【作者】 唐岸柳；

【导师】 沈守荣；

【作者基本信息】 中南大学 ， 内科学， 2012， 博士

【摘要】 [研究背景]非可控性炎症在肿瘤的启动、促进与进展中发挥重要作用。结直肠癌是世界第三大癌,严重威胁人类健康。非可控性的结肠炎症,如溃疡性结肠炎,可显著增加罹患结直肠癌的风险,且结直肠癌的发生与炎症的严重程度、范围等因素呈正相关。结肠炎相关性癌与传统意义上的结直肠癌相似,为一涉及多基因、多步骤、多阶段的复杂过程。而与经典结直肠癌的“腺瘤-腺癌”序列不同,溃疡性结肠炎发展至结直肠癌经历了“炎症-不典型增生-癌症”的序列演进。目前对溃疡性结肠炎相关性结直肠癌发病机制的研究才刚刚起步,尚缺乏针对连续病变阶段的动态组学研究,对其分子机制及其调控网络的认知也不全面。为从全基因组转录水平与蛋白表达水平两个层面全面、深入地探讨“炎-癌链”中的分子事件及其交互作用网络,本研究在构建化学诱导的非可控性结肠炎及其相关性结直肠癌的小鼠模型基础上,采用全基因组表达谱基因芯片技术结合生物信息学分析、炎症因子抗体芯片(蛋白芯片)技术、实时定量PCR以及免疫组织化学法,对“炎症-不典型增生-癌症”序列演进过程的转录组及“炎-癌”调控网络中的关键信号通路与分子的动态变化规律进行分析归纳,旨在为非可控性结肠炎及其相关性结直肠癌的发病机制和临床诊治提供理论依据。[非可控性结肠炎及结肠炎相关性结直肠癌小鼠模型的构建]通过单次小剂量地腹腔注射诱变剂氧化偶氮甲烷,1周后予以循环饮用致炎剂葡聚糖硫酸钠,我们成功构建非可控性结肠炎及其相关性癌小鼠模型。该模型具有与人类溃疡性结肠炎及其相关性癌相似的特征：(1)临床表现上均有腹泻、大便隐血、粘液脓血便、体重不增或下降。(2)病理学观察,给予葡聚糖硫酸钠后小鼠结直肠出现明显的炎症反应,大量炎性细胞浸润,糜烂与浅溃疡从大肠远端向近端发展；随着葡聚糖硫酸钠的循环饮用,形成非可控性的慢性肠道炎症,疾病进展呈现典型的“炎症-不典型增生-腺癌”的序列演进过程；不典型增生以扁平型为主,不典型增生与癌呈多灶性,且发生于炎症较重的部位。(3)小鼠模型与人类溃疡性结肠炎及结肠炎相关性癌有相似的表达谱特征。以上发现证实该模型可较好地模拟人类溃疡性结肠炎与炎症相关性癌,有科学应用价值,可为后续试验提供保障。[非可控性结肠炎癌变过程的转录组动态变化规律]转录水平的变化是基因组遗传信息的集中体现,为了全面地揭示溃疡性结肠炎相关性结直肠癌病变各阶段转录组的动态变化规律,本研究选取“正常-炎症-不典型增生-腺癌”各病理阶段的小鼠肠道黏膜进行全基因组表达谱芯片分析。研究发现,各病理阶段的基因表达呈现一定的规律性,尤以早期炎症期的分子变化最为显著,分别有2245与2294个基因探针在炎症组织中较正常组织发生了上调与下调,形成一阵“转录风暴”。多种聚类分析显示炎症期的样本严格聚为一类,在主成分中占有绝对地位。对基因注释本体(GO)的富集度网络分析发现,该时期变化最显著的为炎症相关基因集,包括直接参与炎症反应的细胞因子、趋化炎症细胞的趋化因子、诱导白细胞粘附的粘附分子、参与组织修复的血管生成因子与基质重建因子、诱导坏死细胞凋亡及促进肠上皮细胞存活与增殖的因子等等,而这些基因集在不典型增生及癌症期表达趋于缓和,表明机体在早期处于积极地防御状态,而至后期,机体逐渐对炎症与肿瘤细胞产生耐受,转录水平改变趋缓；也提示肿瘤微环境对免疫细胞的改造作用。这种早期的剧变可能已经决定了细胞的命运与疾病的走向。在癌症期,仍有部分炎症因子的活跃,主要是促进细胞增殖、抑制凋亡、促进血管新生与基质降解的基因表达上调,充分体现出结直肠癌的恶性行为特征。[信号通路的阶段性活化是连接“炎-癌”的纽带]经基因表达谱芯片的生物信息学分析及免疫组化验证,本研究发现NF-κB、STAT3、p38MAPK及Wnt/β-catenin等信号转导通路在CAC进程中存在阶段性活化。其中NF-κB经典途径(p65)与STAT3两条信号通路在“炎症-不典型增生-癌”的序列演进中呈现持续性活化,p65、磷酸化STAT3(p-STAT3)及其靶基因编码蛋白Bcl-xL、COX-2的表达均呈逐级升高的趋势；NF-κB旁路途径(p52)仅在炎症细胞中活化,且在炎症期最为活跃,而不典型增生期至癌症期表达逐渐下调；p38MAPK信号的活化发生于炎症期及不典型增生期；在肿瘤形成阶段p38MAPK失活,而Wnt/β-catenin通路开始处于明显活化状态。这些结果表明,NF-κB经典途径与旁路途径、STAT3及p38MAPK可能对炎症的启动和维持具有重要作用,参与炎症细胞的趋化、促炎因子的释放；而NF-κB经典途径、STAT3及p38MAPK诱导的持续性炎症反应又对肠上皮细胞的恶性转化产生积极作用,微环境当中的细胞中上述信号的激活导致细胞因子的持续产生,继续作用于转化细胞,促进其存活与生长；随着Wnt/β-catenin通路的激活与p38MAPK的失活(p38MAPK被认为是抑瘤基因),促瘤与抑瘤的平衡终于被打破,在NF-κB经典途径与STAT3信号所营造的炎性背景下,肿瘤稳定形成与进展。[非可控性结肠炎癌变过程动态交互作用网络的关键节点]结合表达谱芯片数据分析及血清炎症因子芯片的检测结果,我们找到非可控性结肠炎癌变过程各病变阶段的交互作用网络关键节点。炎症期,IL-1p、IL-6、COX-2及CEBPB位于上调的分子网络中心,CYP家族成员、紧密连接蛋白occludin与肝细胞核因子4α位于下调的分子网络中心；不典型增生期,上调分子网络的关键节点为CD44和VCAM-1,下调分子网络的关键节点为CYP家族和GSTM家族成员；癌症期,上调分子网络的关键节点包括IL-23a、CXCL2、IL-1β、 LCN2、TIMP1、MMPs、TCF2等,下调分子网络的关键节点则由CYP家族与ALDH家族成员组成。这些关键分子与众多的差异分子发生交互作用,并与NF-κB、STAT3、p38MAPK及Wnt/β-catenin等信号通路紧密相联,形成特色的阶段性的动态调控网络,推动非可控性炎症介导的恶性转化。综上所述,利用小鼠模型研究“非可控性结肠炎-不典型增生-结直肠癌”序列演进过程中不同病理阶段转录组变化规律,并在蛋白水平进行初步验证,为我们进一步探索相关分子机制及其调控网络提供了有价值的理论和实验依据。本研究通过高通量的全基因组表达谱芯片分析发现了基因组的动态转录规律,在炎症早期形成的转录风暴似乎决定了疾病的走向,通过生物信息学分析和免疫组化验证,首次报道了NF-κB、STAT3、p38MAPK及Wnt/β-catenin等关键信号通路的阶段性活化规律；而对血清炎症因子芯片的筛查则对上述结论提供了更有力的支撑。这说明在炎-癌演进的多阶段过程中,关键信号通路及其下游靶基因的阶段性活化发挥主要推动作用。尽管小鼠与人类的同源性很高,对研究结论的推广尚需进行大量临床样本的验证。将人类疾病样本数据与动物模型数据进行整合,有望为临床上对溃疡性结肠炎及其相关性结直肠癌的早期诊治提供新的思路；并提示医务工作者应高度重视对慢性炎症的预防与治疗,阻止炎症的恶性转化。更多还原

【Abstract】 [Background]Nonresolving inflammation is associated with tumor initiation, promotion and progression. Colorectal cancer (CRC)—the third most common cancer in the world—has been endangering human health seriously. Nonresolving colitis, such as ulcerative colitis (UC), is at increased risk of developing CRC, and is associated with the site, extent and duration of inflammation. Evidence is mounting to support a similarity between colitis-associated cancer (CAC) and CRC, which is a multi-gene, multi-step and multi-stage process. However, different from "adenoma-carcinoma" sequence in CRC, colitis-associated carcinogen-nesis experiences a unique sequence of "inflammation-dysplasia-carcinoma". Untill now, the research about the mechanism of CAC has just begun. The dynamic omics study is still lacking, and the recognition of its molecular mechanism and the regulation network is poorly understood. In the present study, we aimed to find the molecular events and its regulation network of the "inflammation-cancer chain" in both transcription level and protein expression level. We performed gene expression array experiments combined with bioinformatic analysis, serum inflammatory antibody array experiments, real-time quantitative PCR and immunohistochemistry detection, based on a chemical induced nonresolving colitis and colitis-associated colorectal cancer mouse model we had established. We dynamically analysed and summarized the changing regularity of molecular events in the process of "inflammation-dysplasia-cancer" sequence, to provide the theoretical basis for the patho-genesis, clinical diagnosis and treatment of nonresolving colitis and CAC. [Establishment of a mouse model of nonresolving colitis and colitis-associated colorectal cancer]We established a nonresolving colitis-CAC mouse model by administration of chemical reagents, azoxymethane (AOM) and dextran sulfate sodium salt (DSS). This model mimicked human UC and CAC closely, for it has the same characteristics as human’s:(1) the same clinical manifestations:diarrhea, stool occult blood, mucopurulent bloody stool, and weight loss;(2) the similar pathology:varying degrees of inflammation, erosions and ulcers were observed with a large number of inflammatory cells infiltrated into the large bowels after DSS administration; later, as the3cycles of DSS administration, nonresolving inflammation established, and the "inflammation-dysplasia-adenocar-cinoma" sequence presented in the gut. What’s more, the dysplasia had a plat form, and multifocal of dysplasia and cancer were presented in the region with more severe inflammation;(3) the same gene expression profiles as human beings. The AOM/DSS induced UC and CAC mouse model we have established has scientific application value. It can provide guarantee for the successor examinations.[Gene expression profiles in different pathology stages of nonresolving colitis-associated colorectal cancer]The transcription level embodies the genome genetic information. In order to fully reveal the gene expression profiles at each stage of CAC, we performed gene expression array experiments on the "normal-inflammatory-dysplastic-cancerous" intestinal mucosa. We found that gene expression at every stage varied with a certain rule. In the early period of the inflammatory phase, molecular change turned to be the most significant. There were2245and2294gene probesets upregulated and downregulated in inflammatory mucosa compared with normal mucosa, respectively, formed a "transcriptional storm". A variety of clustering analysis showed that the inflammatory samples strictly clustered, and hold an absolute dominant position by the principal component analysis. Gene ontology (GO) enrichment network analysis found the predominate differentially expressed gene sets in this phase were the gene sets participate in inflammation, including cytokines, chemokines, adhesion molecules, angiogenic factors and the matrix reconstruction factors, apoptosis inducing factors, survival and proliferation factors, and so on. These gene sets expression changes eased up in the dysplastic and cancerous phases, which indicated that the body was in an active defense state at the beginning, but gradually tolerance to inflammation and tumor cells. These results also imply that the tumor microenvironment has a role in the transformation of the immune cells. The early upheaval may pave the way for the later disease progression, and determine cells fate. In the cancerous phase, part of inflammatory factors were still active, but the gene sets such as cell proliferation promotion, apoptosis inhibition, angiogenesis, and matrix degradation occupied an leading position, which fully reflected the malignant behaviors of colorectal cancer cells.[Dynamic activation of the key pathways:the link between inflammation and cancer]We first time report that the NF-κB, STAT3, p38MAPK and Wnt/β-catenin signaling pathway have a periodic activation in the process of colitis-associated carcinogenesis, through gene expression analysis and immunohistochemistry detection. The canonical NF-κB pathway (p65) and STAT3signaling pathway were persistent activated during the "inflammation-dysplasia-carcinoma" sequence. p65, p-STAT3and their target gene coding proteins Bcl-xL and COX-2were upregulated. The alternative NF-κB pathway (p52) was activated only in the inflammatory cells, especially in the inflammatory phase, while downregulated in the dysplastic and cancerous phases. p38MAPK signaling was activated in the inflammatory phase and dysplastic phase, while inactivated in the cancerous phase. Wnt/(3-catenin signaling pathway was activated from the cancerous phase. These findings indicated the canonical and alternative NF-κB pathway, STAT3and p38MAPK pathway may have an important role in the initiation and maintenance of the inflammation. They involved in the inflammatory cells chemotaxis and proinflammatory factors releasing. The persistent inflammation induced by the canonical NF-κB pathway, STAT3and p38MAPK pathway could play a role in the transformation of the intestinal epithelial cells. Activation of the above signaling in the cells of the microenvironment brought about the persistent cytokine production and promoted the transformed cells survival and growth. At last, with the Wnt/β-catenin signaling pathway activation and p38MAPK (regarded as a tumor suppressor) inactivation, the balance between pro-tumorigenesis and anti-tumorigenesis was broken, and tumor established and stable development in the background of the inflammation induce by the canonical NF-κB and STAT3pathway.[The key nodes of dynamic interaction networks during nonresolving colitis-associated carcinogenesis]Combined with gene expression array data analysis and serum inflammatory antibody array experiments, we found the key nodes of dynamic interaction networks during nonresolving colitis-associated carcinogenesis. In the inflammatory phase, IL-1(3, IL-6, COX-2and CEBPB were in the center of upregulated molecular network, while CYP family members, occludin and HNF4a were in the center of downregulated molecular network. In the dysplastic phase, CD44and VCAM-1were the key nodes of the upregulated molecular network, while CYP and GSTM were the key nodes of the downregulated molecular network. In the cancerous phase, IL-23a, CXCL2, IL-1β, LCN2, TIMP1, MMPs and TCF2were the key nodes of the upregulated molecular network, while CYP and ALDH family member were the key nodes of the downregulated molecular network. These key molecules correlates with many other differentiation expressed molecules, and related to NF-κB, STAT3, p38MAPK and Wnt/β-catenin signaling pathway closely, forming the dynamic regulatory network, which promote the nonresolving inflammation induced malignant trans-formation.In summary, establishment of the gene expression profiles during the "nonresolving colitis-dysplasia-colorectal cancer" sequence by using a mouse model, and validated primarily in the protein level, provide us a valuable theory and experiment evidence to further explore the underlying molecular mechanisms and its regulatory networks. In the present study, we found the dynamic regularity of the genome-wide transcription. The "transcription storm" in the early phase of inflammation seems to determine the end of disease. Through bioinfor-matic analysis and immunohistochemical validation, we first time report the NF-κB, STAT3, p38MAPK and Wnt/β-catenin signaling pathway have a periodic activation, which was confirmed by the serum inflammation antibody array detection. This finding indicates the periodic activation of key signaling pathways have a promotion role in the inflammation-cancer process. Although there is a high homology between mice and human, to expand the results of this research have to be confirmed by clinical experiments. Data integration from both human and the animal model samples will provide new avenues to the early diagnosis and treatment of UC and CAC. What’s more, the conclusions of the study remind medical workers should pay high attention to the prevention and treatment of chronic inflammation, to prevent it from malignant transformation.更多还原