【作者】 沈凯；

【导师】 王杉；

【作者基本信息】 北京大学 ， 外科学， 2008， 博士

【摘要】 背景:结直肠癌是消化道最常见的恶性肿瘤之一,遗传性非息肉病性结直肠癌(Hereditary Nonpolyposis Colorectal Cancer, HNPCC)在遗传性结直肠癌中占很大比例,约占结直肠癌总数的5%-10%。HNPCC为常染色体显性遗传疾病,多具有错配修复(mismatch repair, MMR)基因的突变,而家族内具有此突变基因的成员一生中患结直肠癌或其它肠道外相关肿瘤的风险约为80%。1999年由HNPCC国际协作组(ICG-HNPCC)提出的《Amsterdam II criteria》是传统的诊断标准。但是由于我国计划生育的政策,可能由于家系内成员数量不足而无法满足上述标准。美国国家综合癌症网(National Comprehensive Cancer Network,NCCN)提出了新的筛选流程来筛选HNPCC患者,首先应用筛选标准《Revised Bethesda Guidelines》进行临床病例的筛选,然后通过免疫组织化学和基因测序的方法来诊断HNPCC患者。近期发现,错配修复基因MSH2不仅是错配修复过程的重要成员,还参与细胞的增殖、细胞周期调控和细胞凋亡。目的:研究NCCN推荐流程(V.1.2006)筛选国人HNPCC的临床应用价值及分子生物学基础;分析错配修复蛋白(MSH2和MLH1)在结直肠癌组织中的表达及其与患者临床病理特征和预后的关系;探讨HNPCC的分子生物学基础。材料与方法:一、材料:北京大学人民医院胃肠外科2002年1月至2006年2月经手术治疗的结直肠癌患者419例。二、方法:1.应用NCCN推荐流程(V.1.2006)筛查国人HNPCC患者:(1)应用《Revised Bethesda Guidelines》进行筛选。(2)免疫组织化学法(EnVision二步法)检测结直肠癌组织石蜡切片中错配修复蛋白MLH1和MSH2表达情况(错配修复蛋白缺失者为HNPCC患者)。(3)错配修复蛋白表达缺失者,应用RT-PCR及突变检测MLH1或MSH2突变(4)错配修复蛋白表达正常者,应用《Amsterdam II criteria》进行补充诊断(符合《Amsterdam II criteria》患者为HNPCC)。2.应用《Amsterdam II criteria》筛查国人HNPCC患者3.分析错配修复蛋白表达与结直肠癌患者临床病理指标和预后之间的关系。4.敲减人结肠癌细胞系SW480的错配修复基因MSH2的表达,建立模拟HNPCC细胞模型。5.四唑盐(MTT)比色法,流式细胞术和Transwell法研究小干扰RNA(SiRNA)敲减MSH2基因对结肠癌细胞系SW480增殖,细胞周期与细胞侵袭力的影响。结果:1. NCCN推荐流程(V.1.2006)筛查HNPCC患者21名(1)419例结肠癌患者中有90例患者符合《Revised Bethesda Guidelines》。(2)免疫组织化学法检测到18例患者存在MLH1或MSH2蛋白表达缺失。(3)错配修复蛋白缺失者中,检测到基因突变3例。(4)错配修复蛋白正常者中,《Amsterdam II criteria》补充诊断HNPCC患者3例。(5)NCCN推荐流程(V.1.2006)筛查HNPCC患者21例(错配修复蛋白缺失患者18名+《Amsterdam II criteria》补充诊断3名)2.单纯应用《Amsterdam II criteria》筛查HNPCC患者8名,较NCCN推荐流程少诊断HNPCC患者13名。3.错配修复蛋白表达与肿瘤位置、淋巴结转移情况和肿瘤大小有关,可能是患者预后影响因素之一。4.应用SiRNA敲减MSH2基因,建立MSH2低表达的SW480结肠癌细胞,可能成为模拟HNPCC的细胞模型。5.SiRNA敲减MSH2基因的SW480结肠癌细胞增殖减慢,细胞周期阻滞于G1期,细胞侵袭能力下降。结论:(1)NCCN推荐流程(V.1.2006)可以有效筛查国人HNPCC患者,减少漏诊率,具有良好的临床应用价值。(2)敲减错配修复基因MSH2在结肠癌细胞中的表达可降低结肠癌细胞的增殖和侵袭。更多还原

【Abstract】 Background: Colorectal cancer (CRC) is one of the most common malignant tumors occurring in the gut with an increasing mobidity in China these years. Hereditary nonpolyposis colorectal carcinoma (HNPCC, Lynch syndrome) is the most common hereditary form of colorectal carcinoma and may account for 5-10% of the total CRC burden. HNPCC is characterized genetically by pathogenic germline mutations in one of the DNA mismatch repair (MMR) genes, usually MLH1, MSH2.Clinically, affected individuals display a predisposition for the development of early-onset colorectal cancers and various second primary tumours. In response to the criticism that Amsterdam II criteria (AC-II) were too stringent, NCCN(V.1.2006) recommended the protocol to screen the HNPCC patients. It was recently known that MSH2 played important role not only in mismatch repair but also in cell’s proliferation, apoptosis and cell cycle control.Objective: To investigate the effects of NCCN V.1.2006 recommended protocol: recognition of clinical syndrome characterized with revised Bethesda guidelines, genetic counseling with immunohistochemistry and finally genetic testing to diagnose HNPCC. Evaluate the clinical feartures’diferences between the MMR deficient group and MMR proficient group. Study the role of MMR played in the cell’s proliferation, cell cycle control and cell invasiveness.Methods: Newly diagnosed 419 patients with colorectal cancer from January 1, 2002 to Febrary 28, 2006. Nighty patients fulfilled the revised Bethesda guidelines. Immunohistochemistry was used to detect the expression of MLH1 and MSH2 in the ninety patients.The frozen tissues were collected from the patients who showed loss of MLH1 or MSH2 protein expression, RNA was extracted, and RT-PCR and cDNA sequencing were used to detect the germline mutations of MLH1 and MSH2. Cell proliferation, cell cycle and invasiveness were quantified by MTT, flow cytometry andTranswell respectively.Results:1. Twenty one patients were diagnosed as HNPCC by NCCN recommended protocol.(1) Nighty patients fulfilled the revised Bethesda guidelines.(2) Eighteen patients’tumors exhibiting loss expression of MLH1 or MSH2 protein.(3) Three cases of MLH1 or MSH2 mutation were detected in the patients whose tumors exhibiting loss expression of MLH1 or MSH2 protein. The 3 cases whose cDNA were detected mutations were not fulfilled the Amsterdam II criteria, and finally were diagnosed as HNPCC.(4)Three cases who exhibiting normal expression of MLH1 and MSH2 proteins were diagnosed as HNPCC by the Amsterdam II criteria.(5) Finally 21 patients (18 patients who showed loss expression of MMR protein and 3 patients who fulfilled the Amsterdam II criteria ) were diagnosed as HNPCC.2. Eight patients were diagnosed as HNPCC by Amsterdam II criteria.3. The expression of MSH2 and MLH1 were associated with the tumors’location, size and status of lymph node metastasis. It may be a factor to predict the patients’prognosis.4. A simulated HNPCC cell model was established by knocking down the MSH2 expression in colorectal cancer cell line-SW480 using small interference RNA (SiRNA).5. Proliferation were inhibited, cell cycle was arrested at G0/G1 phase and cell invasiveness was reduced after the colon cancer cell line-SW480 has been knocked down the MSH2 expression by SiRNA.Conclusion:(1) The NCCN(V.1.2006) recommended protocol constitutes a useful approach to identify Chinese patients at risk for HNPCC.(2) Mismatch repair gene-MSH2 play important roles in the cell’s proliferation , cell cycle control and invasiveness.更多还原