，我们推测溃疡性结肠炎脾胃气虚证患者较其它证型患者具有更高的癌变风险。本课题拟从溃疡性结肠炎癌变生物标志物的变化入手，在器官-组织-细胞三个层次上观察患者结肠黏膜的病理形态，在基因及蛋白质水平分别测定结肠黏膜组织中p53、K-ras、APC的表达，并且检定黏膜组织内活性氧的含量，研究PI3K/Akt信号通路的活化情况。通过以上实验结果阐释脾胃气虚证所包涵的病理生理意义，为该证型的判断提供一定的科学依据。同时，这项研究对于参考中医证型评估患者预后，辅助监测病情发展也具有重要意义。

溃疡性结肠炎相关性大肠癌是UC的严重并发症之一，预后差。本课题旨在探讨癌变生物标志物与UC脾胃气虚证的相关性。研究内容：1.收集UC脾胃气虚证和UC湿热内蕴证的住院病例，比较两种证型患者的癌变风险；2.按照异型增生的分级标准，将病例分为6组：UC脾胃气虚证组、UC脾胃气虚证+LGD组（Low Grade Dysplasia，LGD）、UC湿热内蕴证组、UC湿热内蕴证+LGD组、正常阴性对照组和大肠癌阳性对照组（其中包括CAC组和散发性大肠癌组（Sporadic colorectal carcinoma，CRC））；3.免疫组化法观察p53、KRAS、APC蛋白及Ki-67的表达；4.高铁二胺—奥新蓝法观察细胞内黏液类型的变化；5.FFPE组织中提取DNA：①检测4组UC患者的PIK3CA基因突变和p14ARF、MGMT基因甲基化；②检测大肠癌阳性对照组KRAS、BRAF、PIK3CA基因突变和p14ARF、MGMT基因甲基化；6.UC两种证型间的microRNAs差异表达谱芯片分析。结果：1.从病程、病变范围、炎症程度及异型增生的角度，UC脾胃气虚证较UC湿热内蕴证具有更多的癌变危险因素；2.UC脾胃气虚证+LGD组p53蛋白过表达和Ki-67位于隐窝上1/2者的比例均高于UC湿热内蕴证+LGD组；3.UC脾胃气虚证组p14ARF 基因甲基化率较UC湿热内蕴证组高，UC脾胃气虚证组和UC脾胃气虚证+LGD组MGMT基因甲基化率分别高于UC湿热内蕴证组和UC湿热内蕴证+LGD组；4.CAC组KRAS基因突变率和p14ARF甲基化率高于CRC组；5. UC脾胃气虚证组与UC湿热内蕴证组比较有4个miRNAs表达上调，结合靶基因预测和GO-pathway分析，拟选择hsa-miR-193b-5p对应的基因PIK3R2进行靶基因鉴定。综上所述，UC脾胃气虚证与UC湿热内蕴证相比，具有更多的癌变危险因素，前者与p53蛋白、Ki-67指数、高甲基化p14ARF、MGMT基因以及hsa-miR-193b-5p等CAC的生物标志物之间有着更加紧密的联系，UC脾胃气虚证发生癌变的途径可能与p53基因突变，p14ARF、MGMT基因高甲基化和hsa-miR-193b-5p上调及其对应靶基因PIK3R2的异常表达有关。