大肠癌与抑癌基因相关性的研究现状

大肠癌是常见的高危害消化系恶性肿瘤,全球每年新发病例约为120万例.近年来,随着人们生活水平的提高,饮食习惯和结构的改变,我国大肠癌的发病率和死亡率增长迅速,而且,发病年龄明显提前,目前,我国大肠癌中位发病年龄为58岁,比欧美等国家提前12-18年.大肠癌的发生是一个多阶段多步骤的、涉及多个基因改变的复杂过程.许多研究表明,结直肠癌变是一个涉及原癌基因激活、抑癌基因失活等多基因、多阶段、多步骤渐进演化的积累过程.与结直肠癌相关的抑癌基因有p53、APC、DCC、MMR等,原癌基因有k-ras、c-myc等.本文就以上基因改变与大肠癌的发生发展相关性的研究现状作一简单复习.     

饮食因素与大肠癌关系的研究进展

本文综述了近几年来饮食因素对大肠癌发病的影响,提示肉类、高脂肪饮食和饮酒会增加大肠癌发病的几率;而全谷食物、膳食纤维、叶酸、硒和钙减少大肠癌的危险性.可见不合理的饮食对大肠癌的发生影响很大,这从饮食角度为防治肿瘤提供了依据.     

577例大肠癌相关危险因素的研究

目的 探讨惠州地区大肠癌发病的相关危险因素.方法 对惠州地区577例大肠癌患者进行大肠癌危险因素单因素条件Logistics回归分析,纳入多因素条件Logislic回归模型进行多因素分析,计算各危险因素与大肠癌的关联程度OR值及其95％可信区间.结果 对577例大肠癌患者进行单因素条件Logistics回归分析发现,糖尿病病史、既往有大肠腺瘤性息肉病史和一级亲属患大肠腺瘤或大肠癌家族史和大肠癌的发生有关(P＜0.05).根据单因素的分析结果,将有意义的变量引入到多因素条件Logistic回归模型进行分析,结果显示糖尿病病史、既往大肠腺瘤性息肉病史、一级亲属患大肠腺瘤或大肠癌家族史是大肠癌发生的危险因素(P均＜0.05,OR值均＞1).结论 糖尿病病史、既往有大肠腺瘤性息肉病史和一级亲属患大肠腺瘤性息肉或大肠癌家族史增加了患大肠癌的危险性.     

异常隐窝灶与大肠癌

大肠癌是发病率较高的恶性肿瘤,其发生、发展涉及多因素、多阶段和多途径.目前,大肠癌的"正常上皮一增生上皮一腺瘤一癌一转移癌"的序列发病路径已广为接受.异常隐窝灶(aberrant crypt foci,ACF)是目前大肠癌在光镜下可见的最小、最早的黏膜损伤,其在遗传、表观遗传、免疫组化和组织病理等方面的改变都影响着肿瘤发生,可以说是其癌前病变.了解ACF的特征性变化及其与大肠癌发生的关系,对进一步认识和预防大肠癌有重要意义.     

12例青年大肠癌分析

目的 探讨青年人(小于及等于35岁)大肠恶性肿瘤的临床特点、内镜下特征及发病因素.方法 对我院自2006年3月至2011年12月肠镜检查病人的临床资料进行统计分析,并与同期上消化道胃镜检查的病人临床资料进行对比,从青年人大肠癌的临床特点,上下消化道的发病差异、以及青年人大肠癌性别发病差异进行分析.结果 全部青年人患者431例,其中确诊为大肠癌的病人12例,检出率为2.78％,男、女检出率分别为1.13％、5.45％,且青年人结肠癌的检出率明显高于上消化道癌的检出率(上消化道癌的检出率男为0.29％,女为0.93％).发病部位以直肠为多,占58.3％.病程短,发展快,就诊时多为进展期,BorrmanⅢ-Ⅳ型分别为7例、3例.症状多为血便或脓血便.结论 青人大肠癌的检出率虽然明显低于中老年人,但青年人大肠癌的恶性程度高,且结肠癌的检出率明显高于上消化道癌的检出率,女性发病明显多于男性,应引起临床的足够重视.     

青年大肠癌21例临床分析

大肠癌是最常见消化道恶性肿瘤之一。大肠癌发病年龄多在40岁以上。但近年来,大肠癌的发病率和发病年龄有逐渐提高和提前的趋势。我国大肠癌的发病年龄比欧美等西方国家有明显提前。本组病例为1991年至2002年3月我院经大肠镜检查并经病理确诊的大肠癌283例,其中35岁以下(包括35岁)的青年大肠癌21例,我们就其中青年大肠癌的临床症状、镜下形态,病理资料等进行分析,以探讨青年大肠癌的某些临床特征。     

中国青年大肠癌的发病特点及地理分布

为探讨中国青年大肠癌的发病特点及地区分布特点，分析南方医院２０年间病理确诊的１１３５例大肠癌病例和近十年来国内发表的有关资料。结果示青年大肠癌发病率３．５％～２２．７％不等，对近二十年来不同时期大肠癌发病情况调查表明，青年大肠癌占同期大肠癌的比例已明显下降。青年大肠癌以直肠为多（４８％～６３％），右半结肠癌较之同期大肠癌为多。病程多在三个月内，临床症状无特征性，误诊率高达７７．８％～８５．７％。青年大肠癌分化程度较低，分化良好者３２％～６４％，五年生存率为２５．８％～４０．３％之间。本文结果有助于对我国青年大肠癌的发病特点及地理分布的了解，和指导临床防治。     

人巨细胞病毒基因在大肠癌组织的表达及其临床意义

目的:初步探讨人巨细胞病毒(human cytomegalovirus,HCMV)感染相关基因在大肠癌的表达及与大肠癌临床病理特征的关系.方法:巢式PCR方法检测60例大肠癌患者的肿瘤及癌旁正常肠黏膜组织HCMV UL135、UL136、US28及IE1基因;SDSPAGE凝胶电泳分析结合基因测序方法验证巢式PCR结果的准确性,用卡方检验或Fisher确切概率法比较两组间阳性率,Logistic回归分析HCMV感染与大肠癌患者临床病理特征的关系.结果:SDS-PAGE凝胶电泳分析结合基因测序方法验证了巢式PCR结果的准确性.UL135、UL136及US28基因在大肠癌组织表达的阳性率分别为35.0%、15.0%及60.0%,在癌旁正常肠组织的表达阳性率分别为16.7%、1.7%及18.3%,两组具有显著性差异(均P0.05);IE1在大肠癌组织表达的阳性率(13.3%)与癌旁组织表达阳性率(10%)差异无显著意义.Logistic回归分析HCMV基因与大肠癌患者临床病理特征的关系,结果发现UL135、UL136及IE1基因表达与患者性别、年龄、肿瘤大小、病理分化程度、淋巴结转移及Dukes分期无关,而US28基因表达与淋巴结转移及Dukes分期相关,但和性别、年龄、肿块大小及病理分化程度无关.结论:大肠癌组织中UL135、UL136及US28基因表达较癌旁正常组织显著升高,其中US28阳性表达与大肠癌淋巴结转移及Dukes分期相关,提示HCMV某些基因表达可能参与大肠癌的发展.     

大肠癌的常规诊断

随着人们生活水平的提高和医疗条件的改善,诸多急性传染病均得到了有效的控制.然而,一些与环境因素密切相关的慢性非传染性疾病,如心、脑血管疾病和恶性肿瘤的发病率有了明显上升.大肠癌便是一个突出的例子.欧美和日本等大肠癌高发国家的研究证明,长年的高蛋白、高脂肪和低纤维素饮食习惯是促发大肠癌的重要环境因素(外因),患者遗传上的缺陷,如多种病相关基因的突变又是大肠癌发病的内在因素,这些相关的内外因素结合在一起可能也是近年我国大肠癌发病率上升的重要原因之一.预防为主是我国一贯的卫生工作方针,也是大肠癌防治工作的重点,纠正不良的饮食习惯,深入研究大肠癌发病的分子机制,从病因上着手防病是大肠癌预防工作的一级预防,而提高早癌和癌前疾病的检出率则是大肠癌的二级预防.众所周知,大肠癌有两个特点:一是有明确的癌前病变(93％的大肠癌来源于腺瘤);二是从癌前病变发展为癌有一较长的过程(平均7a).我们可以利用这些特点,通过普查发现癌前病变和早期癌,经过内镜的微创治疗,预防大肠癌的发生,提高早癌治愈率.下面几篇论文将围绕大肠癌早诊、早治的问题进行初步讨论,希望能引起同行专家的兴趣,提出更深入的见解.     

大肠癌相关基因对大肠癌的筛检

随着人们生活水平的提高和医疗条件的改善,诸多急性传染病均得到了有效的控制.然而,一些与环境因素密切相关的慢性非传染性疾病,如心、脑血管疾病和恶性肿瘤的发病率有了明显上升.大肠癌便是一个突出的例子.欧美和日本等大肠癌高发国家的研究证明,长年的高蛋白、高脂肪和低纤维素饮食习惯是促发大肠癌的重要环境因素(外因),患者遗传上的缺陷,如多种病相关基因的突变又是大肠癌发病的内在因素,这些相关的内外因素结合在一起可能也是近年我国大肠癌发病率上升的重要原因之一.预防为主是我国一贯的卫生工作方针,也是大肠癌防治工作的重点,纠正不良的饮食习惯,深入研究大肠癌发病的分子机制,从病因上着手防病是大肠癌预防工作的一级预防,而提高早癌和癌前疾病的检出率则是大肠癌的二级预防.众所周知,大肠癌有两个特点:一是有明确的癌前病变(93％的大肠癌来源于腺瘤);二是从癌前病变发展为癌有一较长的过程(平均7a).我们可以利用这些特点,通过普查发现癌前病变和早期癌,经过内镜的微创治疗,预防大肠癌的发生,提高早癌治愈率.下面几篇论文将围绕大肠癌早诊、早治的问题进行初步讨论,希望能引起同行专家的兴趣,提出更深入的见解.     

Mad2 and p53 expression profiles in colorectal cancer and its clinical significance

AIM: To investigate the expression of tumor suppressor gene p53 and spindle checkpoint gene Mad2, and to demonstrate their expression difference in colorectal cancer and normal mucosa and to evaluate its clinical significance.METHODS: Westemn blot and immunohistochemistry methods were used to analyze the expression of Mad2 in colorectal cancer and its corresponding normal mucosa. The expression of p53 was detected by immunohistochemistry method in colorectal cancer and its corresponding normal mucosa.RESULTS: Mad2 was significantly overexpressed in colorectal cancer compared with corresponding normal mucosa (P＜0.001), and it was not related to the differentiation of adenocarcinoma and other dinical factors (P＞0.05).The ratio of Mad2 protein in cancer tissue (C) to that in its normal mucosa tissue (N) was higher than 2, which was more frequently observed in patients with lymph gland metastasis (P＜0.05). p53 protein expression was not observed in normal mucosa. The rate of p53 positive expression in adenocarcinomas was 52.6%. There was a significant difference between adenocarcinomas and normal mucosa(P＜0.001), which was not related to the differentiation degree of adenocarcinoma and other clinical factors (P＞0.05).CONCLUSION: Defect of spindle checkpoint gene Mad2and mutation of p53 gene are involved mainly in colorectal carcinogenesis and C/N＞2 is associated with prognosis of colorectal cancer.     

Upregulation of nemo-like kinase is an independent prognostic factor in colorectal cancer

AIM: To investigate the expression and oncogenic role of nemo-like kinase(NLK) in colorectal cancer.METHODS: Expression of NLK protein was assessed by immunohistochemistry in tissue specimens from 56 cases of normal colorectal mucosa, 51 cases of colorectal adenoma, and 712 cases of colorectal cancer. In addition, NLK expression was knocked down using a lentivirus carrying NLK small hairpin RNA in colorectal cancer cells. Cell viability methylthiazoletetrazolium assays, colony formation assays, flow cytometry cell cycle assays, Transwell migration assays, and gene expression assays were performed to explore its role on proliferation and migration of colorectal cancer.RESULTS: Expression of NLK protein progressively increased in tissues from the normal mucosa through adenoma to various stages of colorectal cancer. Overexpression of NLK protein was associated with advanced tumor-lymph node-metastasis stages, poor differentiation, lymph node and distant metastases, and a higher recurrence rate of colorectal cancer(P 0.05). Multivariate analyses showed that NLK expression was an independent prognostic factor to predict overallsurvival(hazard ratio 2.57, 95% confidence interval: 1.66-3.98; P 0.001) and disease-free survival(hazard ratio 1.96, 95% confidence interval: 1.40-2.74: P 0.001) of colorectal cancer patients. Furthermore, knockdown of NLK expression in colorectal cancer cell lines reduced cell viability, colony formation, and migration, and arrested tumor cells at the G0/G1 phase of the cell cycle. At the gene level, knockdown of NLK expression inhibited matrix metalloproteinase-2 expression in colorectal cancer cells. CONCLUSION: NLK overexpression is an independent prognostic factor in colorectal cancer and knockdown of NLK expression inhibits colorectal cancer progression and metastasis.     

hClock基因mRNA及其蛋白在结直肠肿瘤中表达的研究

目的探讨人类生物钟基因hClockmRNA及其蛋白在不同Dukes分期结直肠肿瘤中的表达,研究它们的表达与结直肠肿瘤的侵袭及转移的关系。方法采用原位杂交检测结直肠癌与相应癌旁组织中hClock基因mRNA的表达,并采用免疫组织化学检测相应标本中hCloek基因蛋白产物（CLOCK蛋白）的表达。结果21例结直肠肿瘤中hCloekmRNA弱阳性表达率47．62％,中或强阳性表达率52．38％,且与Dukes分期相关（P〈0．05）;CLOCK蛋白均呈中或强阳性表达。相应癌旁组织中hClockmRNA及蛋白呈弱阳性表达（P〈0．01）。结论hCloek基因可能与结直肠肿瘤的发生、发展及侵袭、转移有相关性。     

Gene expression analysis in colorectal cancer using practical DNA array filter

PURPOSE: We examined the usability of a newly developed, compact-sized DNA array filter for studying the gene expression pattern of individual colorectal cancer. METHODS: Complementary DNA probes were prepared from mRNA extracted from colonic cancer specimens and adjacent normal mucosa and then were labeled with chemiluminescence. These labeled probes were allowed to bind to the gene fragments on the filter. A specialized scanning charge-coupled device camera measured the intensity of each chemiluminescent spot, which is an indicator of the degree to which a specific gene is expressed. Gene expression image was quantified into intensity of signals by using computer software. RESULTS: Characteristic gene expression patterns were obtained from the colonic cancer cell line, RPMI4788, and the leukemia cell line, HL60, by using this compact-sized DNA array filter in the preliminary experiment. Up-regulation of nm23, TIMP1, VEGF, and cyclin E and down-regulation of some tumor suppressor genes (p53, TOSO, and SIVA), beta-catenin, and metallothionein were observed in colonic cancer specimen when compared with those of normal mucosa. CONCLUSIONS: We have obtained unique gene expression patterns from colorectal cancer and normal tissue by using a newly developed compact-sized DNA array filter system. Collecting, storing, and analyzing of gene expression data from many samples of colorectal cancer will enable us to identify distinct subsets of patients based on molecular characteristics in the near future.     

Mad2 and p27 expression profiles in colorectal cancer and its clinical significance

AIM: To investigate the expression of tumor suppressor gene p27 and spindle checkpoint gene Mad2 and to demonstrate their expression difference in colorectal cancer and normal mucosa and to evaluate its clinical significance. METHODS: Immunohistochemical staining was used for detection of expression of Mad2 and p27 in colorectal cancer and its corresponding normal mucosa. RESULTS: Mad2 was significantly overexpressed in colorectal cancer compared with corresponding normal mucosa (P<0.01, chi(2) = 7.5), and it was related to the differentiation of adenocarcinoma, lymph node metastasis and survival period after excision (P<0.05, chi(2) = 7.72, chi(2) = 4.302, chi(2) = 6.234). The rate of p27 positive expression in adenocarcinomas and normal mucosa was 40% and 80% respectively. There was a significant difference in p27 expression between adenocarcinomas and normal mucosa (P<0.001, chi(2) = 13.333), which was related to the differentiation degree of adenoca rcinoma and lymph node metastasis (P<0.05, chi(2) = 8.901 chi(2) = 4). The positive expression of p27 was not correlated with survival period after excision. CONCLUSION: Defect of spindle checkpoint gene Mad2 and mutation of p27 gene are involved mainly in colorectal carcinogenesis and associated with prognosis of colorectal cancer.     

Construction of humanized carcinoembryonic antigen specific single chain variable fragment and mitomycin conjugate

AIM： To construct a new target-oriented conjugate of humanized carcinoembryonic antigen （CEA） specific single chain variable fragment （scFv） and mitomycin （MMC） against colorectal cancer, and to investigate its influence on the growth and apoptosis of colorectal cancer cells. METHODS： The primer was designed according to the gene sequence described in reference 16, which respectively contains restriction enzyme cleavage sites BamH I and EcoR I in its upstream and downstream. PCR was performed with the plasmid as template containing genes of humanized anti-CEA scFv. The product was digested by BamH I and EcoR I, and connected to an expression vector which also has the restriction enzyme cleavage sites BamH I and EcoR. Expression of the reaction was induced by isopropy-β -D-thiogalactoside （IPTG）. Then the expression product was covalently coupled with MMC by dextran T-40. The immunoreactivity of the conjugate against colorectal cancer cells as well as CEA was measured by enzyme linked immunosorbent assay （ELISA）. The inhibiting ratio of conjugate on the growth of colorectal cancer cells was also measured by ELISA. The effect of conjugate on the apoptosis of colorectal cancer cells was determined by flow cytometry （FCM）. RESULTS： Restriction endonuclease cleavage and gene sequencing confirmed that the expression vector was successfully constructed. Sodium dodecyl sulfate polyacrylamide gel electropheresis （SDS-PAGE） confirmed that this vector correctly expressed the fusion protein. ELISA confirmed that the conjugate had quite a strong immunoreactivity against colorectal cancer cells and CEA. The conjugate had inhibitory effects on colorectal cancer cells in a concentration-dependent manner and could induce apoptosis of colorectal cancer cells in a concentration-dependent manner.CONCLUSION： The CEA-scFv-MMC conjugate can be successfully constructed and is able to inhibit the growth and induce apoptosis of colorectal cancer cells.     

Induction of HSF1 expression is associated with sporadic colorectal cancer

AIM: To explore the activation of signal transduction pathways related with the carcinogenesis of sporadic colon cancers. METHODS: A gene array monitoring the activation of 8 signal transduction pathways (PathwayFinder GEArray) was used to screen the differentially expressed genes between colorectal cancer and normal colon tissues. The differentially expressed genes were further analyzed by RT-PCR, using RNA derived from colorectal cancer and normal colon tissue of 35 patients. RESULTS: The expression of HSF1, HSF27, HSP90 and iNOS was increased in colon cancer tissues compared to normal colon tissue using PathwayFinder GEArray. The RT-PCR results showed that the expression of HSF1 was increased in 86% (30/35) patients and the expression of iNOS was increased in 63% (22/35) patients. CONCLUSION: The induction of HSF1 gene expression is associated with sporadic colon cancer. HSF1 induces heat shock stress signaling pathway, which might play a role in the carcinogenesis of sporadic colorectal cancer.     

Survivin基因沉默对结直肠癌PTTG mRNA的影响

目的探讨重组载体介导的Survivin小干扰RNA(siRNA)对人结肠癌细胞株Lovo中PTTG mRNA的影响。方法前期成功构建的Survivin特异性siRNA真核表达载体转染结肠癌Lovo细胞后,采用实时荧光定量PCR检测PTTG mRNA的表达水平。结果 Survivin基因沉默后24～72 h,与正常对照组相比,Survivin mRNA表达明显降低(P<0.05);基因沉默24和48 h后,pGenesil-2-Survivin 1组和pGenesil-2-Survivin 2组PT-TG mRNA表达显著低于正常对照组(P<0.05)。结论 Survivin基因沉默后结直肠癌细胞PTTG mRNA表达显著下调,提示Survivin和PTTG可能相互促进共同参与结直肠癌的发生发展。