【作者】 杨夏彤；

【导师】 张亚历；

【作者基本信息】 南方医科大学 ， 内科学， 2011， 硕士

【摘要】 研究背景和目的：大肠癌(colorectal cancer, CRC)是最常见的恶性肿瘤之一,目前对其癌变途径和分子机制的认识尚不十分清楚。传统腺瘤(traditional adenomas, TA)-腺癌途径是公认的CRC发生最重要的途径,2/3以上的CRC由此进展而来。近年来提出的“锯齿状癌变途径”,即增生性息肉(hyperplastic polyp, HP)-锯齿状腺瘤(serrated adenomas, SA)-腺癌途径,是另外一条受到广泛重视的癌变通路,可以解释7.5%-17.5%近侧CRC的发生。锯齿状癌变途径的提出,推翻了过去通常认为的HP不发生癌变的观点,为CRC的防治研究提出了新的课题,也为CRC发病机制的研究提供了新的线索。锯齿状息肉主要包括：部分增生性息肉(hyperplastic polyp, HP)、无蒂锯齿状腺瘤(sessile serrated adenomas, SSA)、传统锯齿状腺瘤(traditional serrated adenomas, TSA)。其中,近年提出的SSA代表了形态学居于HP和TSA之间的一种中间类型,并被认为是微卫星不稳定CRC的前期病变,因而受到广泛重视。目前国外关于SSA的诊断和鉴别诊断目前仍是难点,缺乏操作性强的统一标准。尤其值得注意的是,以往诊断为HP的病例中,有相当一部分是SSA,两者的鉴别诊断较为困难。虽然SSA没有明显的细胞学异型性,但由于其恶变潜能,目前推荐将其作为腺瘤处理和随访。然而,目前国内对SA的认识不足,更缺乏完整的研究。因此,初步探索国内SA的诊断标准和鉴别诊断要点,显得尤为必要。虽然锯齿状腺瘤的概念已被广泛接受,并且关于SSA的研究也有很多,但其确切的分子机制尚不清楚。SA的癌变机制是否确实不同于TA,尚有待进一步确定。Wnt信号通路激活是大部分CRC发生过程中的重要早期事件,但该通路在SA中的作用尚不清楚。近来有文献报道了Wnt信号通路在SA中的激活情况,但结果互相矛盾,一些研究发现SA中APC突变和β-catenin核转位常见,而其它报道则认为Wnt通路在SA中是失活的。因些,有必要进一步试验明确Wnt通路在锯齿状息肉中的作用。结直肠肿瘤中Wnt信号通路的激活几乎无例外地为两个关键基因的互斥性突变所致,80%为APC基因突变,10%为β-catenin突变。但是,APC基因突变在SA中罕见,文献报道仅为3.8%(1/26)。这提示：基因突变可能不是SA中Wnt通路激活的主要原因。CpG岛甲基化表型是锯齿状癌变途径的重要特征,以基因启动子区的CpG岛广泛甲基化为特征,在其发病机制中起重要作用。很多学者认为,DNA甲基化可能替代基因突变,成为沉默APC的替代机制。但目前尚未见SA中APC甲基化状态的研究报道。细胞增殖和分化的严格调控需要细胞粘附和基因转录之间的协同作用。Wnt信号通路的关键分子β-catenin,是这个过程中的重要整合分子,在细胞粘附及基因转录中都发挥着重要作用[10]。β-catenin在粘附与转录功能之间的适当选择对正常发育至关重要,其调节失常导致细胞间粘附功能丢失,Wnt信号通路目的基因转录增强,参与形成恶性肿瘤。近来研究发现,β-catenin在SA中的浆表达率低于传统腺瘤,未见核转位,这提示我们：在SA中,β-catenin没有进入核内形成转录复合体；β-catenin\α-catenin\E-cadherin粘附复合体可能是锯齿状腺瘤中β-catenin发挥作用的主要形式,主要参与细胞粘附作用。但这种推测尚需要进一步实验证实。本课题的研究目的和意义在于：(1)初步探索我国锯齿状息肉的诊断标准和鉴别诊断要点,指导临床决策,避免漏诊和误诊；(2)阐明Wnt信号通路在锯齿状癌变途径中的作用,深入认识其分子基础；(3)揭示锯齿状癌变途径中Wnt通路激活的主要机制,为锯齿状息肉的防治提供新的干预靶点。(4)揭示β-catenin的粘附与转录调节功能在锯齿状息肉发病机制中的作用,为临床防治提供理论依据。研究材料和方法：1、标本收集：根据Snover等人2005年提出的诊断标准,分析我科病理室2007年2月-2009年7月之间存档的97例大肠锯齿状息肉石蜡包埋标本(其中HP 32例、SSA 28例、TSA 37例),并收集我科内镜室2008年12月-2009年7月之间行息肉切除术和内镜检查的51例新鲜TA标本、10例正常的大肠组织。其中28例SSA标本中含3例混合型SSA-HP,37例TSA标本中含5例混合型TSA-SSA。新鲜标本分两部分：一部分立即置入组织保存液(大连宝生物工程有限公司),-80度低温冰箱保存；另一部分10%福尔马林固定,石蜡包埋。石蜡标本室温保存。临床病理数据从南方医院的病例中获得,均得到了病人的知情同意。2、HE染色和免疫组织化学技术：锯齿状息肉标本行HE染色,一部分新鲜标本固定、脱水、包埋、制片成石蜡标本后,所有石蜡标本切片、免疫组化染色、光镜下观察。免疫组化采用二步法染色,抗原修复采用微波热修复,抗原修复液用枸橼酸(PH=6.0)、EDTA (PH=8.0),用不加一抗的组织切片作阴性对照,已知抗原表达的组织切片作为阳性对照,检测P-catenin、α-catenin、E-cadherin及APC在HP、SSA、TSA各组病例中以及TA、NC中的表达情况。3、DNA提取以及PCR扩增后-直接测序：按照TIANGEN公司的新鲜组织提取DNA试剂盒说明书,从10例NC、51例TA新鲜组织中提取DNA。按照OMEGA公司的石蜡标本专用试剂盒说明书,提取32例HP、28例SSA、37例TSA石蜡组织标本中的DNA,提取成功后均置于-20度冰箱保存。根据APC第15外显子和β-catenin第3外显子突变多发区用Primer 5.0设计引物(表2-1)。PCR直接扩增DNA片段,扩增产物以2.5%琼脂糖凝胶电泳观察以确保目的片段扩增成功,然后采用DNA产物纯化试剂盒(TIANGEN公司提供)将目的片段纯化回收,后送公司测序。将碱基序列与正常序列比对,以发现异常的碱基序列,如发现有序列变化者,重新扩增该片段并从正反两方向进行测序,统计各组样本的突变情况,最后进行统计学分析。表2-1.APC第15外显子和β-catenin第3外显子的引物序列Table 2-1 The primer sequences of APC exon 15 andβ-catenin exon 3统计分析：采用SPSS 13.0统计软件进行分析,计量资料组间比较经方差齐性检验,多样本均数比较方差齐性者采用one-way ANOVA,多重比较用Bonferroni test,分类变量组间比较和率的比较用Chi-square检验、R×C表资料的x2检验或Fisher’s精确检验,统计结果以P<0.05表示差异有统计学意义。结果：1、结直肠锯齿状息肉的临床病理特征根据我们的诊断实践,结合国外文献,初步拟定了各型锯齿状息肉的鉴别诊断要点。SSA区别于HP的要点：1、隐窝中上部有丝分裂活性增加；2,隐窝浅表部位的细胞核以圆形、椭圆形为主；3,锯齿状结构起始于隐窝基底部；4,其它结构特征：水平隐窝、分支隐窝、倒置隐窝；5,50%以上的隐窝发育不成熟；6,细胞成熟异常的其它特征：隐窝基底部可见杯状细胞,浅表部位的杯状细胞扩张、充满粘液。TSA区别于SSA的要点：1、核深染；2,核拉伸；3,核分层；4,核仁突出；5,胞浆强嗜酸性。2、β-catenin为核心的粘附复合物及APC在HP、SSA、TSA、TA、NC中的表达情况。在10例NC中,全部都有β-catenin、α-catenin、E-cadherin、APC的正常膜表达。然而,仅有33.3%(17/51)的TA中有E-cadherin的正常表达,仅有27.5%(14/51)的TA有α-catenin的正常表达,有31.4%(16/51)的TA中有APC的正常表达。仅有29.4%(15/51)的TA中有β-catenin的膜表达,有51.0%(26/51)的TA中有β-catenin的浆表达,有19.6%(10/51)的TA中有β-catenin的核表达。E-cadherin的正常膜表达在HP、SSA、TSA中的比例明显高于TA中的E-cdherin的正常膜表达率(33.3%,17/51, P=0.000,χ2=5.029)。α-catenin在HP、SSA、TSA中的正常膜表达率明显高于TA中a-catenin的正常膜表达率(27.5%,14/51, P=0.000,χ2=56.568). APC在HP、SSA、TSA中的正常膜表达率也明显高于TA中的APC的正常膜表达率(31.4%,16/51,P=0.000,x2=69.240)。P-catenin的正常膜表达出现在HP、SSA、TSA中的频率明显高于TA中P-catenin的正常膜表达率(29.4%,15/51,P<0.001)。另外,P-catenin在HP、SSA、TSA中的浆表达率明显低于P-catenin在TA中的浆表达率(51%,26/51, P=0.000,χ2=52.004)。β-catenin广泛或局灶性的核表达出现在19.6%(10/51)的TA中,但是在HP、SSA、TSA中均没有β-catenin的核表达(P=0.000,x2=22.398)。3、APC、β-catenin的突变分析APC第15外显子和β-catenin第3外显子突变测序结果发现：仅有1例TSA(2.7%,1/37)中出现APC的突变。APC突变在HP、SSA、TSA中出现的频率明显低于在TA中的频率(51.0%,26/51,P=-0.000,x2=58.216)。在所有的HP、SSA、TSA、TA、NC中均没有检测出β-catenin的突变。结论：1、Wnt信号通路在锯齿状癌变途径中并不起主要作用,锯齿状癌变途径不同于传统腺瘤-癌途径。2、β-catenin在锯齿状癌变途径中主要起细胞粘附作用,而不是参与Wnt信号通路的激活。3、基因突变不是锯齿状癌变途径中Wnt信号通路适度激活的主要原因,可能存在另一种替代机制。更多还原

【Abstract】 Background and aimsColorectal cancer (CRC) is one of the most commonly occurring malignancies. A well-defined pathogenic pathway, the adenoma-adenocarcinoma sequence, is the predominant pathogenic pathway in colorectal tumorigenesis,and accounts for upwards of two thirds of all CRCs. Recently, an alternative "serrated pathway"of colorectal carcinogenesis with a hyperplastic polyp (HP)-serrated adenoma (SA)-adenocarcinoma sequence has been proposed, which may explain at least 7.5% of all and up to 17.5% of proximal CRCs. The propose of the serrated pathway of colorectal carcinogenesis have overthrew the view that HPs are non-neoplastic lesions, and raised new issues for the prevention and treatment of CRCs, also provide new clues for the pathogenesis of CRCs.The serrated polyps including:HP(hyperplastic polyp)、SSA(Sessile serrated adenoma)、TSA(Traditional serrated adenoma). The concept of SSA was recently introduced, representing a morphological intermediate between HP and TSA. More and more evidences indicate that SSA might be the precursor lesion for some cases of microsatellite unstable colorectal carcinoma. The diagnosis and differential diagnosis of SSAs are still unclear, lacking uniform standards. It is worth noting that a considerable part of which diagnosed SSAs before may be HPs. It is still difficult to distinguish HPs from SSAs.SSAs are now treated as adenomas for their malignant potential. However, a thorough study and a clear understanding on SAs is still lacking in China.So a preliminary study on the diagnosis and differential diagnosis of SAs seems very important.Although the concept of SA has been generally accepted, there have been several reports on SAs, the result is still conflicting, Whether the canceration of SAs the same as TAs is still unclear. It has been widely accepted that activation of the Wnt signalling pathway is a key early event involved in the multi-step process of colorectal tumorigenesis, but the role in SAs is not clear. Recently, some studies have reported the action of Wnt signalling in SAs,but the result is opposite.Some found that APC mutation andβ-catenin nuclear expression is frequent in SAs,while others reported that Wnt signalling is inactivated in SAs So further study is needed to make clear on the role of Wnt signalling in SAs.Aberrant activation of the Wnt signalling pathway in CRC occurs almost invariably through mutation of the two key regulators:adenomatous polyposis coli (APC) andβ-catenin. Up to 80% of CRCs have APC mutations, whereas 10% of CRCs haveβ-catenin mutations. However, it is reported that infrequent APC mutation has been reported in SAs.,only 3.8%(1/26). It implys that gene mutation may not play a key role on Wnt signalling in SAs. CpG island methylator phenotype, which characterized by a wide range of promoter methylation,plays an important role in the "serrated pathway"of colorectal carcinogenesis. Many reaserchers thought DNA methylation may provide an alternative mechanism to gene mutation for silencing APC, and could bridge the mutational gap. We can conlude from it that high frequency of DNA methylation may be the main reason of the reduction of APC expression.There still have no reports on the APC methylation status in SAs. Make sure that the two processes of cell adhesion and nuclear signaling is tightly coordinated and interrelated important for cell proliferation and differentiation. The key regulator of Wnt signalling,β-catenin, plays both a critical structural role in cadherin-based adhesions and also an essential activator of gene transcription. The control of the dual functions ofβ-catenin in cellular adhesion and in transcriptional regulation is crucial for maintaining normal cellular function,either function of P-catenin de-regulated will result in activation of Wnt sigalling,then will lead to human malignancies. Recently, some reasearchers found that the cytoplasm expression ofβ-catenin is infrequent in SAs than in TAs, and no nuclear expression was found in SAs. It implies us thatβ-catenin assembles with a-catenin and E-cadherin into adhension complexes without acting in nuclear transcription in SAs. Whetherβ-catenin that favors adhesion function rather than the function in transcription in SAs still needs further study.The object and significance of this article is that:first, preliminary study on the diagnosis and differential diagnosis of SAs, to guide clinical decision-making and avoid misdiagnosis; second, to make sure whether Wnt signalling acts in SAs, in order to understand the molecular basis deeply; third, to reveal the molecular mechanism of Wnt signaling activation in serrated pathway; forth, to reveal the adhension and transcription function ofβ-catenin in serrated pathogenesis, which will provide theoretical basis for clinical prevention and treatment.Materials and Methods:1. Sample collection: according to the diagnostic criteria of Snover etal in 2005, retrospectively analysis 97 paraffin serrated polyps specimen(includig 32 HP samples, 28 SSA samples,37 TSA samples) archived in our pathology department, and collect 51 fresh TA samples and 10 fresh normal colorectal tissue from patients who did 111 endoscopy polypectomy and polypectomy in our endoscopy department from February 2007 to July 2009. Among the 28 SSA sections,3 samples are mixed SSA-HP, among the 37 TSA sections,5 samples are mixed TSA-SSA. Each specimen was divided into two parts:one was frozen immediately in liquid nitrogen and stored at -80℃until required, the other was fixed in 10% formalin and embedded in paraffin. Paraffin specimen are maintained at room temperature. Clinical pathological datas were got from Nanfang Hospital cases, and informed consent was received from all patients.2、HE staining and immunohistochemistry:Each fresh sample was divided into two parts, one part was fixed in 10% formalin and embedded in paraffin, then all formalin specimen sliced、HE staining、immunohistochemistry and observed under microscope. Immunohistochemistry using two-step staining, heated-repairing, citrate antigen retrieval solution, specimens without adding antigen were used as an negative control, and specimens which known antigen were used as a positive control, thus allowing direct comparison within the same tissue specimen, then detected the expression of APC、β-catenin、α-catenin、E-cadherin in HPs、SSAs、TSAs and control groups of TAs、NCs.3. DNA extraction and PCR:According to the fresh tissue genomic DNA Kit manual provided by TIANGEN company, we extracted DNA from 10 NCs and 51 TAs. According to EZNA FFPE DNA Kit of OMEGA company, we extracted DNA from 32 HPs、28 SSAs、37 TSAs paraffin specimen. All DNA samples were preserved in -20℃. According to exon 15 of APC MCR region and exon 3 ofβ-catenin mutation region design primers using Primer 5.0 software. PCR amplification DNA section, amplified products by 2.5% agarosegelelectrophoresis to ensure that the ob servation fragment was successfullyamplifed, after succeeded amplified, purify the PCR production(using TIANGEN quick Midi Purification Kit),then send to company for sequencing. Assessment genotype with chromas 2.0 and lastcontrasted the gene bank data to find abnormal sequence, if we find mutation, then re-amplify the fragment and sequencing from both directions, then statistical analysis each mutation sample. Table 2-1 The primers sequence of APC exon 15 (MCR region) and (3-catenin exon 3 Statistical analysis:Data are presented as mean and standard deviation for continuous variables and as proportions for categorical variables. Data were analysed using one-way ANOVA, followed by Bonferroni test for multiple comparisons. Differences in categorical variables were determined by the Chi-square or Fisher’s exact tests, as appropriate. Differences were considered significant if P<0.05. All significance tests were two-tailed. All statistical tests were performed using SPSS software Version 13.0 (SPSS Inc, Chicago, IL, USA).Results:1.Clinical and pathological features:According to our practial diagnosis and combines with foreign literature, initially identified the differential diagnosis of all kinds of serrated polyps. SSAs differ from HPs in:1.mitotic activity increases in the upper and middle part of crypts; 2. most nucleus of the superficial parts of crypts is round or oval; 3.the serrated structure starts from the base of the crypts; 4.other features:have flat crypts, branch crypts, inverted crypts; 5.50% of the crypts are immature; 6. other feature of abnormal mature cell:goblet cells can be seen at the base of crypts, goblet cells which on superficial parts of the crypts are outstretched and full of mucus. TSAs differ from SSAs in:1. stained nuclear; 2. stretched nuclear; 3. layered nuclear; 4. prominent nucleolus; 5. strong eosinophilic cytoplasm.2. APC,β-catenin, a-catenin and E-cadherin expression in HPs,SSAs,TSAs,TAs and NCs.Expression of E-cadherin,α-,β-catenin and APC was clearly evident at the cell-cell boundaries of all 10 normal colonic tissues and normal mucosa adjacent to tumors. However, membranous expression of E-cadherin,a-catenin and APC was only seen in 33.3%(17/51),27.5%(14/51) and 31.4%(16/51) of all investigated TAs, respectively.Membranous expression of beta-catenin was demonstrated in 29.4%(15/51) of TAs. Cytoplasmic accumulation and widespread or focal nuclear expression of beta-catenin was observed in 60.8%(31/51) and 19.6%(10/51) of all TAs, respectively. Mesenchymal tissue surrounding the epithelial cells did not express E-cadherin or any of the catenins.The frequence of E-cadherin membranous expression in HPs,SSAs and TSAs was significantly higher than that of TAs (33.3%,17/51, P=0.000, x2=5.029).The frequence of a-catenin membranous expression in HPs, SSAs, and TSAs was significantly higher than that of TAs(27.5%,14/51,P=0.000,χ2=56.568).The rate of APC membranous expression in HPs,SSAs and TSAs was significantly higher than that of TAs,too (31.4%,16/51, P=0.000,χ2=69.240).The frequence ofβ-cadherin membranous expression in HPs, SSAs and TSAs was significantly higher than that of TAs(29.4%, P<0.001). The rate of cytoplasmic accumulation of P-catenin in HPs, SSAs, and TSAs was significantly lower than that in TAs (60.8%, P<0.001). Widespread or focal nuclear staining for beta-catenin was observed in 19.6%(10/51) of TAs, while none of the HPs, SSAs, or TSAs showed nuclear labeling for beta-catenin (P<0.001). 3. APC andβ-catenin mutationThe result of APC andβ-catenin mutation is that:APC mutation was detected in only one TSA. The frequence of APC mutation in HPs, SSAs, and TSAs was significantly lower than that in TAs(51.0%,26/51,P=0.000,χ2=51.003). None ofβ-catenin mutation was observed in HPs, SSAs, TSAs and TAs.Conclusion:1. Wnt signaling pathway plays a less active role in the development of colorectal serrated polys, the tumorigenesis of the SAs is distinct from that of TAs.2. P-catenin participates in adhesion function in serrated pathway rather than the function in Wnt signaling activation.3. Gene mutation can not be the reason that the moderate activation of Wnt signaling in serrated pathway, maybe there is another alternative mechanism.更多还原