散发性结直肠癌患者18号染色体高频杂合缺失的研究

目的：探讨散发性结直肠癌患者18号染色体上抑癌基因相关的杂合缺失（LOH）情况，并探索新的抑癌基因位点。方法：对83例散发性结直肠癌患者基因组DNA用14个不同荧光标记的高度多态性微卫生引物，扩增相应的微卫星位点，平均距离为10厘摩（centi-morgan,cM）。用ABI PRISM377测序仪进行基因扫描，统计各位点杂合缺失率。结果：在12个获得有效数据的微卫星位点中，平均杂合缺失率为36．78％,18p中最高为D18S53（38．09％），18q中最高为D18S474（55．74％）。4位患者的18号染色体所有杂合位点都存在缺失，30位患者的杂合缺失位点不少于50％（平均6个／人）；缺失位点少于50％的有53人（平均1个／人）。结论：结直肠癌患者18号染色体存在高频的LOH，并以整体缺失为特点。存在高频LOH的区域定位有转化生长因子（TGF）信号传导相关基因、结直肠癌缺失基因（DCC）、Rb结合蛋白8(RbBP8），特别是TGF信号传导相关基因MADH2、4、转化生长因子－β1反应元件（TGF－β1）等的缺失可能对结直肠癌的发生有重要影响。18p也有存在未知抑癌基因的可能。     

散发性结直肠癌相关半胱氨酸甘氨酸富集蛋白1抑癌基因的筛选

目的 本课题组前期研究对染色体1q31.1-32.1区域进行杂合缺失精细定位,发现D1S413-D1S2622区域存在高频杂合缺失现象,提示可能有抑癌基因的存在.本研究在此基础上,对该区域与散发性结直肠癌发生相关的抑癌基因开展筛选研究.方法 构建包含上述区域基因的基因芯片,对19例散发性结直肠癌标本进行基因芯片扫描,并与其临床病理特征进行统计学分析,筛选该区域与结直肠癌相关的未知抑癌基因,然后对筛选出的候选基因采用Real-time PCR进行初步验证.结果 根据前期实验结果,通过检索,挑选了25个基因进行散发性结直肠癌相关基因的筛选.结果发现半胱氨酸甘氨酸富集蛋白1 (cysteine and glycine-rich protein 1,CSRP1)、LMOD1 、PPP1R12B和CFHL3 4个基因表达显著下调.这4个基因表达情况与结直肠癌患者的临床病理特征无关.通过生物信息学分析,推测CSRP1基因可能是该区域中与结直肠癌相关的抑癌基因.通过Real-time PCR验证结果也发现CSRP1基因在结直肠癌组织中表达显著下调,与芯片结果相符.结论 CSRP1基因可能是一个与结直肠癌发生相关的新的抑癌基因.     

散发性结直肠癌微卫星不稳定与hMHL1和hMSH2基因突变的研究

目的:探讨散发结直肠癌组织微卫星不稳定性与错配修复基因hMHL1和hMSH2蛋白表达的关系。方法:结直肠癌患者病理标本和正常肠壁组织(距肿瘤边缘10cm取材各40例)。选取微卫星位点(D2S123、BAT-26、D17S261、D17S799)进行PCR,PCR产物行毛细管电泳法检测。用免疫组化染色方法分析错配修复基因hMHL1和hMSH2在肿瘤组织的蛋白表达情况。结果:1)4个位点(D2S123、BAT26、D17S261、D17S799)的微卫星不稳定性检出率分别为:12.5%、17.5%、10%、7.5%。总的微卫星不稳定率为9/40(22.5%)。MSI-H表达为7例,均表现BAT26位点不稳定。MSI-L表达2例。2)所有标本错配修复基因hMSH2检测均正常表达。错配修复基因hMHL1表达阴性11份,结肠比直肠hMLH1蛋白的阴性表达率高(P0.01)。3)hMLH1表达阴性,是出现MSI的重要分子因素,hMLH1不表达和MSI相关显著(P0.01)。结论:1)错配修复基因突变引起微卫星不稳定性是散发性结肠癌发生的重要机制;2)部分微卫星不稳定性是由错配修复基因hMLH1不表达引起,其余的微卫星不稳定性可能涉及到其它错配修复基因。     

多发性结直肠癌中抑癌基因p53的表达与突变

目的探讨抑癌基因p53与多发性结直肠癌的关系。方法实验组为同时多发性结直肠癌19个癌灶和结直肠再发癌12个癌灶,对照组为散发性结直肠癌17个癌灶。分别采用免疫组织化学抗生物素蛋白生物素过氧化物酶复合物(ABC)方法和单链DNA构像多态性(PCRSSCP)方法对癌组织和癌旁正常组织进行p53蛋白以及p53基因第5、7、8外显子突变的检测,比较各组蛋白表达和基因突变的不同。结果在同时多发癌组、再发癌组以及对照组中p53蛋白阳性表达率分别为73.7%、75%、35.3%,3组比较差异有统计学意义(χ2=6.952,P<0.05);突变率3组分别为52.6%、41.7%、29.4%,差异均无统计学意义。癌旁正常组织均未检出突变;但蛋白表达在同时性三发性癌患者中为阳性。结论多发性结直肠癌中p53基因突变率和蛋白表达高于散发性结直肠癌。p53过度表达的结直肠癌患者要警惕多发癌的发生。     

散发性结直肠癌染色体7q21-22区杂合性缺失精细定位

目的 研究散发性结直肠癌7号染色体杂合性缺失,对7q21-22区精细定位,寻找新的结直肠癌抑癌基因.方法 采用15对微卫星DNA标记7号染色体,在高频杂合缺失区另取5对微卫星标记对83例结直肠癌病例的肿瘤和正常组织进行PCR反应.PCR产物在ABI Prism 377自动荧光测序仪进行电泳3 h,以GeneScan3.1和Genotyper 2.1软件进行基因分型.结果 在7号染色体上发现1个高频杂合缺失区即7q21-22区.对该区再用5对微卫星标记引物行精细定位,界定了1个跨越D7S657、D7S646位点精细的高频杂合缺失区域.结论 通过精细杂合缺失作图的研究,在7号染色体发现了1个跨越D7S657、D7S646位点的精细杂合缺失区,该区很可能存在1个或多个与结直肠癌相关的新的抑癌基因.     

散发性胆管癌患者染色体3p21.3区段微卫星位点不稳定性分析

目的研究散发性胆管癌患者染色体3p21．3区段的微卫星不稳定性（MSI）及杂合性缺失（LOH），探讨染色体3p21．3区段遗传不稳定性与散发性胆管癌发生发展的关系，定位该区段上散发性胆管癌相关肿瘤基因。方法用聚合酶链反应一单链构象多态性分析（PCR—SSCP）方法检测24例散发性胆管癌患者染色体3p21．3区段上D3S1568、D3S1621、D3S1578和D3S1289四个微卫星位点的MSI和LOH发生率，分析其与临床病理因素之间的关系。结果24例散发性胆管癌组织中，4个微卫星位点的MSI和LOH平均发生率分别为7．23％和15．63％。其中D3S1621位点的LOH最高（45．83％，11／24），并与TNM分期、是否伴有局部／淋巴结转移相关（P〈0．05）。结论染色体3p21．3区段133S1621位点高频率杂合性缺失，提示3p21．3区段定位有散发性胆管癌的候选抑癌基因，并在散发性胆管癌的发生发展过程中发挥重要作用。     

P53，C—erbB—2和bcl—2基因在膀胱癌中的表达及意义

目的：研究癌基因和抑癌基因蛋白产物在膀胱移行细胞癌组织中异常表达与肿瘤病理分级和临床分期之间的关系。方法：应用免疫组织化学方法检测96例膀胱移行细胞癌标本中P53,C－erbB-2和bcl-2基因的表达水平。结果：96例膀胱移行细胞癌标本中P53,C－erbB-2,bcl-2基因的阳性表达率分别为60．4％,66．7％和81．3％,P53,C－erbB-2和bcl-2异常表达与膀胱癌的病理分级和临床分期之间的差异有统计学意义（P＜0．01）,结论：P53,C－erbB-2和bcl-2基因异常表达在膀胱癌发生发展中起重要作用。肿瘤的多基因分析比单基因分析更有价值,为临床病理诊断及估计预后和复发提供了参考指标。     

结直肠癌中抑癌基因ING1的表达研究

目的 探讨抑癌基因ING1及其蛋白p33／ING1在结直肠癌中的表达。方法 用RT-PCR检测35例散发性结直肠癌中ING1 mRNA的表达水平，并应用S-P法检测60例散发性结直肠癌及正常黏膜组织中p33／ING1蛋白的表达。结果 结直肠癌组织、正常黏膜组织中p33／ING1蛋白阳性表达率分别为43．3％（26／60）、100％（60／60），两者比较差异有统计学意义（P＜0．01）。p33／ING1在无淋巴结转移组及淋巴结转移组中的阳性表达率分别为57．6％（19／33）、25．9％（7／27），两者比较差异有统计学意义（P〈0．05）。在Dukes A、B期和Dukes C、D期患者癌组织中p33／ING1的阳性表达率分别为56．7％（17／30）、30．0％（9／30），两者比较差异有统计学意义（P＜0．05）。结论 ING1及p33／ING1蛋白的低表达与散发性结直肠癌发生、发展密切相关，临床分期越差者表达水平越低。     

结直肠癌中错配修复基因杂合性缺失的初步分析

目的探讨散发性结直肠癌中错配修复基因杂合性缺失的发生情况。方法利用微卫星分析法对30例散发性结直肠癌6个错配修复基因(mismatch repair genes，MMR)的杂合性缺失(loss of heterozygosity，LOH)进行了分析。结果6个MMR基因LOH的发生率由高到低依次分别为hMSH3(30％)、hPMS2(25％)、hMLH1(30．0％)、hMSH2／hMSH6(23．07％)，hPMS1基因未发现杂合性缺失。结论在中国人散发性结直肠癌中，MMR基因通过等位基因杂合性缺失是结直肠癌发生的重要分子遗传途径之一。     

错配修复、微卫星不稳定性与散发性结肠直肠癌

近年来研究发现ＤＮＡ修复基因突变引起ＤＮＡ错配修复系统的功能降低或丧失 ,从而引起遗传物质不稳定 ,主要表现为微卫星不稳定性 ,进而导致肿瘤的发生。我们采用ＰＣＲ技术对 48例散发性结肠直肠癌检测了错配修复基因ｈｍｓｈ3、ｈｍｓｈ6和 4个位点的微卫星不稳定性 ,现将结果报告如下。1.材料与方法 :(1)临床资料 :我院1997～ 1998年经手术切除的新鲜结、直肠癌标本 48例 ,其中男女各 2 4例 ;年龄2 6～ 72岁 ,平均 5 3岁 ;结肠癌 2 9例 (右半结肠癌 15例 ,左半结肠癌 14例 ) ,直肠癌 19例 ;高分化癌 19例 ,中分化癌 2 2例 ,低分化癌 7例…     

多重荧光PCR方法对结直肠癌进行微卫星不稳定检测及其临床价值

目的探讨多重荧光PCR方法检测结直肠癌微卫星不稳定（MSI）及其临床意义。方法将2004-2005年间进行手术治疗的110例结直肠癌患者建立队列，以多重荧光PCR方法检测结直肠癌MSI，并对MSI和微卫星稳定（MSS）结直肠癌患者的临床病理特点进行比较。结果多重荧光PCR方法扩增出所有患者的5个微卫星序列。其中MSI．H10例（8．1％），MSI-L13例（11．8％），MSS为87例（79．1％）。共检测BAT．26变异9例（8．2％）、BAT．25变异11例（10．0％）、D2S123变异11例（10．0％）、D5S346变异6例（5．5％）和D17S250变异8例（7．3％）。MSI-L、MSI．H和MSS组结直肠癌患者年龄比较，差异有统计学意义（P〈0．05）；其他临床病理特点差异无统计学意义（P〉0．05）。结论多重荧光PCR方法检测MSI结果稳定，宜于临床应用；MSI和MSS结直肠癌患者临床病理特点比较未见差异。     

Comparison of microsatellite instability and chromosomal instability in predicting survival of patients with T3N0 colorectal cancer

BACKGROUND: At least 2 apparently independent mechanisms, microsatellite instability (MSI) and chromosomal instability, are implicated in colorectal tumorigenesis. Their respective roles in predicting clinical outcomes of patients with T3N0 colorectal cancer remain unknown. METHODS: Eighty-eight patients with a sporadic T3N0 colon or rectal adenocarcinoma were followed up for a median of 67 months. For chromosomal instability analysis, Ki-ras mutations were determined by single-strand polymerase chain reaction, and p53 protein staining was studied by immunohistochemistry. For MSI analysis, DNA was amplified by polymerase chain reaction at 7 microsatellite targets (BAT25, BAT26, D17S250, D2S123, D5S346, transforming growth factor receptor II, and BAX). RESULTS: Overall 5-year survival rate was 72%. p53 protein nuclear staining was detected in 39 patients (44%), and MSI was detected in 21 patients (24%). MSI correlated with proximal location (P <.001) and mucinous content (P <.001). In a multivariate analysis, p53 protein expression carried a significant risk of death (relative risk = 4.0, 95% CI = 1.6 to 10.1, P =.004). By comparison, MSI was not a statistically significant prognostic factor for survival in this group (relative risk = 2.2, 95% CI = 0.6 to 7.3, P =.21). CONCLUSIONS: p53 protein overexpression provides better prognostic discrimination than MSI in predicting survival of patients with T3N0 colorectal cancer. Although MSI is associated with specific clinicopathologic parameters, it did not predict overall survival in this group. Assessment of p53 protein expression by immunocytochemistry provides a simple means to identify a subset of T3N0 patients with a 4-times increased risk for death.     

Infrequent microsatellite instability in urothelial cell carcinoma of the bladder in young patients

OBJECTIVE: Defects in the DNA mismatch repair result in microsatellite instability (MSI), which characterise most tumours related to the hereditary non-polyposis colorectal cancer syndrome and some sporadic tumours. Several studies have reported the occurrence of MSI in urothelial cell carcinoma (UCC) of the bladder with a particularly high incidence in tumours from young patients. In this study, we have evaluated the occurrence of MSI in primary bladder UCC arising in seventeen young patients selected for being below 45 years of age at diagnosis. METHODS: Microsatellite analysis has been performed using the panel of five quasimonomorphic mononucleotide repeats (BAT-25, BAT-26, NR-21, NR-24, NR-27) recently recommended to detect MSI tumours. The original Bethesda panel including BAT-25, BAT-26 and three dinucleotide repeats (D2S123, D5S346, D17S250) has further been studied in 10 UCC samples. RESULTS: MSI has been observed in only one of the 17 bladder UCC studied. Using the original Bethesda panel, identical results were obtained, indicating that the panel of five mononucleotide markers adequately detected MSI in UCC tumours. CONCLUSIONS: Our data indicate that classical MSI affecting mono- or di-nucleotides are rarely involved in bladder UCC developing in young patients. Further studies using gold standard criteria would help clarifying the involvement of MSI in the pathogenesis of bladder UCC.     

Low frequency of microsatellite instability in hereditary prostate cancer

OBJECTIVE: To investigate whether there is widespread microsatellite instability (MSI) in families with hereditary prostate cancer (HPC). PATIENTS AND METHODS: Eighty-four prostate tumours from 80 Swedish men in 35 families with HPC were screened for genetic instability at microsatellite marker loci BAT-25, BAT-26, BAT-34C4, D2S123 and D17S250. RESULTS: MSI was detected in only five individuals from different families. Three tumours (4%) were unstable at more than two MSI loci and hence classified as high-frequency MSI (MSI-H) according to a previous definition. Interestingly, two of the MSI-H tumours were from patients in families with both HPC and familial colon cancer. CONCLUSIONS: Widespread MSI is a rare event in hereditary prostate cancer, indicating that defective DNA mismatch repair is not an important element in the genesis of HPC.     

应用荧光多重聚合酶链反应法检测结直肠癌微卫星不稳定

目的建立一种敏感、稳定、高通量的结直肠癌微卫星不稳定(MSI)检测技术。方法105例散发性结直肠癌患者取其新鲜癌组织及正常肠黏膜,DNA提取,荧光多重聚合酶链反应(PCR)扩增5个微卫星位点,应用GeneScan方法分析PCR产物。结果105例结直肠癌患者26例(24.7%)存在微卫星不稳定,其中MSI-H 14例(13.3%),MSI-L 12例(11.4%),MSS 79例(75.3%)。D5S346、BAT26、BAT25、D17S250、D2S123突变率分别为5.6%、8.6%、10.5%、8.6%、10.5%。结论应用荧光多重PCR可以高通量检测结直肠癌微卫星不稳定,敏感性高,结果可靠,适合临床应用。     

Microsatellite instability is not a common feature in medullary breast cancer

Microsatellite instability (MSI) is a form of genomic instability associated with defective DNA mismatch repair in tumors. MSI is found in 85-90% of hereditary nonpolyposis colorectal cancer cases; however, its occurrence in breast carcinogenesis still remains to be clarified. In addition, data are limited on the incidence of MSI in the medullary subtype. The purpose of this study was to investigate the occurrence of MSI in medullary breast cancer (MBC). The study included a total of 16 patients with MBC, nine with typical and seven with atypical histology. The incidence of MSI in five microsatellite loci (D2S123, D3S1611, D17S807, D17S796 and Xq11-12) was determined by comparing paired normal and tumor tissue DNA after PCR amplification from paraffin-embedded tissues. All 16 tumors showed stability at five loci. Although the number of microsatellite markers and DNA samples may limit the value of our results, we conclude that the MSI phenotype is uncommon in human MBC.     

Angiogenesis and p53 at the invading tumor edge: prognostic markers for colorectal cancer beyond stage

BACKGROUND: Angiogenesis has emerged as a major prognostic factor in many human malignancies and it is a prospective target for cancer therapy. MATERIALS AND METHODS: In this study, we investigated immunohistochemically the angiogenic activity and the expression of p53 and bcl-2 proteins in a series of 170 operable colorectal carcinomas, stage B and C. RESULTS: A high vascular density at the invading tumor front was directly related to nuclear p53 accumulation, and inversely to cytoplasmic expression of bcl-2. Furthermore, high angiogenic activity was significantly associated with lymph node metastasis. Survival analysis showed that Dukes stage and vascular density were the most important and independent prognostic factors in colorectal cancer. DISCUSSION: It is believed that angiogenesis at the invading tumor edge can be used as an independent prognostic marker to identify subgroups of colorectal cancer patients with an unfavorable post-operative outcome.     

肝细胞癌微卫星不稳定和杂合性缺失分析

目的 通过检测肝细胞癌(hepatocellular carcinoma,HCC)微卫星不稳定（microsatellite instability,MSI)和杂合性缺失（loss of heterozygosity,LOH)发生频率,探讨上述遗传学改变与HCC临床病理的关系。方法 选择5个微卫星多态性标记对32例HCC进行了MSI与LOH分析。结果 32例HCC中有14例出现MSI,其中有6例出现2个位点MSI,后者癌肿转移率明显低于其他的组（P＝0．001）,在所检5个位点中D1S484和TP53 MSI发生频率最高,而D9S1604昨D8S555遗传学改变多为LOH且发生频率较低。结论 MSI是部分HCC发生、发展进程中一个重要的遗传改变,尤其在在无癌肿转移的肿瘤中。D1S484和TP53是HCC中对MSI敏感的检测位点。