Ack1、Mawbp在炎症性肠病中的表达与活性
摘要
研究背景与目的
炎症性肠病(inflammatory bowel disease, IBD)包括克罗恩病(Crohn's disease, CD)和溃疡性结肠炎(ulcerative colitis, UC)。目前研究认为发病与基因、肠道菌群、环境因素有关。
IBD在西方及北欧的发病率较高,近年我国的发病率呈现逐年增高的趋势。目前关于IBD的研究主要集中在诊断和治疗上,对其分子机制尚缺乏系统研究。
一般情况下UC和CD可以根据临床、内镜、病理综合分析判断。但是如果结肠广泛病变,并且内镜、病理表现不典型时,UC和CD有时难以鉴别；如果IBD内镜下炎症表现较轻,无典型内镜下改变,还需要与肠道感染性疾病相鉴别。CD、肠结核(intestinal tuberculosis, ITB)、肠道淋巴瘤(intestinal lymphoma,IL)因为存在诸多相似的地方,三者之间的鉴别也是临床的一个难题。
ITB多由活动期肺结核病人吞下含结核杆菌的痰,结核杆菌穿透肠黏膜定植在肠道引起。原发性胃肠淋巴瘤(primary gastrointestinal lymphoma, PGL)是非霍奇金氏淋巴瘤中具有特征性并所占比例较少(11%-34%)的一类,在原发性胃肠淋巴瘤中,IL约占15%-20%。目前病因未明。ITB和IL近年来在发达国家和发展中国家都有上升趋势。
CD、ITB、IL临床表现(如腹痛、腹泻)缺乏特异性,内窥镜下多累及回盲部、手术及病理有很多相似之处,如肠壁的慢性非特异性炎症、肉芽肿、溃疡和节段性病变等。而治疗方案却有巨大差异性。所以误诊会带来严重的后果。这几种疾病的鉴别对临床医生来说是一项重大挑战。
目前,临床上尚未有简单易行的实验室方法来鉴别诊断IBD。
血液学指标如白细胞计数、血沉(erythrocyte sedimentation rate, ESR)和C-反应蛋白(C-reactive protein, CRP)均无特异性。
诊断ITB最可靠的是从肠道组织中发现结核杆菌,但是抗酸染色特异性、敏感性都不够。分枝杆菌的培养要耗时,而结果通常是阴性的(25%-35%)。PPD皮试阳性作为ITB的诊断并不可靠。诊断性抗结核治疗时间长,会对病人健康产生影响。
而活检取材多浅小,只能取到黏膜层和部分黏膜下层,并不能观察肠壁全貌,病理黏膜非特异性炎症的检出率常较高,因此一些特征性的病变难以被发现,如属于全壁性炎症的CD的特异性病理表现非干酪样坏死,IL的淋巴瘤细胞以黏膜下浸润为主,而隐窝脓肿不仅见于UC,也见于CD和ITB。所以IL常常需要手术切除后行免疫组化才能最后确诊。
最近关于ITB诊断的研究进展还包括对ITB粘膜活检组织进行“DNA原位PCR”技术。PCR检测的速度快,准确性高,与组织中是否有干酪样坏死性肉芽肿无关,但敏感度低,且受组织样本大小以及样品和甲醛接触时间的影响。
Wolfgang Tillinger最近证明血清血指标CD64可以用来鉴别IBD、感染性肠炎及功能性胃肠病。近来有人在IBD患者粪便中发现钙卫蛋白升高,但后来发现,感染性肠炎中也存在,诊断特异性不高。
抗酵母菌抗体(antisaccharomyces antibodies, ASCA)和抗中性粒细胞胞浆抗体(perinuclear anti-neutrophil cytoplasmic antibody, pANCA)对鉴别CD敏感度较高,但特异性低。ASCA无法鉴别CD和ITB。最近有研究发现,ASCA和pANCA对中国和日本人的敏感性较低。另有学者认为从CD患者组织提取物中测定特异性的RAN序列,对临床诊断的CD活检组织进行检测,发现阳性率80%,其作为组织学标记物的潜在应用价值值得进一步研究。
综上所述,目前仍无一种简单易行的可以鉴别IBD的金标准。它们的鉴别需要从临床、影像学、内镜、病理学表现等多方面综合考虑。近几年出现的一些新的检查方法虽很有前途,但尚需更多的临床试验进行验证。
我们课题组运用蛋白质组学技术,筛选出UC与正常人肠道黏膜差异蛋白若干,其中包括活化的cdc42结合蛋白激酶1(activited Cdc42 kinase1, Ack1)和Mawb结合蛋白(Mawb binding protein, Mawbp)。
Ackl体内发挥的作用及其具体机制尚不十分清楚。目前仅有少量研究认为它参与细胞生长、抑制细胞凋亡,促进血管生成、细胞浸润和转移,在晚期肿瘤的转移中发挥作用。因为它与胞内外信号通路核心蛋白Cdc42结合,参与和炎症及肿瘤均相关的MAPK、NF-kB等信号通路,于是我们推测它在细胞内可能发挥重要作用。
目前关于Mawbp的文献仅有3篇,Zhang J通过比较原发型胃癌与周围黏膜蛋白表达的差异,认为Mawbp是胃癌相关蛋白。WesthoffTH研究发现Mawbp在低血压大鼠血液中高表达,推测其有调节血压的功能。Kurokawa分析肝癌组织与癌旁组织的基因差异表达发现Mawbp在肝癌中高表达,因此他推测Mawbp在肝癌形成中可能发挥重要的作用。
目前国内外尚未见Ackl和Mawbp与IBD相关文献报道。我们课题组的前期工作证明Ack1和Mawbp在UC肠道黏膜较正常人肠道黏膜含量差异明显。这几个蛋白的改变是UC特有还是炎症的普遍改变,目前尚不清楚。又如前文所示,Ack1和Mawbp与某些消化系统肿瘤相关,那么我们推想Ack1可能参与UC的癌变。UC癌变不同于普通的散发性结肠癌。UC相关性结肠癌常发生于平坦型不典型增生或有不典型增生的病灶(dysplasia-assoeiated lesiono mass, DALM),而散发性大肠癌多由散发性腺瘤性息肉(sporadic adenoma, SA)发展而来,按照腺瘤-不典型增生-大肠癌-转移的途径发生。它们的分子机制有诸多不同,如p53突变为UC癌变的早期事件,APC突变及其失活导致的Wnt途径激活在结直肠癌发生初期具有重要作用,但在IBD相关结直肠癌正好相反。UC-associated SAs和non-UC-associated SAs的p53、APC表达与DALM相反。从分子水平上证明DALM与SAs的区别。Ack1、Mawbp在这几种组织结构中的表达是否有差异性,它们能否作为UC癌变区别于普通大肠癌的标志物,目前不清楚。
因此,本次实验的目的是希望通过对IBD、ITB、IL、普通肠道炎症及息肉、DALM中进行Ack1、Mawbp分子标志的系统检测,观察相对完整的Ackl1、Mawbp在这几种疾病中的表达情况,从而推测：(a)它们是否能将IBD病人与ITB、IL、感染性结肠炎、慢性结肠炎相鉴别；(b)它们是否与炎症相关；(c)它们能否鉴别non-UC-associated SAs、UC-associated SAs、DALMs以及炎性息肉。以期找出IBD发生发展的分子学基础及其与传统的大肠腺瘤-癌变途径的差异。
材料和方法
1.完全随机收集78例UC、27例CD、20例感染性肠炎、26例慢性结肠炎、60例肠道黏膜正常病人、15例ITB、6例IL、6例UC-associated SAs、4例DALMs、10例non-UC-associated SAs、10例UC相关性炎症性息肉、10例散发性炎症性息肉；
2.比较UC组和CD组之间临床病理学资料的差异,包括患者性别、年龄、病变累及部位、白细胞、血小板、血沉、C反应蛋白等；
3.运用免疫组织化学染色技术观察Ack1、Mawbp在上述各组中的表达情况；
4.统计分析：采用SPSS 13.0统计软件进行分析,用Independent-Samples T Test测定组和组之间患者年龄的差异;用One way ANOVA测定多组计量资料之间的差异性；用Chi-square tes、Mann-Whitney U test测定两组之间其他因素的差异(包括性别、血清学指标及各个分子的染色情况等)；用Kruskal Wallist测定多组等级资料之间的差异；用Spearman rank-order correlation coefficient测定各等级资料之间或者等级资料与计量资料之间的相关性。所有分析结果取双侧P值,P<0.05认为差异具有显著性。
结果
1.78例UC有33例发生于直肠(43.4%),23例发生于左半结肠(30.3%)。25例CD有20例发生于小肠及回末(74.1%)。
2.CD病人的CRP显著高于UC组(10.95vs1.8,P<0.05)和正常对照组(10.95vs5,P<0.05)。临床活动度为中重度的UC病人的CRP高于正常人(6.4vs5,P<0.05)和临床活动度为轻度的UC患者(2.14±1.44),PLT、ESR、APTT、PT、CEA、CA199指标CD和UC之间,IBD与正常人之间无显著差异(P>0.05)；两组在性别、年龄等方面无显著差异(P>0.05)。不论是在UC组(r=0.604,P<0.05)还是在CD组(r=0.652,P<0.05),CRP均与ESR呈正相关关系,且相关关系密切。UC组临床分级与CRP呈正相关且关系密切(r=0.563,P<0.05),而与ESR呈正相关但关系不密切(r=-0.409,P<0.05)。CD组中临床分级与CRP、ESR无关(P>0.05)。
3.在CD和UC组,Ack1的表达与临床活动度呈正相关性(r=0.693,P<0.05)；在UC组,Ack1胞浆表达与Mawbp的胞浆表达具有负相关性(r=-0.503,P<0.05)；在CD组,Mawbp与CEA的胞浆表达呈负相关性(r=-0.607,P<0.05),Ack1的胞浆表达与Mawbp的胞浆表达无关(P>0.05)。
4.感染性肠炎中Mawbp表达与炎症病理分级呈正相关(P<0.01),但相关关系均不密切(r<0.5)。Ack1表达与慢性结肠炎、感染性肠炎炎症病理分级无关(P>0.05)。CD和UC组Ack1和Mawbp的胞浆表达与病理分级无关(P>0.05)。
5.以正常人为对照组,在UC组Ack1+的敏感性(88.9%)和符合率(84.06%)较高,特异性(78.8%)较低；而在CD组,Ack1+的特异性(90%)和符合率(83.33%)较高,敏感性(66.6%)较低。以正常组+慢性结肠炎+感染性肠炎为对照组,在UC组Ack1+的敏感性(65.31%)和特异性(83.70%)较高,符合率(73.91%)稍低；而在CD组,Ack1+的特异性(92.30%)较高,敏感性(32.00%)和符合率(68.75%)较低。
6.Ack1在本组所有ITB(15/15)和IL(6/6)中均高表达(3+),Mawbp表达量中等(2+)。ITB和IL表达无显著性差异(P>0.05)。
7.Ack1在SAs(2+或3+)中表达明显高于炎性息肉(-或+)。相同程度不典型增生的DALM和UC-associated-SAs或non-UC-associated-SAs染色无显著性差别(P>0.05)。
结论
Ack1、Mawbp在IBD、ITB、IL、感染性肠炎、慢性结肠炎中均有表达,广泛参与炎症反应,与炎症具体关系复杂,不是IBD特异性标志物。Ack1不是UC癌变有别于普通大肠癌的特异性指标,可能通过促生长和抗凋亡在结肠癌的发展过程中发挥作用,应只是推动作用而不是起始因子的作用。
Background and aims
Inflammatory bowel diseases (IBD) are chronic inflammatory disorders affecting the gastrointestinal tract. The two main forms of IBD are Crohn's disease (CD) and ulcerative colitis (UC). These conditions are thought to be genetically determined, overaggressive immune responses of the intestinal mucosa to normal resident bacterial constituents and environmental factors.
The prevalence of IBD in the Western Britain and Northern Europe is high and it shows increasing trend in China. Recent studies were mainly focused on endoscopic diagnosis and treatment, while little is know about its molecular mechanism. Nowadays, the diagnosis of IBD is still established by the endoscopic, histologic, and radiologic methods and clinical manifestations.
There is a close resemblance in clinical, radiological, endoscopic, surgical and histological features among IBD, intestinal tuberculosis (ITB) and intestinal lymphoma (IL). Patients with activity tuberculosis swallow sputum containing Mycobacterium tuberculosis through intestinal mucosa. Mycobacterium tuberculosis colonizated in the intestinal mucosa and ITB may occur. IL accounts for about 15% to 20% of primary gastrointestinal lymphoma. It is reported that nearly 20 years, the incidence of both IL and ITB has increased around the world.
These diseases appear quite stereotyped including diarrhea, abdominal pain, fever and degradation of the general physical condition. The endoscopic features show they all have a predilection for the small bowel, particularly the terminal ileum, although any part of the gastrointestinal tract may be affected. And both ITB and CD are chronic granulomatous disorders.However, the treatments of these conditions are quite different, and the morbidity and mortality resulting from a delayed diagnosis or misdiagnosis is considerably high.Thus differential diagnosis of these conditions remains a major challenge to clinicians.
There is currently no easy diagnostic tool as biomarks for these pathologies. The routine blood tests looking at the cell counts and fractionation of white blood cells and other conventional tests, such as the erythrocyte sedimentation rate and C-reactive protein, are too nonspecific which can change during inflammatory states. And do not aid in the work-up of these patients.
The most reliable differentiation method of ITB is to find evidence of M. tuberculosis in the intestinal tissues. Unfortunately, acid-fast bacilli's staining lacks sensitivity and specificity. In addition, the biopsy culture for M. tuberculosis is time consuming (3-8 weeks) and results are frequently negative (accuracy ranging from 25 to 35%). The specific pathology of ITB is caseous granulomatous, and CD the whole wall inflammation, non-caseous granulomas. Primary intestinal lymphoma was more central lesions, lymphoma cells to the submucosa infiltrated. However, due to the Unreliable of endoscopic biopsy, typical pathological lesions are not always Available.And for PIL, sometimes surgery and immunohistochemistry is needed.
The PPD skin test is not,because in areas with a very low incidence of active TB (<10 per 100,000 per year), a positive TST will more likely be a false positive, and the false-positive rate is very high in areas of the world where the BCG vaccination is still given. New finding such as "in situ PCR" is very promising, but there are many variables that may hamper the diagnostic value of the test, such as the tissue sample size and the duration of exposure of the sample to formalin and other technical aspects that need to be improved to gain a high yield.and "in situ PCR" is not sensitive enough and confusingly may occasionally detect mycobacterial signals from diagnosed CD tissues.
More recently, the fecal protein marker calprotectin has been shown to closely correlate with macroscopic and histologic evidence of inflammation. Tibble et al. demonstrated that calprotectin is specific in differentiating between patients with irritable bowel syndrome and IBD.However, these measures are hampered by a low sensitivity and specificity for intestinal inflammation, and do not adequately reflect disease activity.As elevated concentrations can be due to other conditions, including colon cancer, colitis induced by nonsteroidal antiinflammatory agents, or intestinal infections. Notably, intestinal infections are a major issue, as acute exacerbations of IBD may require immunosuppressive therapy, which could be detrimental in patients with infectious diseases. Therefore, a marker that readily differentiates between acute IBD and intestinal infections is highly desirable for clinical decision-making. Wolfgang Tillinger recently demonstrated that CD64 could serve as a valuable tool to discriminate between IBD, infectious enterocolitis, and functional intestinal disorders.
A recent series of studies showed that the use of different serological markers, such as the anti-neutrophil cytoplasmic antibodies, the peri-nuclear and the cytoplasmic variants (p-ANCA and c-ANCA), and the IgA and IgG subtypes of
Anti- Saccharomyces cerevisiae antibodies (ASCA).ASCA and ANCA are the only available commercial tests that can be helpful for CD and UC discrimination. Although, they show a quite good specificity, their sensitivity is rather low and they are therefore not recommended for broad clinical practice.However ASCA and ANCA had no significant diagnostic value in discriminating between ITB and CD.
Taken together, laboratory markers are useful and should be part of the global management of our IBD patients. They are however not magic and until more data become available, the use of CRP and other laboratory markers should be seen as an additive tool to clinical observation and physical examination rather than a replacement.Therefore, initial diagnosis still relies on the combination of several biological and morphological tests, including gastrointestinal endoscopies and histology, and is based on standardized validated diagnostic criteria.
Thus, a rapid test that is sensitive and specific in its ability to differentiate IBD, ITB, PIL and intestinal bacterial infections would be desirable.
UC-cancer is different from the normal intestinal cancer, and they show different molecular mechanisms. UC-associated epithelial dysplasia is defined as unequivocally neoplastic epithelium. Currently, it is the most important marker of an increased risk of malignancy in UC. Dysplasia may grow as a flat lesion or as a dysplasia-associated lesion or mass (DALM). In patients with chronic UC, polypoid dysplastic lesions (PDLs) are morphologically similar to sporadic adenomas (SAs), but may be biologically distinct from them and are managed differently. One specific subtype of DALM that poses a diagnostic challenge to clinicians and pathologists is the isolated, discrete dysplastic nodule or polyp that pathologically resembles a sporadic colonic adenoma. Despite the histologic similarity between these two pathogenetically distinct neoplasms, the clinical distinction between UC- PDLs and SAs that arise in patients with UC is extremely important. The former lesion arises as a result of UC, and its presence is usually an indication for colectomy, whereas the treatment of SAs, whether UC-associated or not, is a simple polypectomy. On routine histologic evaluation, distinction between these two neoplasms is difficult. p53 mutations have been shown to occur at an earlier phase in the progression of UC-associated neoplasia when compared with sporadic colon carcinogenesis. In contrast, APC gene mutations are common and occur at an earlier stage in the development of SA.βcatenin is a cell membrane protein that accumulates in the nucleus of colon cancer cells in response to APC gene mutations. No differences were observed in p53 orβcatenin staining, between CUC-associated and non-CUC-associated SAs. Neither p53 norβcatenin expression correlated with any clinical or pathologic features, including size and degree of dysplasia of the polyps. CUC-associated PDLs and CUC-associated SAs may have a different molecular genotype. In patients with CUC, the combination of strong p53 expression and absent or weakβcatenin expression is evidence in favor of a UC-associated PDL in diagnostically difficult lesions. Furthermore, UC-associated and non-UC-associated SAs have a similar p53 andβcatenin immunophenotype and thus provide evidence that they are pathogenetically related neoplasms regardless of the presence or absence of colitis. Therefore, there is great interest in understanding more fully the molecular pathogenesis of these lesions and in detecting markers that may be used to differentiate them.
Our search group has found several proteins present in UC unhealth mucosal that are differert from normal mucosal. activated cdc42 kinasel (Ackl) and Mawb binding protein (Mawbp) are two among them and they show significant difference than others.
Nowadays, Ackl, a specific downstream effector of Cdc42, have show to be involved in cell growth, inhibit apoptosis, promoting angiogenesis, cell invasion and metastasis. Because it is conbined with Cdc42,the internal and external cellular signaling pathways core protein binding, which has involved with MAPK, NF-kB and other signaling pathways the inflammation associated with Which are not only tumor-related,but also inflammation-related, we guess it may play an important role in cells.
The current literatures on the Mawbp are only three.By comparing protein expressions between the primary gastric cancer and the surrounding normal gastric mucosa of differences, Zhang J found that Mawbp are related with in gastric cancer. Westhoff TH found high expression of Mawbp in mice blood is associated with low blood pressure, suggesting its function of blood pressure regulater. Kurokawa analize the different expression genes of liver cancer and adjacent liver tisssues.
Currently, there is no study about Ackl, Mawbp associated with IBD. The preliminary work of our group has found that the expression levers of Ackl and Mawbp in UC are significantly higher than that of normal intestinal mucosa. That the changes are UC specific or merely a general change in inflammation remains unclear. And just as noted above, the two proteins are associated with digestive tract tumors, we suspect wether they are involved in the formation of CUC-cancer and colon cancer.
The aim of this study was to investigated the utility of Ackl、Mawbp as laboratory biomarkers of IBD to address:(1)whether these markers can differentiate IBD patients from non-IBD gastrointestinal inflammationthe(enterocolitis, chronic colitis);(2)whether they correlate with severity of inflammation; and (3)compare the performance of Ackl, Mawbp in different kind of polys. So we can identify the molecular basis for the development of IBD, and the difference between colorectal adenoma-carcinoma and UC-cancer.
Materials and Methods
1. A total of 212 colorectal specimens were collected randomly from individuals who underwent endoscopic resection under total colonoscopy. The specimens consisted of 78 UCs,27 CDs,15ITBs,6IL,20 Infectious colitis,26chronic colitis, 10 UC-associated SAs,10 non-UC-associated SAs,10 non-UC-associated inflammatory polyps,10 UC-associated inflammatory polyps and 60 healthy controls.
2. The following indexes were obtained from all the patients: age, gender, course of disease, colitis location, serological tests(white blood cells, erythrocyte sedimentation rate, serum albumin, C-reactive protein、Platelet etc); endoscopic feature, and pathological feature.
3. Expression of Ackl,Mawbp was examined by immunohistochemistry in all these specimens.
4. The performance of each marker with reference to inflammatory activity and clinical activity was assessed by computing correlations, and sensitivity and specificity.
Results
1. The majority of UCs present with rectum colitis (43.4%) and CDs with small intestine and Ileum (74.1%);
2. CRP of CDs was higher than that of UCs(10.95vs5, P<0.05); CRP of active UCs was higher than normal group, there was no significant difference of other indicators among normal, CDs and UCs. Both in the UCs or in the CDs, CRP and ESR were positively and closely correlated. In UCs, the clinical stage was positively and correlated with CRP and ESR. But in CDs, the clinical stage has nothing to do with CRP and ESR.
3. In CDs and UCs, Ackl expression and clinical disease activity was positively correlated; in UCs, Ackl cytoplasm cytoplasmic expression and Mawbp were negative correlation; Mawbp and CEA in the CD group was negatively correlated with the cytoplasmic expression sex, Ackl the cytoplasm cytoplasmic expression and Mawbp expression.
4. Mawbp, but not Ackl expression of Chronic colitis and infectious colitis was positively correlated with inflammatory pathology grading (P<0.01), but not closely correlated (correlation coefficient<0.5). In CDs and UCs, Ackl and Mawbp showed no correlated with inflammatory pathology grading.
5. For differentiation between IBD and healthy controls,Ackl+ in UCs sensitivity (88.9%) and compliance rate (84.06%)was high, but specificity(78.8%)low; the CDs Ackl+ specifity(90%)and compliance rate(83.33%)was high, but sensitivity(66.6%)low.For differentiation between IBD, healthy controls and non-IBD gastrointestinal inflammationthe,UCs Ackl+ specificity(83.70%)was higher, but sensitivity (65.31%) slightly lower; in CDs,Ackl+specificity(92.30%) was higher,while sensitivity (32.00%) and compliance rate (68.75%)was low.
6. In all samples of ITB (15/15) and IL (6/6) Ackl show high expression (3+) and Mawbp expression Medium (2+).
7. The expression of Ackl in SAs (2+or3+) were significantly higher than that in inflammatory polyps (-or+).DALM, UC-associated-SAs, non-UC-associated-SAs staining had a similar Ackl immunophenotype.
Conclusions
In the present study, we have shown that Ackl, Mawbp protein expression is increased not only in IBD mucosa and ITB, IL but also in bacterial enterocolitis, chronic colitis, using Immunohistochemistry. None of these three markers is consistently superior in its ability to differentiate active IBD from other colitis.On the other hand, it may be noted in this study that colitic cancers develop in only a limited number of patients with UC, although Ackl is ubiquitously expressed in the inflamed UC mucosa. Thus Ackl, Mawbp may play a role in the UC-cancer sequence as one of the growth promoting as well as antiapoptotic factors, and function in colitic cancer development not as a inflammation/tumour initiator but as a inflammation/tumour promoter.
炎症性肠病(inflammatory bowel disease, IBD)包括克罗恩病(Crohn's disease, CD)和溃疡性结肠炎(ulcerative colitis, UC)。目前研究认为发病与基因、肠道菌群、环境因素有关。
IBD在西方及北欧的发病率较高,近年我国的发病率呈现逐年增高的趋势。目前关于IBD的研究主要集中在诊断和治疗上,对其分子机制尚缺乏系统研究。
一般情况下UC和CD可以根据临床、内镜、病理综合分析判断。但是如果结肠广泛病变,并且内镜、病理表现不典型时,UC和CD有时难以鉴别；如果IBD内镜下炎症表现较轻,无典型内镜下改变,还需要与肠道感染性疾病相鉴别。CD、肠结核(intestinal tuberculosis, ITB)、肠道淋巴瘤(intestinal lymphoma,IL)因为存在诸多相似的地方,三者之间的鉴别也是临床的一个难题。
ITB多由活动期肺结核病人吞下含结核杆菌的痰,结核杆菌穿透肠黏膜定植在肠道引起。原发性胃肠淋巴瘤(primary gastrointestinal lymphoma, PGL)是非霍奇金氏淋巴瘤中具有特征性并所占比例较少(11%-34%)的一类,在原发性胃肠淋巴瘤中,IL约占15%-20%。目前病因未明。ITB和IL近年来在发达国家和发展中国家都有上升趋势。
CD、ITB、IL临床表现(如腹痛、腹泻)缺乏特异性,内窥镜下多累及回盲部、手术及病理有很多相似之处,如肠壁的慢性非特异性炎症、肉芽肿、溃疡和节段性病变等。而治疗方案却有巨大差异性。所以误诊会带来严重的后果。这几种疾病的鉴别对临床医生来说是一项重大挑战。
目前,临床上尚未有简单易行的实验室方法来鉴别诊断IBD。
血液学指标如白细胞计数、血沉(erythrocyte sedimentation rate, ESR)和C-反应蛋白(C-reactive protein, CRP)均无特异性。
诊断ITB最可靠的是从肠道组织中发现结核杆菌,但是抗酸染色特异性、敏感性都不够。分枝杆菌的培养要耗时,而结果通常是阴性的(25%-35%)。PPD皮试阳性作为ITB的诊断并不可靠。诊断性抗结核治疗时间长,会对病人健康产生影响。
而活检取材多浅小,只能取到黏膜层和部分黏膜下层,并不能观察肠壁全貌,病理黏膜非特异性炎症的检出率常较高,因此一些特征性的病变难以被发现,如属于全壁性炎症的CD的特异性病理表现非干酪样坏死,IL的淋巴瘤细胞以黏膜下浸润为主,而隐窝脓肿不仅见于UC,也见于CD和ITB。所以IL常常需要手术切除后行免疫组化才能最后确诊。
最近关于ITB诊断的研究进展还包括对ITB粘膜活检组织进行“DNA原位PCR”技术。PCR检测的速度快,准确性高,与组织中是否有干酪样坏死性肉芽肿无关,但敏感度低,且受组织样本大小以及样品和甲醛接触时间的影响。
Wolfgang Tillinger最近证明血清血指标CD64可以用来鉴别IBD、感染性肠炎及功能性胃肠病。近来有人在IBD患者粪便中发现钙卫蛋白升高,但后来发现,感染性肠炎中也存在,诊断特异性不高。
抗酵母菌抗体(antisaccharomyces antibodies, ASCA)和抗中性粒细胞胞浆抗体(perinuclear anti-neutrophil cytoplasmic antibody, pANCA)对鉴别CD敏感度较高,但特异性低。ASCA无法鉴别CD和ITB。最近有研究发现,ASCA和pANCA对中国和日本人的敏感性较低。另有学者认为从CD患者组织提取物中测定特异性的RAN序列,对临床诊断的CD活检组织进行检测,发现阳性率80%,其作为组织学标记物的潜在应用价值值得进一步研究。
综上所述,目前仍无一种简单易行的可以鉴别IBD的金标准。它们的鉴别需要从临床、影像学、内镜、病理学表现等多方面综合考虑。近几年出现的一些新的检查方法虽很有前途,但尚需更多的临床试验进行验证。
我们课题组运用蛋白质组学技术,筛选出UC与正常人肠道黏膜差异蛋白若干,其中包括活化的cdc42结合蛋白激酶1(activited Cdc42 kinase1, Ack1)和Mawb结合蛋白(Mawb binding protein, Mawbp)。
Ackl体内发挥的作用及其具体机制尚不十分清楚。目前仅有少量研究认为它参与细胞生长、抑制细胞凋亡,促进血管生成、细胞浸润和转移,在晚期肿瘤的转移中发挥作用。因为它与胞内外信号通路核心蛋白Cdc42结合,参与和炎症及肿瘤均相关的MAPK、NF-kB等信号通路,于是我们推测它在细胞内可能发挥重要作用。
目前关于Mawbp的文献仅有3篇,Zhang J通过比较原发型胃癌与周围黏膜蛋白表达的差异,认为Mawbp是胃癌相关蛋白。WesthoffTH研究发现Mawbp在低血压大鼠血液中高表达,推测其有调节血压的功能。Kurokawa分析肝癌组织与癌旁组织的基因差异表达发现Mawbp在肝癌中高表达,因此他推测Mawbp在肝癌形成中可能发挥重要的作用。
目前国内外尚未见Ackl和Mawbp与IBD相关文献报道。我们课题组的前期工作证明Ack1和Mawbp在UC肠道黏膜较正常人肠道黏膜含量差异明显。这几个蛋白的改变是UC特有还是炎症的普遍改变,目前尚不清楚。又如前文所示,Ack1和Mawbp与某些消化系统肿瘤相关,那么我们推想Ack1可能参与UC的癌变。UC癌变不同于普通的散发性结肠癌。UC相关性结肠癌常发生于平坦型不典型增生或有不典型增生的病灶(dysplasia-assoeiated lesiono mass, DALM),而散发性大肠癌多由散发性腺瘤性息肉(sporadic adenoma, SA)发展而来,按照腺瘤-不典型增生-大肠癌-转移的途径发生。它们的分子机制有诸多不同,如p53突变为UC癌变的早期事件,APC突变及其失活导致的Wnt途径激活在结直肠癌发生初期具有重要作用,但在IBD相关结直肠癌正好相反。UC-associated SAs和non-UC-associated SAs的p53、APC表达与DALM相反。从分子水平上证明DALM与SAs的区别。Ack1、Mawbp在这几种组织结构中的表达是否有差异性,它们能否作为UC癌变区别于普通大肠癌的标志物,目前不清楚。
因此,本次实验的目的是希望通过对IBD、ITB、IL、普通肠道炎症及息肉、DALM中进行Ack1、Mawbp分子标志的系统检测,观察相对完整的Ackl1、Mawbp在这几种疾病中的表达情况,从而推测：(a)它们是否能将IBD病人与ITB、IL、感染性结肠炎、慢性结肠炎相鉴别；(b)它们是否与炎症相关；(c)它们能否鉴别non-UC-associated SAs、UC-associated SAs、DALMs以及炎性息肉。以期找出IBD发生发展的分子学基础及其与传统的大肠腺瘤-癌变途径的差异。
材料和方法
1.完全随机收集78例UC、27例CD、20例感染性肠炎、26例慢性结肠炎、60例肠道黏膜正常病人、15例ITB、6例IL、6例UC-associated SAs、4例DALMs、10例non-UC-associated SAs、10例UC相关性炎症性息肉、10例散发性炎症性息肉；
2.比较UC组和CD组之间临床病理学资料的差异,包括患者性别、年龄、病变累及部位、白细胞、血小板、血沉、C反应蛋白等；
3.运用免疫组织化学染色技术观察Ack1、Mawbp在上述各组中的表达情况；
4.统计分析：采用SPSS 13.0统计软件进行分析,用Independent-Samples T Test测定组和组之间患者年龄的差异;用One way ANOVA测定多组计量资料之间的差异性；用Chi-square tes、Mann-Whitney U test测定两组之间其他因素的差异(包括性别、血清学指标及各个分子的染色情况等)；用Kruskal Wallist测定多组等级资料之间的差异；用Spearman rank-order correlation coefficient测定各等级资料之间或者等级资料与计量资料之间的相关性。所有分析结果取双侧P值,P<0.05认为差异具有显著性。
结果
1.78例UC有33例发生于直肠(43.4%),23例发生于左半结肠(30.3%)。25例CD有20例发生于小肠及回末(74.1%)。
2.CD病人的CRP显著高于UC组(10.95vs1.8,P<0.05)和正常对照组(10.95vs5,P<0.05)。临床活动度为中重度的UC病人的CRP高于正常人(6.4vs5,P<0.05)和临床活动度为轻度的UC患者(2.14±1.44),PLT、ESR、APTT、PT、CEA、CA199指标CD和UC之间,IBD与正常人之间无显著差异(P>0.05)；两组在性别、年龄等方面无显著差异(P>0.05)。不论是在UC组(r=0.604,P<0.05)还是在CD组(r=0.652,P<0.05),CRP均与ESR呈正相关关系,且相关关系密切。UC组临床分级与CRP呈正相关且关系密切(r=0.563,P<0.05),而与ESR呈正相关但关系不密切(r=-0.409,P<0.05)。CD组中临床分级与CRP、ESR无关(P>0.05)。
3.在CD和UC组,Ack1的表达与临床活动度呈正相关性(r=0.693,P<0.05)；在UC组,Ack1胞浆表达与Mawbp的胞浆表达具有负相关性(r=-0.503,P<0.05)；在CD组,Mawbp与CEA的胞浆表达呈负相关性(r=-0.607,P<0.05),Ack1的胞浆表达与Mawbp的胞浆表达无关(P>0.05)。
4.感染性肠炎中Mawbp表达与炎症病理分级呈正相关(P<0.01),但相关关系均不密切(r<0.5)。Ack1表达与慢性结肠炎、感染性肠炎炎症病理分级无关(P>0.05)。CD和UC组Ack1和Mawbp的胞浆表达与病理分级无关(P>0.05)。
5.以正常人为对照组,在UC组Ack1+的敏感性(88.9%)和符合率(84.06%)较高,特异性(78.8%)较低；而在CD组,Ack1+的特异性(90%)和符合率(83.33%)较高,敏感性(66.6%)较低。以正常组+慢性结肠炎+感染性肠炎为对照组,在UC组Ack1+的敏感性(65.31%)和特异性(83.70%)较高,符合率(73.91%)稍低；而在CD组,Ack1+的特异性(92.30%)较高,敏感性(32.00%)和符合率(68.75%)较低。
6.Ack1在本组所有ITB(15/15)和IL(6/6)中均高表达(3+),Mawbp表达量中等(2+)。ITB和IL表达无显著性差异(P>0.05)。
7.Ack1在SAs(2+或3+)中表达明显高于炎性息肉(-或+)。相同程度不典型增生的DALM和UC-associated-SAs或non-UC-associated-SAs染色无显著性差别(P>0.05)。
结论
Ack1、Mawbp在IBD、ITB、IL、感染性肠炎、慢性结肠炎中均有表达,广泛参与炎症反应,与炎症具体关系复杂,不是IBD特异性标志物。Ack1不是UC癌变有别于普通大肠癌的特异性指标,可能通过促生长和抗凋亡在结肠癌的发展过程中发挥作用,应只是推动作用而不是起始因子的作用。
Background and aims
Inflammatory bowel diseases (IBD) are chronic inflammatory disorders affecting the gastrointestinal tract. The two main forms of IBD are Crohn's disease (CD) and ulcerative colitis (UC). These conditions are thought to be genetically determined, overaggressive immune responses of the intestinal mucosa to normal resident bacterial constituents and environmental factors.
The prevalence of IBD in the Western Britain and Northern Europe is high and it shows increasing trend in China. Recent studies were mainly focused on endoscopic diagnosis and treatment, while little is know about its molecular mechanism. Nowadays, the diagnosis of IBD is still established by the endoscopic, histologic, and radiologic methods and clinical manifestations.
There is a close resemblance in clinical, radiological, endoscopic, surgical and histological features among IBD, intestinal tuberculosis (ITB) and intestinal lymphoma (IL). Patients with activity tuberculosis swallow sputum containing Mycobacterium tuberculosis through intestinal mucosa. Mycobacterium tuberculosis colonizated in the intestinal mucosa and ITB may occur. IL accounts for about 15% to 20% of primary gastrointestinal lymphoma. It is reported that nearly 20 years, the incidence of both IL and ITB has increased around the world.
These diseases appear quite stereotyped including diarrhea, abdominal pain, fever and degradation of the general physical condition. The endoscopic features show they all have a predilection for the small bowel, particularly the terminal ileum, although any part of the gastrointestinal tract may be affected. And both ITB and CD are chronic granulomatous disorders.However, the treatments of these conditions are quite different, and the morbidity and mortality resulting from a delayed diagnosis or misdiagnosis is considerably high.Thus differential diagnosis of these conditions remains a major challenge to clinicians.
There is currently no easy diagnostic tool as biomarks for these pathologies. The routine blood tests looking at the cell counts and fractionation of white blood cells and other conventional tests, such as the erythrocyte sedimentation rate and C-reactive protein, are too nonspecific which can change during inflammatory states. And do not aid in the work-up of these patients.
The most reliable differentiation method of ITB is to find evidence of M. tuberculosis in the intestinal tissues. Unfortunately, acid-fast bacilli's staining lacks sensitivity and specificity. In addition, the biopsy culture for M. tuberculosis is time consuming (3-8 weeks) and results are frequently negative (accuracy ranging from 25 to 35%). The specific pathology of ITB is caseous granulomatous, and CD the whole wall inflammation, non-caseous granulomas. Primary intestinal lymphoma was more central lesions, lymphoma cells to the submucosa infiltrated. However, due to the Unreliable of endoscopic biopsy, typical pathological lesions are not always Available.And for PIL, sometimes surgery and immunohistochemistry is needed.
The PPD skin test is not,because in areas with a very low incidence of active TB (<10 per 100,000 per year), a positive TST will more likely be a false positive, and the false-positive rate is very high in areas of the world where the BCG vaccination is still given. New finding such as "in situ PCR" is very promising, but there are many variables that may hamper the diagnostic value of the test, such as the tissue sample size and the duration of exposure of the sample to formalin and other technical aspects that need to be improved to gain a high yield.and "in situ PCR" is not sensitive enough and confusingly may occasionally detect mycobacterial signals from diagnosed CD tissues.
More recently, the fecal protein marker calprotectin has been shown to closely correlate with macroscopic and histologic evidence of inflammation. Tibble et al. demonstrated that calprotectin is specific in differentiating between patients with irritable bowel syndrome and IBD.However, these measures are hampered by a low sensitivity and specificity for intestinal inflammation, and do not adequately reflect disease activity.As elevated concentrations can be due to other conditions, including colon cancer, colitis induced by nonsteroidal antiinflammatory agents, or intestinal infections. Notably, intestinal infections are a major issue, as acute exacerbations of IBD may require immunosuppressive therapy, which could be detrimental in patients with infectious diseases. Therefore, a marker that readily differentiates between acute IBD and intestinal infections is highly desirable for clinical decision-making. Wolfgang Tillinger recently demonstrated that CD64 could serve as a valuable tool to discriminate between IBD, infectious enterocolitis, and functional intestinal disorders.
A recent series of studies showed that the use of different serological markers, such as the anti-neutrophil cytoplasmic antibodies, the peri-nuclear and the cytoplasmic variants (p-ANCA and c-ANCA), and the IgA and IgG subtypes of
Anti- Saccharomyces cerevisiae antibodies (ASCA).ASCA and ANCA are the only available commercial tests that can be helpful for CD and UC discrimination. Although, they show a quite good specificity, their sensitivity is rather low and they are therefore not recommended for broad clinical practice.However ASCA and ANCA had no significant diagnostic value in discriminating between ITB and CD.
Taken together, laboratory markers are useful and should be part of the global management of our IBD patients. They are however not magic and until more data become available, the use of CRP and other laboratory markers should be seen as an additive tool to clinical observation and physical examination rather than a replacement.Therefore, initial diagnosis still relies on the combination of several biological and morphological tests, including gastrointestinal endoscopies and histology, and is based on standardized validated diagnostic criteria.
Thus, a rapid test that is sensitive and specific in its ability to differentiate IBD, ITB, PIL and intestinal bacterial infections would be desirable.
UC-cancer is different from the normal intestinal cancer, and they show different molecular mechanisms. UC-associated epithelial dysplasia is defined as unequivocally neoplastic epithelium. Currently, it is the most important marker of an increased risk of malignancy in UC. Dysplasia may grow as a flat lesion or as a dysplasia-associated lesion or mass (DALM). In patients with chronic UC, polypoid dysplastic lesions (PDLs) are morphologically similar to sporadic adenomas (SAs), but may be biologically distinct from them and are managed differently. One specific subtype of DALM that poses a diagnostic challenge to clinicians and pathologists is the isolated, discrete dysplastic nodule or polyp that pathologically resembles a sporadic colonic adenoma. Despite the histologic similarity between these two pathogenetically distinct neoplasms, the clinical distinction between UC- PDLs and SAs that arise in patients with UC is extremely important. The former lesion arises as a result of UC, and its presence is usually an indication for colectomy, whereas the treatment of SAs, whether UC-associated or not, is a simple polypectomy. On routine histologic evaluation, distinction between these two neoplasms is difficult. p53 mutations have been shown to occur at an earlier phase in the progression of UC-associated neoplasia when compared with sporadic colon carcinogenesis. In contrast, APC gene mutations are common and occur at an earlier stage in the development of SA.βcatenin is a cell membrane protein that accumulates in the nucleus of colon cancer cells in response to APC gene mutations. No differences were observed in p53 orβcatenin staining, between CUC-associated and non-CUC-associated SAs. Neither p53 norβcatenin expression correlated with any clinical or pathologic features, including size and degree of dysplasia of the polyps. CUC-associated PDLs and CUC-associated SAs may have a different molecular genotype. In patients with CUC, the combination of strong p53 expression and absent or weakβcatenin expression is evidence in favor of a UC-associated PDL in diagnostically difficult lesions. Furthermore, UC-associated and non-UC-associated SAs have a similar p53 andβcatenin immunophenotype and thus provide evidence that they are pathogenetically related neoplasms regardless of the presence or absence of colitis. Therefore, there is great interest in understanding more fully the molecular pathogenesis of these lesions and in detecting markers that may be used to differentiate them.
Our search group has found several proteins present in UC unhealth mucosal that are differert from normal mucosal. activated cdc42 kinasel (Ackl) and Mawb binding protein (Mawbp) are two among them and they show significant difference than others.
Nowadays, Ackl, a specific downstream effector of Cdc42, have show to be involved in cell growth, inhibit apoptosis, promoting angiogenesis, cell invasion and metastasis. Because it is conbined with Cdc42,the internal and external cellular signaling pathways core protein binding, which has involved with MAPK, NF-kB and other signaling pathways the inflammation associated with Which are not only tumor-related,but also inflammation-related, we guess it may play an important role in cells.
The current literatures on the Mawbp are only three.By comparing protein expressions between the primary gastric cancer and the surrounding normal gastric mucosa of differences, Zhang J found that Mawbp are related with in gastric cancer. Westhoff TH found high expression of Mawbp in mice blood is associated with low blood pressure, suggesting its function of blood pressure regulater. Kurokawa analize the different expression genes of liver cancer and adjacent liver tisssues.
Currently, there is no study about Ackl, Mawbp associated with IBD. The preliminary work of our group has found that the expression levers of Ackl and Mawbp in UC are significantly higher than that of normal intestinal mucosa. That the changes are UC specific or merely a general change in inflammation remains unclear. And just as noted above, the two proteins are associated with digestive tract tumors, we suspect wether they are involved in the formation of CUC-cancer and colon cancer.
The aim of this study was to investigated the utility of Ackl、Mawbp as laboratory biomarkers of IBD to address:(1)whether these markers can differentiate IBD patients from non-IBD gastrointestinal inflammationthe(enterocolitis, chronic colitis);(2)whether they correlate with severity of inflammation; and (3)compare the performance of Ackl, Mawbp in different kind of polys. So we can identify the molecular basis for the development of IBD, and the difference between colorectal adenoma-carcinoma and UC-cancer.
Materials and Methods
1. A total of 212 colorectal specimens were collected randomly from individuals who underwent endoscopic resection under total colonoscopy. The specimens consisted of 78 UCs,27 CDs,15ITBs,6IL,20 Infectious colitis,26chronic colitis, 10 UC-associated SAs,10 non-UC-associated SAs,10 non-UC-associated inflammatory polyps,10 UC-associated inflammatory polyps and 60 healthy controls.
2. The following indexes were obtained from all the patients: age, gender, course of disease, colitis location, serological tests(white blood cells, erythrocyte sedimentation rate, serum albumin, C-reactive protein、Platelet etc); endoscopic feature, and pathological feature.
3. Expression of Ackl,Mawbp was examined by immunohistochemistry in all these specimens.
4. The performance of each marker with reference to inflammatory activity and clinical activity was assessed by computing correlations, and sensitivity and specificity.
Results
1. The majority of UCs present with rectum colitis (43.4%) and CDs with small intestine and Ileum (74.1%);
2. CRP of CDs was higher than that of UCs(10.95vs5, P<0.05); CRP of active UCs was higher than normal group, there was no significant difference of other indicators among normal, CDs and UCs. Both in the UCs or in the CDs, CRP and ESR were positively and closely correlated. In UCs, the clinical stage was positively and correlated with CRP and ESR. But in CDs, the clinical stage has nothing to do with CRP and ESR.
3. In CDs and UCs, Ackl expression and clinical disease activity was positively correlated; in UCs, Ackl cytoplasm cytoplasmic expression and Mawbp were negative correlation; Mawbp and CEA in the CD group was negatively correlated with the cytoplasmic expression sex, Ackl the cytoplasm cytoplasmic expression and Mawbp expression.
4. Mawbp, but not Ackl expression of Chronic colitis and infectious colitis was positively correlated with inflammatory pathology grading (P<0.01), but not closely correlated (correlation coefficient<0.5). In CDs and UCs, Ackl and Mawbp showed no correlated with inflammatory pathology grading.
5. For differentiation between IBD and healthy controls,Ackl+ in UCs sensitivity (88.9%) and compliance rate (84.06%)was high, but specificity(78.8%)low; the CDs Ackl+ specifity(90%)and compliance rate(83.33%)was high, but sensitivity(66.6%)low.For differentiation between IBD, healthy controls and non-IBD gastrointestinal inflammationthe,UCs Ackl+ specificity(83.70%)was higher, but sensitivity (65.31%) slightly lower; in CDs,Ackl+specificity(92.30%) was higher,while sensitivity (32.00%) and compliance rate (68.75%)was low.
6. In all samples of ITB (15/15) and IL (6/6) Ackl show high expression (3+) and Mawbp expression Medium (2+).
7. The expression of Ackl in SAs (2+or3+) were significantly higher than that in inflammatory polyps (-or+).DALM, UC-associated-SAs, non-UC-associated-SAs staining had a similar Ackl immunophenotype.
Conclusions
In the present study, we have shown that Ackl, Mawbp protein expression is increased not only in IBD mucosa and ITB, IL but also in bacterial enterocolitis, chronic colitis, using Immunohistochemistry. None of these three markers is consistently superior in its ability to differentiate active IBD from other colitis.On the other hand, it may be noted in this study that colitic cancers develop in only a limited number of patients with UC, although Ackl is ubiquitously expressed in the inflamed UC mucosa. Thus Ackl, Mawbp may play a role in the UC-cancer sequence as one of the growth promoting as well as antiapoptotic factors, and function in colitic cancer development not as a inflammation/tumour initiator but as a inflammation/tumour promoter.
引文
[1]Price AB.Overlap in the spectrum of non-specific inflammatory bowel disease: colitis indeterminate.J Clin Pathol 1978; 31:567-77
[2]Gan HT, Chen YQ, Ouyang Q et al. Differentiation between intestinal tuberculosis and Crohn's disease in endoscopic biopsy specimens by polymerase chain reaction. Am J Gastroenterol 2002; 97:1446-51
[3]Koch P, del Valle F, Berdel WE, et al. Primary gastrointestinal non-Hodgkin's lymphoma:I. Anatomic and histologic distribution, clinicalfeatures, and survival data of 371 patients registered in the Germanmulticenter study GIT. J Clin Oncol,2001,19:3861-3873
[4]Amer MH, el-Akkad S. Gastrointestinal lymphoma in adults:clinical features and management of 300 cases[J]. Gastroenterology,1994,106:846-858.
[5]Otter R, Gerrits WB, vd Sandt MM, et al. Primary extranodal andnodal non-Hodgkin's lymphoma. A survey of a population-based registry. EurJ Cancer Clin Oncol,1989,25:1203-1210
[6]毛永平,杨云生,王淑芳,等.原发性小肠淋巴瘤临床病理分析23例.世界华人消化杂志.2005；13：2711-2713
[7]irsch R, Pentecost M, Hall PM et al. Role of colonoscopic biopsy in distinguishing between Crohn's disease and intestinal tuberculosis.J Clin Pathol 2006; 59:840-4
[8]Witte J, Shivananda S, Lennard-Jones JE, et al. Disease outcome in inflammatory bowel disease:mortality, morbidity and therapeutic management of a 796-person inception cohort in the European Collaborative Study on Inflammatory Bowel Disease (EC-IBD).Scand J Gastroenterol,2000,35 (12):1272-1277.
[9]中国炎症性肠病协作组.3100例溃疡性结肠炎住院病例回顾分析.中华消化杂志；2006；26：368-372.
[10]Liu TH. Crohn's disease-differential diagnosis with intestinal tuberculosis. Zhonghua Nei Ke Za Zhi 1981; 20:211-5.
[11]Arnold C,Moradpour D,Blum HE.Tuberculosis colitis mimicking Crohn's disease.Am J Gastroenterol.1998,93;2294-2296
[12]Al Jahdali H, Memish ZA, Menzies D. The utility and interpretation of tuberculin skin tests in the Middle East. Am J Infect Control.2005; 33:151-6.
[13]Rangaka MX, Wilkinson KA, Seldon R et al. Effect of HIV-1 infection on T-Cell-based and skin test detection of tuberculosis infection. Am J Respir Crit Care Med 2007; 175:514-20.
[14]李佩文,贾立群,朱世杰.恶性肿瘤误诊误治与防范.北京：科学技术文献出版社；2001：280-283.
[15]Pulimood AB, Peter S, Rook GW et al. In situ PCR for Mycobacterium tuberculosis in endoscopic mucosal biopsy specimens of intestinal tuberculosis and Crohn disease. Am J Clin Pathol 2008; 129:846-51
[16]Majid Abdulrahman Almadi, Subrata Ghosh, Abdulrahman Mohamed Aljebreen et al. Differentiating Intestinal Tuberculosis from Crohn's Disease:A Diagnostic Challenge. Am J Gastroenterol 2009; 104:1003-1012
[17]Expression of the High-Affi nity IgG Receptor FcRI (CD64) in Patients With Infl ammatory Bowel Disease:A New Biomarker for Gastroenterologic Diagnostics
[18]Solem C A, Jr Loftus E V, Tremaine W J, et al. Correlation of C-reactive protein with clinical, endoscopic, histologic, and radiographic activity in inflammatory bowel disease.[J]. Inflamm Bowel Dis,2005,11(8):707-712.
[19]Tibble J A, Sigthorsson G, Foster R, et al. Use of surrogate markers of inflammation and Rome criteria to distinguish organic from nonorganic intestinal disease.[J]. Gastroenterology,2002,123(2):450-460.
[20]Ghoshal UC, Ghoshal U, Singh H et al. Anti- Saccharomyces cerevisiae antibody is not useful to differentiate between Crohn's disease and intestinal tuberculosis in India. J Postgrad Med 2007; 53:166-70.
[21]Makharia GK, Sachdev V, Gupta R et al. Anti- Saccharomyces cerevisiae antibody does not differentiate between Crohn's disease and intestinal tuberculosis. Dig Dis Sci 2007; 52:33-9.
[22]Vermeire S, Joossens S, Peeters M, et al. Comparative study of ASCA (Anti-Saccharomyces cerevisiae antibody) assays in inflammatory bowel disease.[J]. Gastroenterology,2001,120(4):827-833.
[23]Garrard S M, Capaldo C T, Gao L, et al. Structure of Cdc42 in a complex with the GTPase-binding domain of the cell polarity protein, Par6.[J]. EMBO J, 2003,22(5):1125-1133.
[24]Linseman D A, Heidenreich K A, Fisher S K. Stimulation of M3 muscarinic receptors induces phosphorylation of the Cdc42 effector activated Cdc42Hs-associated kinase-1 via a Fyn tyrosine kinase signaling pathway.[J]. J Biol Chem,2001,276(8):5622-5628.
[25]Yang W, Cerione R A. Cloning and characterization of a novel Cdc42-associated tyrosine kinase, ACK-2, from bovine brain.[J]. J Biol Chem, 1997,272(40):24819-24824.
[26]Mackeigan J P, Murphy L O, Blenis J. Sensitized RNAi screen of human kinases and phosphatases identifies new regulators of apoptosis and chemoresistance.[J]. Nat Cell Biol,2005,7(6):591-600.
[27]Guo W, Giancotti F G. Integrin signalling during tumour progression.[J]. Nat Rev Mol Cell Biol,2004,5(10):816-826.
[28]Mercurio A M, Bachelder R E, Chung J, et al. Integrin laminin receptors and breast carcinoma progression. J Mammary Gland Biol Neoplasia,2001, 6(3):299-309.
[29]Manser E, Leung T, Salihuddin H, et al. A non-receptor tyrosine kinase that inhibits the GTPase activity of p21cdc42. Nature,1993,363(6427):364-367.
[30]van der Horst E H, Degenhardt Y Y, Strelow A, et al. Metastatic properties and genomic amplification of the tyrosine kinase gene ACK1. Proc Natl Acad Sci U S A,2005,102(44):15901-15906.
[31]Zhang J, Kang B, Tan X, et al. Comparative analysis of the protein profiles from primary gastric tumors and their adjacent regions:MAWBP could be a new protein candidate involved in gastric cancer. J Proteome Res,2007, 6(11):4423-4432.
[32]Westhoff T H, Scheid S,Tolle M, et al. A physiogenomic approach to study the regulation of blood pressure.Physiol Genomics,2005,23(1):46-53.
[33]Kurokawa Y, Matoba R, Nakamori S, et al. PCR-array gene expression profiling of hepatocellular carcinoma. J Exp Clin Cancer Res,2004, 23(1):135-141
[34]Judge T A, Lewis J D, Lichtenstein G R. Colonic dysplasia and cancer in inflammatory bowel disease. Gastrointest Endosc Clin N Am, 2002,12(3):495-523.
[35]Viscido A, Bagnardi V, Sturniolo G C, et al. Survival and causes of death in Italian patients with ulcerative colitis. A GISC nationwide study. Dig Liver Dis, 2001,33(8):686-692.
[36]Rhodes J M, Campbell B J. Inflammation and colorectal cancer:IBD-associated and sporadic cancer compared. Trends Mol Med,2002,8(1):10-16.
[37]Odze R D, Farraye F A, Hecht J L, et al. Long-term follow-up after polypectomy treatment for adenoma-like dysplastic lesions in ulcerative colitis.[J]. Clin Gastroenterol Hepatol,2004,2(7):534-541.
[38]Walsh S V, Loda M, Torres C M, et al. P53 and beta catenin expression in chronic ulcerative colitis--associated polypoid dysplasia and sporadic adenomas: an immunohistochemical study. Is J Surg Pathol,1999,23(8):963-969
[39]Hussain S P, Amstad P, Raja K, et al. Increased p53 mutation load in noncancerous colon tissue from ulcerative colitis:a cancer-prone chronic inflammatory disease. Cancer Res,2000,60(13):3333-3337.
[40]Seo M, Kada M, Yao T, et al. An index of disease activity in patients with ulcerative colitis. Am J Gastroenterol,1992,87(1):971-976
[41]Arnold C, Moradpour D, Blum HE.Tuberculosis colitis mimicking Crohn's disease.Am J Gastroenternl.1998,93:2294-2296
[42]Differing acute phase responses in Crohn's disease and ulcerative colitis.
[43]内镜及病理学检查在克罗恩病和肠结核鉴别诊断中的价值
[44]Al Karawi MA, Mohamed AE, Yasawy MI, et al.Protean manifestation of gastrointestinal tuberculosis:report on 130 patients.J Clin Gastroenterol,1995, 20:225-232
[45]Dubinsky MC, Lin YC, Dutridge D, et al. Serum immune responses predict rapid disease progression among children with Crohn's disease:immune responses predict disease progression. Am J Gastroenterol 2006; 101:360-367
[46]Mow WS, Vasiliauskas EA, and Lin YC, et al. Association of antibody responses to microbial antigens and complications of small bowel Crohn's disease. Gastroenterology 2004; 126:414-424
[47]Papadakis KA, Yang H, Ippoliti A, et al. Anti-flagellin (CBirl) phenotypic and genetic Crohn's disease associations. Inflamm Bowel Dis 2007; 13:524-530
[48]Spivak J, Landers CJ, Vasiliauskas EA, et al. Antibodies to 12 predict clinical response to fecal diversion in Crohn's disease. Inflamm Bowel Dis 2006; 12: 1122-1130
[49]Papp M, Altorjay I, Norman GL, et al. Seroreactivity to microbial components in Crohn's disease is associated with ileal involvement, noninflammatory disease behavior and NOD2/CARD15 genotype, but not with risk for surgery in a Hungarian cohort of IBD patients. Inflamm Bowel Dis 2007; 13:984-992
[2]Gan HT, Chen YQ, Ouyang Q et al. Differentiation between intestinal tuberculosis and Crohn's disease in endoscopic biopsy specimens by polymerase chain reaction. Am J Gastroenterol 2002; 97:1446-51
[3]Koch P, del Valle F, Berdel WE, et al. Primary gastrointestinal non-Hodgkin's lymphoma:I. Anatomic and histologic distribution, clinicalfeatures, and survival data of 371 patients registered in the Germanmulticenter study GIT. J Clin Oncol,2001,19:3861-3873
[4]Amer MH, el-Akkad S. Gastrointestinal lymphoma in adults:clinical features and management of 300 cases[J]. Gastroenterology,1994,106:846-858.
[5]Otter R, Gerrits WB, vd Sandt MM, et al. Primary extranodal andnodal non-Hodgkin's lymphoma. A survey of a population-based registry. EurJ Cancer Clin Oncol,1989,25:1203-1210
[6]毛永平,杨云生,王淑芳,等.原发性小肠淋巴瘤临床病理分析23例.世界华人消化杂志.2005；13：2711-2713
[7]irsch R, Pentecost M, Hall PM et al. Role of colonoscopic biopsy in distinguishing between Crohn's disease and intestinal tuberculosis.J Clin Pathol 2006; 59:840-4
[8]Witte J, Shivananda S, Lennard-Jones JE, et al. Disease outcome in inflammatory bowel disease:mortality, morbidity and therapeutic management of a 796-person inception cohort in the European Collaborative Study on Inflammatory Bowel Disease (EC-IBD).Scand J Gastroenterol,2000,35 (12):1272-1277.
[9]中国炎症性肠病协作组.3100例溃疡性结肠炎住院病例回顾分析.中华消化杂志；2006；26：368-372.
[10]Liu TH. Crohn's disease-differential diagnosis with intestinal tuberculosis. Zhonghua Nei Ke Za Zhi 1981; 20:211-5.
[11]Arnold C,Moradpour D,Blum HE.Tuberculosis colitis mimicking Crohn's disease.Am J Gastroenterol.1998,93;2294-2296
[12]Al Jahdali H, Memish ZA, Menzies D. The utility and interpretation of tuberculin skin tests in the Middle East. Am J Infect Control.2005; 33:151-6.
[13]Rangaka MX, Wilkinson KA, Seldon R et al. Effect of HIV-1 infection on T-Cell-based and skin test detection of tuberculosis infection. Am J Respir Crit Care Med 2007; 175:514-20.
[14]李佩文,贾立群,朱世杰.恶性肿瘤误诊误治与防范.北京：科学技术文献出版社；2001：280-283.
[15]Pulimood AB, Peter S, Rook GW et al. In situ PCR for Mycobacterium tuberculosis in endoscopic mucosal biopsy specimens of intestinal tuberculosis and Crohn disease. Am J Clin Pathol 2008; 129:846-51
[16]Majid Abdulrahman Almadi, Subrata Ghosh, Abdulrahman Mohamed Aljebreen et al. Differentiating Intestinal Tuberculosis from Crohn's Disease:A Diagnostic Challenge. Am J Gastroenterol 2009; 104:1003-1012
[17]Expression of the High-Affi nity IgG Receptor FcRI (CD64) in Patients With Infl ammatory Bowel Disease:A New Biomarker for Gastroenterologic Diagnostics
[18]Solem C A, Jr Loftus E V, Tremaine W J, et al. Correlation of C-reactive protein with clinical, endoscopic, histologic, and radiographic activity in inflammatory bowel disease.[J]. Inflamm Bowel Dis,2005,11(8):707-712.
[19]Tibble J A, Sigthorsson G, Foster R, et al. Use of surrogate markers of inflammation and Rome criteria to distinguish organic from nonorganic intestinal disease.[J]. Gastroenterology,2002,123(2):450-460.
[20]Ghoshal UC, Ghoshal U, Singh H et al. Anti- Saccharomyces cerevisiae antibody is not useful to differentiate between Crohn's disease and intestinal tuberculosis in India. J Postgrad Med 2007; 53:166-70.
[21]Makharia GK, Sachdev V, Gupta R et al. Anti- Saccharomyces cerevisiae antibody does not differentiate between Crohn's disease and intestinal tuberculosis. Dig Dis Sci 2007; 52:33-9.
[22]Vermeire S, Joossens S, Peeters M, et al. Comparative study of ASCA (Anti-Saccharomyces cerevisiae antibody) assays in inflammatory bowel disease.[J]. Gastroenterology,2001,120(4):827-833.
[23]Garrard S M, Capaldo C T, Gao L, et al. Structure of Cdc42 in a complex with the GTPase-binding domain of the cell polarity protein, Par6.[J]. EMBO J, 2003,22(5):1125-1133.
[24]Linseman D A, Heidenreich K A, Fisher S K. Stimulation of M3 muscarinic receptors induces phosphorylation of the Cdc42 effector activated Cdc42Hs-associated kinase-1 via a Fyn tyrosine kinase signaling pathway.[J]. J Biol Chem,2001,276(8):5622-5628.
[25]Yang W, Cerione R A. Cloning and characterization of a novel Cdc42-associated tyrosine kinase, ACK-2, from bovine brain.[J]. J Biol Chem, 1997,272(40):24819-24824.
[26]Mackeigan J P, Murphy L O, Blenis J. Sensitized RNAi screen of human kinases and phosphatases identifies new regulators of apoptosis and chemoresistance.[J]. Nat Cell Biol,2005,7(6):591-600.
[27]Guo W, Giancotti F G. Integrin signalling during tumour progression.[J]. Nat Rev Mol Cell Biol,2004,5(10):816-826.
[28]Mercurio A M, Bachelder R E, Chung J, et al. Integrin laminin receptors and breast carcinoma progression. J Mammary Gland Biol Neoplasia,2001, 6(3):299-309.
[29]Manser E, Leung T, Salihuddin H, et al. A non-receptor tyrosine kinase that inhibits the GTPase activity of p21cdc42. Nature,1993,363(6427):364-367.
[30]van der Horst E H, Degenhardt Y Y, Strelow A, et al. Metastatic properties and genomic amplification of the tyrosine kinase gene ACK1. Proc Natl Acad Sci U S A,2005,102(44):15901-15906.
[31]Zhang J, Kang B, Tan X, et al. Comparative analysis of the protein profiles from primary gastric tumors and their adjacent regions:MAWBP could be a new protein candidate involved in gastric cancer. J Proteome Res,2007, 6(11):4423-4432.
[32]Westhoff T H, Scheid S,Tolle M, et al. A physiogenomic approach to study the regulation of blood pressure.Physiol Genomics,2005,23(1):46-53.
[33]Kurokawa Y, Matoba R, Nakamori S, et al. PCR-array gene expression profiling of hepatocellular carcinoma. J Exp Clin Cancer Res,2004, 23(1):135-141
[34]Judge T A, Lewis J D, Lichtenstein G R. Colonic dysplasia and cancer in inflammatory bowel disease. Gastrointest Endosc Clin N Am, 2002,12(3):495-523.
[35]Viscido A, Bagnardi V, Sturniolo G C, et al. Survival and causes of death in Italian patients with ulcerative colitis. A GISC nationwide study. Dig Liver Dis, 2001,33(8):686-692.
[36]Rhodes J M, Campbell B J. Inflammation and colorectal cancer:IBD-associated and sporadic cancer compared. Trends Mol Med,2002,8(1):10-16.
[37]Odze R D, Farraye F A, Hecht J L, et al. Long-term follow-up after polypectomy treatment for adenoma-like dysplastic lesions in ulcerative colitis.[J]. Clin Gastroenterol Hepatol,2004,2(7):534-541.
[38]Walsh S V, Loda M, Torres C M, et al. P53 and beta catenin expression in chronic ulcerative colitis--associated polypoid dysplasia and sporadic adenomas: an immunohistochemical study. Is J Surg Pathol,1999,23(8):963-969
[39]Hussain S P, Amstad P, Raja K, et al. Increased p53 mutation load in noncancerous colon tissue from ulcerative colitis:a cancer-prone chronic inflammatory disease. Cancer Res,2000,60(13):3333-3337.
[40]Seo M, Kada M, Yao T, et al. An index of disease activity in patients with ulcerative colitis. Am J Gastroenterol,1992,87(1):971-976
[41]Arnold C, Moradpour D, Blum HE.Tuberculosis colitis mimicking Crohn's disease.Am J Gastroenternl.1998,93:2294-2296
[42]Differing acute phase responses in Crohn's disease and ulcerative colitis.
[43]内镜及病理学检查在克罗恩病和肠结核鉴别诊断中的价值
[44]Al Karawi MA, Mohamed AE, Yasawy MI, et al.Protean manifestation of gastrointestinal tuberculosis:report on 130 patients.J Clin Gastroenterol,1995, 20:225-232
[45]Dubinsky MC, Lin YC, Dutridge D, et al. Serum immune responses predict rapid disease progression among children with Crohn's disease:immune responses predict disease progression. Am J Gastroenterol 2006; 101:360-367
[46]Mow WS, Vasiliauskas EA, and Lin YC, et al. Association of antibody responses to microbial antigens and complications of small bowel Crohn's disease. Gastroenterology 2004; 126:414-424
[47]Papadakis KA, Yang H, Ippoliti A, et al. Anti-flagellin (CBirl) phenotypic and genetic Crohn's disease associations. Inflamm Bowel Dis 2007; 13:524-530
[48]Spivak J, Landers CJ, Vasiliauskas EA, et al. Antibodies to 12 predict clinical response to fecal diversion in Crohn's disease. Inflamm Bowel Dis 2006; 12: 1122-1130
[49]Papp M, Altorjay I, Norman GL, et al. Seroreactivity to microbial components in Crohn's disease is associated with ileal involvement, noninflammatory disease behavior and NOD2/CARD15 genotype, but not with risk for surgery in a Hungarian cohort of IBD patients. Inflamm Bowel Dis 2007; 13:984-992