【作者】 赵芯梅；

【导师】 智发朝；

【作者基本信息】 南方医科大学 ， 内科学， 2013， 博士

【摘要】 背景、目的溃疡性结肠炎(ulcerative colitis,UC)是炎症性肠病(inflammatory bowel disease,IBD)的一个主要类型,是一种原因不明的慢性非特异性肠道炎症’。病变主要限于结肠的黏膜层和粘膜下层,以溃疡和隐窝脓肿为主要特点,多累及直肠和乙状结肠,也可遍及整个结肠2。临床表现主要是腹泻、粘液脓血便、腹痛。病情轻重不等,多呈反复发作的慢性病程。本病可以发生在任何年龄,多见于20-40岁5,亦可见于儿童或老年。男女发病率无显著差别。病理特点是粘膜的弥漫性炎症,固有膜内弥漫性淋巴细胞、浆细胞、单核细胞等炎细胞浸润,活动期有大量中性粒细胞和嗜酸性粒细胞浸润,可发生隐窝炎和隐窝脓肿。上世纪六十年代英国科学家Morson从病理学特征上将它与克罗恩病(Crohn’s disease,CD)加以区分并将其界定为独立的疾病。该病在欧美国家较常见,欧洲20个中心调查显示UC的年发病率为11.2/10万,以往发病率较低的东方国家,近年也呈现增高趋势。我国1981-2000年间共计报道UC病例10218,近10年该病报道数上升3.1倍6。我国目前暂缺乏大规模的流行病学调查,但粗略估计UC发病率为11.6/10万7,而且有证据表明成逐年上升趋势8-10。而且,溃疡性结肠炎是一种癌前病变,与结肠癌的发病有关,且病程长,病变程度轻重各异,由于病因不明,临床上常常表现反复发作而治愈难度大,被世界卫生组织列为现代难治病之一。要达到UC治愈的目的,早期发现及发病机制的研究至关重要。尽管深入到分子生物学、基因水平甚至蛋白水平的研究方法,使得溃疡性结肠炎的发病机制的研究取得了突飞猛进的发展；我们必须明确溃疡性结肠炎是多因素参与的复杂疾病,单基因或蛋白质标记物无法全面而准确地反映其复杂的发病机制。因此我们需要运用基因组、转录组和蛋白质组等高通量技术来寻找由多基因或蛋白质组成的复合型标记物,以期更准确、敏感地预测疾病,并深入阐释其发生发展的分子机制。我们都知道基因组水平的研究,主要集中在微卫星不稳定性和基因的扩增、缺失,转录组的研究重点是在mRNA水平检测基因的表达变化,可以高通量地检测组织或细胞样本中全基因的mRNA水平,建立基因表达谱,从而确定一组表达量与疾病相关的基因(Gene signature),用于疾病预测和预后评估。与基因组水平的研究相比,Gene signature更具实用性。而蛋白质组则不仅研究基因的蛋白产物丰度,还注重检测蛋白质翻译后修饰及其对蛋白功能的影响。这三个层次的研究并不孤立,都反映了差异基因的丰度和表达变化,三个层次的研究结果可以相互印证,互为补充。前期我们首先运用双向电泳和质谱技术,比较12例溃疡性结肠炎和12例正常组织的蛋白质表达谱,从中鉴定出30种差异表达蛋白质。进一步利用生物信息学方法,找到11种与P38MAPK通路相关的差异表达蛋白质,并将这11种蛋白与该通路的关键蛋白质组成一个差异蛋白质簇。我们用Western Blot技术在上述样本中初步验证了此差异蛋白质簇与溃疡性结肠炎的相关性。然后从该蛋白质簇中选取了两种蛋白,Galectin-3和MAWBP,用免疫组化技术在56例溃疡性结肠炎和正常样本中检测其丰度。发现Galectin-3和MAWBP在溃疡性结肠炎组织中表达显著降低(χ2=31.404,P=0.000；χ2=20.541,P=0.000),并与炎症程度特异相关(χ=12.315,P=0.002；χ2=8.726,P=0.018).本研究中,我们拟在前期试验结果的基础上,进一步探讨P38MAPK与溃疡性结肠炎的相关性和调控机制,并进一步扩大样本量研究差异蛋白簇中抽提出的蛋白质印记作为溃疡性结肠炎分子标志物的可能性。针对此蛋白印迹中的一种与溃疡性结肠炎密切相关的新蛋白MAWBP,我们对此产生了浓厚的兴趣,利用基因组学和多种分子生物学技术结合对其生物学功能及与溃疡性结肠炎的关系进行了研究。我们还进一步利用基因组学分析了溃疡性结肠炎反复迁延不愈的复杂机制,找到了重要的肿瘤相关基因,从而为溃疡性结肠炎癌前病变提供了候选标志物及有希望的新治疗靶点。方法1、运用Western Blot和ELISA技术,在巨噬细胞系RAW264.7中验证前期蛋白组学得到的差异蛋白簇受P38MAPK信号通路的调控,用免疫组化技术检测118例样本中差异蛋白簇磷酸化P38、Galectin-3和MAWBP的表达并分析其与溃疡性结肠炎临床特点的相关性和作为复合标志物的可能性。2、运用Microarray和多种分子生物学方法揭示差异蛋白MAWBP在溃疡性结肠炎中的作用、功能和分子机制。首先通过免疫组化和Western Blot检测MAWBP结合蛋白MAWD在溃疡性结肠炎中的表达和定位,进而通过免疫共沉淀实验确定两者相互结合。通过构建真核表达载体转染Caco-2细胞来分别及共同高表达MAWBP和MAWD,Microarray技术分析其基因表达谱,进而通过Real-time PCR和Western Blot验证差异基因。最后通过干扰RNA技术敲低MAWBP和MAWD的表达,进一步从反方向验证MAWBP和MAWD与ERK通路的关系。3、运用DASL技术在转录水平对溃疡性结肠炎活动期和静止期的基因表达谱进行分析,挑选同一病人同一部位的活动期和静止期内镜下标本共29例,对差异基因进行生物信息学分析,进一步通过Real-time PCR和免疫组织化学染色方法验证差异基因的表达。4、统计学分析：我们用SPSS13.0软件来进行所有的数据分析；Pearson chi-square检验和Fisher’s确切概率法来分析蛋白质表达与疾病病理特征之间的关系；方差分析及后续的基于方差分析的多重比较方法来分析Real-time PCR结果和基因组学差异基因结果；P<0.05被认为差异有统计学意义；结果的相关性我们用Pearson相关性检验,r>0.5为强相关。结果1、我们用Western Blot技术验证了与P38MAPK通路相关的差异蛋白质簇与溃疡性结肠炎的相关性。细胞水平进一步验证了差异蛋白簇受P38MAPK通路的调控。然后从该蛋白质簇中选取了三种可以指示P38MAPK通路活化状态的蛋白,磷酸化P38,Galectin-3和MAWBP,免疫组化结果显示这三种蛋白质与溃疡性结肠炎特异相关(χ2=41.509, P<0.001; χ2=62.668, P<0.001;χ2=68.997,P<0.001),其组成的蛋白质印记以高敏感性(94.83%)和特异性(98.33%)可以对溃疡性结肠炎进行正确判定,这种蛋白质印记是一种评估溃疡性结肠炎炎症程度的复合标志物。2、生物信息学分析显示MAWBP为MAWD的结合蛋白,免疫组化和Western Blot结果显示MAWD同MAWBP一样也在溃疡性结肠炎组织中表达降低。免疫共沉淀实验显示了两种蛋白可以相互结合形成复合物。Real-time PCR和Western Blot结果显示MAWBP和MAWD真核表达载体成功转染入Caco-2细胞。对Microarray结果进行生物信息学分析发现MAWBP和MAWD可导致ERK1通路活化,癌基因TPX2被激活高表达,而炎症因子和趋化因子能够被抑制。Real-timePCR验证了炎症因子IL8、TSC1、CXCL2和IFIT1及癌基因TPX2的表达(P<0.05)。Microarray和Real-time PCR结果强相关(r>0.72)。Western Blot验证了MAWBP和MAWD高表达能够激活ERK通路,以及干扰RNA敲低MAWBP和MAWD的表达后能够抑制ERK通路。3、运用DASL对溃疡性结肠炎活动期和静止期的基因表达谱进行分析,共发现上调基因909个,下调基因823个,其中上调组基因主要涉及了炎症反应、结直肠肿瘤癌基因和EMT,下调组基因主要涉及了结直肠肿瘤抑癌基因和PPAR通路。通过Real-time PCR验证了结直肠肿瘤相关癌基因MMP1、MMP3、CHI3L1和CHRDL2,抑癌基因ABCG2和PLA2G12B在UC活动期中有显著的差异表达(P<0.05),进一步通过免疫组化检测发现MMP1、CHI3L1和ABCG2在溃疡性结肠炎、结肠癌和正常组织三组之间的表达均有统计学差异(χ2=8.418, P=0.015; χ2=6.995, P=0.030;χ2=6.285,P=0.043),提示了UC的高癌变风险。结合蛋白组学及Western Blot、Real-time PCR结果发现间质标志物Vimentin在UC中升高,上皮标志物E-cadherin在UC中减低。结论1、运用整合蛋白组学得到一个由三种差异蛋白组成的蛋白质印记,磷酸化P38,Galectin-3和MAWBP,是一种评估溃疡性结肠炎炎症程度的复合标志物。2、运用整合基因组学发现MAWBP及其结合蛋白MAWD可激活ERK通路、活化癌基因TPX2,并在溃疡性结肠炎中有抑制炎症的作用。3、运用整合基因组学发现癌基因和抑癌基因在溃疡性结肠炎未发生癌变时已经发生显著变化,提示UC的高癌变风险。4、运用整合蛋白组学和基因组学发现溃疡性结肠炎中有EMT发生。更多还原

【Abstract】 Background and ObjectiveUlcerative colitis (UC), as one category of inflammatory bowel disease, is a chronic, spontaneously relapsing, debilitating idiopathic, non-specific inflammation. UC is characterized by crypt abscess and ulceration of the colon mucosa and submucosa, the key features are diffuse mucosal inflammation that extends proximally from the rectum to a varying degree. The main clinical manifestations of UC are diarrhea, abdominal pain and bloody mucous stools with pus. UC is an idiopathic chronic disease of the colon characterized by periods of active disease followed by periods of remission. UC can be found in all range ages, also chidren and elder, especially20-40years old. There is no significance of incidence between male and female. The pathology feature are the diffuse inflammation of mucous membrane and the diffuse infiltration of lymphocytes, plasma cells and mononuclear cells within lamina propria, the activity has a large number of neutrophils and eosinophils infiltration, inflammation can occur fossae and crypt abscesses. In the1960s the British scientist Morson sorted it with crohn’s disease depends on pathological features and defined it as an independent disease. UC is very common in European and North American countries, the investigation from20centres in European indicated that the incidence of UC is11.2/105population/year, recently there is convincing evidence of rising trend of UC in oriental countries which have lower incidence in old days.1981-2000, a total of10218reported cases of UC in our country, which increased3.1times in a decade. Temporary lack of large-scale epidemiological investigation in our country, but a rough estimate incidence of UC as of11.6/105, and there is evidence that a rising trend year by year. Moreover, ulcerative colitis is a kind of precancerous lesions, associated with colon cancer, and UC usually has a long duration, with different pathological changes degree and unknown etiologylt recrudesces time after time and relates to colon cancer. Because UC recurs frequently and is hard to cure completely, it ranks among refractory diseases of modern times by world health organization (WHO). To achieve the purpose of cure UC, the early detection and the pathogenesis research are very important.Although the research methods which deep into the molecular biology, gene level and protein level, makes the research of the pathogenesis of ulcerative colitis has developed by leaps and bounds, we must be clear of ulcerative colitis is multiple factors involved in complex diseases, single gene or protein markers cannot fully and accurately reflect its complex pathogenesis. So we need to use the genome, transcriptome and proteome high throughput technologies to search the compound marker genes or proteins, in order to more accurately and sensitively predict disease, and explain the molecular mechanism of development. We all know that the genome level research, mainly concentrated in the micro satellite instability and gene amplification, missing. Transcriptome research is focused on detection of Gene expression changes in mRNA level, high flux to test samples of tissues or cells in Gene mRNA level, and to establish the Gene expression patterns, so as to determine the amount of disease related genes expressed in a set of (Gene signature), used for disease evaluation and prognosis. Compared with the genome level of research, Gene signature is more practical. While the proteomic not only investigate the expression of protein products of genes, but also pay attention to the detection of protein modification after translation and its influence on protein function. The three levels of study are not isolated, which reflect the abundance of differential gene and the expression change, three levels of the research results can confirm each other, complement each other.Early we first used two-dimensional electrophoresis and mass spectrometry, compared12cases of ulcerative colitis and12cases of normal tissue protein expression spectrum, and identified30differential expressed proteins. Using bioinformatics methods, we further found11kinds differential proteins related with P38MAPK pathway, and we combined the11kind proteins with the key proteins of this pathway as a differential protein cluster. We used Western Blot technique to validate the correlation of differential protein cluster with ulcerative colitis in the above sample. And then chose two kinds of protein from the protein clusters, Galectin-3and MAWBP, we used immunohistochemical technique to test its abundance in58cases of ulcerative colitis and normal samples. We found that the expression of Galectin3and MAWBP significantly reduced in ulcerative colitis group (χ2=31.404, P=0.000, χ2=20.541, P=0.000) and specifically associated with the degree of inflammation (x2=12.315, P=0.002, χ2=8.126, P=0.018).In this study, we further explored the correlation and the regulatory mechanism of P38MAPK signaling pathway with ulcerative colitis on the basis of the preliminary test results, and further expand the sample size and investigated the the possibility of protein signature, extracted from differential protein cluster, as the biomarker of unicerative colitis. We were very interested in the MAWBP, a new protein of the protein signature, which was closely associated with ulcerative colitis.So we used genomics combined with a variety of molecular biology techniques to investigate its biological function and its relationship with ulcerative colitis. We also further analyzed the complicated mechanism of ulcerative colitis delay, using genomics and found an important tumor related genes, and thus provides the candidate biomarkers of precancerous lesions in ulcerative colitis and promising new treatment targets.Methods1. Using Western Blot and ELISA technology, we verify the previous proteomics differential protein clushter regulated by P38MAPK signaling pathway in the macrophage cell line RAW264.7, then we used immunohistochemistry technique to investigate the expression of three different proteins cluster, phosphorylated P38, Galectin3and MAWBP in118cases samples and analyzed the correlation with clinical features of ulcerative colitis and the possibility of compound biomarkers.2. Using Microarray and molecular biology method to reveal the role, function and molecular mechanism of the differential protein MAWBP in ulcerative colitis. First we detect the expression and localization of MAWBP binding protein (MAWD) by immunohistochemistry and Western Blot in ulcerative colitis, and then we confirmed whether they can combine with each other by immune co-precipitation experiment. Then we construct the eukaryotic expression vector of MAWBP and MAWD, and respectively or simultaneously transfect into Caco-2cells, we analyze the gene expression profile through Microarray technology, and validate the differential genes by Real-time PCR and Western Blot. Finally we knock down MAWBP and MAWD expression by RNA interference technology and further from the opposite direction to verify the correlation of MAWBP and MAWD with ERK pathway.3. Using DASL technology to analyze the gene expression profile of active ulcerative colitis and inactive ulcerative colitis at transcription level, choose the same patient at the same site active and inative stage endoscopic specimens, a total of29cases, bioinformatics analysis of differential genes, further validated by Real-time PCR and IHC in differential gene expression.4. Statistical analysis:we use SPSS13.0software to do all the data analysis. Pearson chi square test and Fisher’s exact probability method to analyze the relationship between the pathological characteristics of protein expression and disease. Analysis of variance and subsequent multiple comparison method based on the analysis of variance to analyze Real-time PCR results and differential genes and proteins of composite omics, multiple test P values need to correct with Bonferroni method. P<0.05was considered statistically significant; We used Pearson correlation test to detect the correlation of two results, r>0.5was considered strong correlation.Results1. A differential protein cluster involved in p38mitogen-activated protein kinase (MAPK) pathway was deduced and validated by Western blot. Furthermore, three proteins elicited from the protein cluster, phosphorylated p38, MAWBP and galectin-3, as a molecular signature, was analyzed by immunohistochemistry. Increased expression of P-p38and down-regulated MAWBP and/or galectin-3were detected in UC compared to normal samples (χ2=41.509, P<0.001; χ2=62.668, P<0.001;χ2=6%.991, P<0.001), and classified UC risk with high sensitivity (94.83%) and specificity (98.33%). These results indicate that molecular signature of P38MAPK pathway might be a potential biomarker for evaluating UC risk.2. Bioinformatics analysis indicated that MAWBP is MAWD binding protein; immunohistochemistry and Western Blot showed MAWD also decreased expressed in ulcerative colitis tissues such as MAWBP. Immune co-precipitation experiment showed that the two proteins can be combined each other to form compounds. Real-time PCR and Western Blot showed MAWBP and MAWD eukaryotic expression vector successfully transfected into Caco-2cells. Bioinformatics analysis revealed MAWBP and MAWD can result in ERK1pathway activation, oncogene TPX2high expressed, and inflammation factors and chemokines can be suppressed. Real-time PCR verified the inflammation factor IL8, TPX2TSC1, CXCL2, IFIT1and oncogene TPX2(P<0.05). Microarray and Real-time PCR results strong correlated (r>0.72). Western Blot validated that MAWBP and MAWD can activate ERK pathway, they can inhibit ERK pathway through knock down MAWBP and MAWD expression by RNA interference RNA3. We applied DASL to elucidate the differential gene profile of UCative and UCJnactive. A total of909up-regulated differential genes were obtained and823down-regulated differential genes. We categorized up-regulated genes into several groups, including inflammation, colorectal adenoma related oncogene, EMT, and the down-regulted genes involved in the PPAR pathway, tumor-suppressor genes related to colorectal adenoma. We validated colon cancer related oncogenes MMP1and MMP3and CHI3L1and CHRDL2, tumor suppressor genes ABCG2and PLA2G12B by Real-time PCR in UC_active and UC_inactive which have significant difference (P<0.05),we further found that MMP1and CHI3L1and ABCG2had sigficant difference between UC and normal and colon caner (χ2=8.418, P=0.015;χ=6.995, P=0.030; χ2=6.285, P=0.043), which indicatead that UC had high risk of canceration. Combination of proteomics and Western Blot, Real-time PCR results found that mesenchymal markers Vimentin up-regulated in UC, epithelial markers E-cadherin down-regulated in UC.Conclusion1. The molecular signature obtained from the integrate proteomics consisting of three differential proteins, the phosphorylated P38, Galectin3and MAWBP, might be a potential biomarker for evaluating UC risk.2. Using the integrate genomics found that MAWBP and its binding protein MAWD can activate the ERK pathway and oncogene TPX2, and inhibit the role of inflammation in ulcerative colitis.3. Using the integrate genomics discovered oncogenes and tumor suppressor genes in ulcerative colitis without carcinogenesis have differential expression.4. Applying integrate proteomics and genomics found that ulcerative colitis might have the EMT process.更多还原