双色荧光原位杂交检测苯系物暴露工人精子9、18号染色体数目畸变

为研究苯系物暴露工人精子常染色体数目畸变.用地高辛标记的9号染色体探针(D9Zl)和生物素标记的18号染色体探针(D18Z1)进行双色荧光原位杂交,测定精子9、18号染色体非整倍体率.车间空气苯时间加权平均浓度(TWA)为86.49mg/m3,高于国家卫生标准1倍,苯暴露工人尿粘糠酸(ttMA)显著高于对照组.共计数14名暴露工人136401条精子,16名对照工人156955条精子.暴露组9、18染色体双体精子率(分别为0.168%±0.063%、0.055%±0.031%)和二倍体精子率(0.073%±0.045%)均高于对照组相应数值(分别为0.050%±0.030%、0.033%±0.025%和0.040%±0.036%);暴露组9、18号染色体缺体率(分别为0.206%±0.047%,0.068%±0.044%)高于对照组值(0.067%±0.037%、0.048%±0.034%).总数目畸变率(0.570%±0.144%)亦高于对照组值(0.218%±0.071%).实验表明接触较高浓度苯可引起长期暴露者精子常染色体非整倍体率增高.     

荧光原位杂交技术检测骨髓增生异常综合征染色体异常的临床意义

目的:探讨荧光原位杂交(FISH)技术检测骨髓增生异常综合征(MDS)染色体异常的敏感性,特异性及临床意义。方法:采用细胞遗传学分析(CCA)和组合探针CSF1R/D5S23,D5S721(5q33),EGR1/D5S23,D5S721(5q31),D7S486/CSP7(7q31),D7S522/CSP7(7q31),D20S108/CSP8(20q12/CSP8)检测45例MDS患者骨髓细胞的染色体异常,并比较检测结果。结果:两种方法共检出染色体异常26例(58%),染色体数目异常9例,占34.6%;染色体结构异常13例,占50%;复杂核型4例。CCA检出+8和20q-各3例,7q-2例;FISH检出7号染色体异常8例占17.8%(8/45),两组间比较差异有统计学意义(P=0.0441713)。FISH检出+8和20q-各5例,5q-异常4例。7号染色体异常和复杂核型组与核型正常组比较转白率高。结论:组合探针检出MDS中5q-,-7/7q-,+8,20q-核型异常高于CCA,CCA结合FISH技术能提高MDS染色体异常的检出率,对于疾病诊断,判断预后具有重要价值。     

多色荧光原位杂交检测苯系物接触工人精子1,18号染色体畸变率

为研究苯系物暴露对工人精子染色体的损伤 ,用 4条DNA探针与间期精子核染色体进行多色荧光原位杂交 ,同时检测精子 1号、18号染色体数目畸变和 1号染色体结构畸变 (末端缺失与重复 )。作业车间空气中苯的时间加权浓度 (TWA)为 4 2 2 9mg m3,高于国家卫生标准 (6mg m3)。暴露组工人尿粘糠酸 (ttMA)高于对照组。共计数15例暴露组工人 14 4 2 82条精子 ,14例对照组工人 135 937条精子 ,杂交效率为 99 85 %。非整倍体测定结果 :暴露组精子 1号、18号染色体双体率 (分别为 0 0 88%± 0 0 4 1% ,0 0 87%± 0 0 4 9% )显著高于对照组 1号、18号双体率(0 0 4 5 %± 0 0 2 4 % ,0 0 5 3%± 0 0 2 8% ) ;暴露组 1号、18号染色体缺体率分别为 (0 11%± 0 0 5 9% ,0 0 75 %±0 0 35 % )显著高于对照组相应数值 (0 0 4 8%± 0 0 18% ,0 0 4 5 %± 0 0 2 4 % ) ;而二倍体精子率 ,两组差别无显著性。结构畸变测定结果 :暴露组 1号染色体的末端重复率、末端缺失率 (分别为 0 16 %± 0 0 37% ,0 14 %± 0 0 5 3% )显著高于对照组数值 (分别为 0 0 82 %± 0 0 2 3% ,0 0 6 9%± 0 0 2 8% ) ;暴露组 1号染色体着丝粒重复率及着丝粒缺失率(0 10 %± 0 0 35 % ,0 10 %± 0 0 4 1% )显著性高于对     

应用多重RT—PCR技术检测食管癌患者外周血多种肿瘤基因标志物的研究

目的:建立多重RT-PCR同步检测技术,联合检测黑色素瘤抗原MAGEA3基因、人类斯钙素hsTC1基因、上皮细胞角质蛋白CK20、CK19基因及癌胚抗原CEA基因mRNA在食管癌患者外周血中的表达。方法:在分析MAGEA3、hSTC1、CK20、CK19、CEA和β-actin六种基因mRNA全长序列资料的基础上,自行设计并筛选出6对引物及6条寡核苷酸探针,利用多重RT-PCR技术并结合基因芯片对样本进行检测。结果:全部样品均可检出β-actin,55例食管癌患者外周静脉血中MAGEA3、hSTC1、CK20、CK19和CEA的阳性表达率分别为25.45%、47.27%、49.09%、34.55%和52.73%;多重RT-PCR技术同时检测MAGEA3、hSTC1、CK20、CK19和CEA基因,如果至少表达其中一种即可作为阳性表达者,其阳性表达率可达72.73%。而10例食管癌组织中均为阳性,28例正常健康捐献者的外周血中有2例CEA表达阳性,27例非肿瘤病人的外周血中有3例CK20阳性,3例CK19阳性,3例CEA阳性。测序结果证实RT-PCR产物确为MAGEA3、hSTC1、CK20、CK19和CEA基因。结论:联合检测MAGEA3、hSTC1、CK20、CK19和CEA可以增加外周血循环癌细胞检测的敏感性,本研究建立的多重RT-PCR技术可以做为一种快速、灵敏的检测手段。     

应用多重RT-ORC技术检测食管癌患者外周血多种肿瘤基因标志物的研究

目的:建立多重RT-PCR同步检测技术,联合检测黑色素瘤抗原MAGEA3基因、人类斯钙素hSTC1基因、上皮细胞角质蛋白CK20、CK19基因及癌胚抗原CEA基因mRNA在食管癌患者外周血中的表达.方法:在分析MAGEA3、hSTC1、CK20、CK19、CEA和B-actin六种基因mRNA全长序列资料的基础上,自行设计并筛选出6对引物及6条寡核苷酸探针,利用多重RT-PCR技术并结合基因芯片对样本进行检测.结果:全部样品均可检出B-actin,55例食管癌患者外周静脉血中MAGEA3、hSTC1、CK20、CK19和CEA的阳性表达率分别为25.45%、47.27%、49.09%、34.55%和52.73%;多重RT-PCR技术同时检测MAGEA3、hSTC1、CK20、CK19和CEA基因,如果至少表达其中一种即可作为阳性表达者,其阳性表达率可达72.73%.而10例食管癌组织中均为阳性,28例正常健康捐献者的外周血中有2例CEA表达阳性,27例非肿瘤病人的外周血中有3例CK20阳性,3例CK19阳性,3例CEA阳性.测序结果证实RT-PCR产物确为MAGEA3、hSTC1、CK20、CK19和CEA基因.结论:联合检测MAGEA3、hSTC1、CK20、CK19和CEA可以增加外周血循环癌细胞检测的敏感性,本研究建立的多重RT-PCR技术可以做为一种快速、灵敏的检测手段.     

用三色荧光原位杂交(Three-Color FISH)检测小鼠精子中染色体数目异常的研究

用小鼠X、Y和8号染色体特异的DNA探针,与经DTT（dithiotreitol）和LIS（lithium-3,5-diiodosalicylicacid）解聚的小鼠附单精子进行三色荧光原位杂交（fluoresceoceinsituhybridization,FISH）,以检测精子中的染色体数目异常,并与MMⅡ染色体分析比较．结果表明精子三色FISH具有以下优点和特点：（1）方法敏感稳定,且简便快速;（2）在每一个体至少分析10000尾精子的基础上计算非整倍体单,因此结果更为准确;（3）能检测多倍体即减数分裂停止的发生率及停止的时期;（4）不仅能测定发生于试数分裂Ⅰ（MI）的染色体分离异常,还能检测发生于减数分裂Ⅱ（MII）的不分离和丢失．并对探针的选用、分析标准的建立以及三色FISH用于精于染色体分析的必要性等进行了讨论．     

食管鳞癌患者ERCC1、TYMS、TUBB3的表达特征及其与化疗效果的相关性分析

探讨食管鳞癌患者ERCC1、TYMS、TUBB3的表达特征,以及其与化疗效果的相关性。选择2013年8月~2014年8月我科收治的食管癌患者78例,于术后采用液相芯片技术对食管癌组织ERCC1、TYMS、TUBB3基因m RNA表达水平进行检测,所有患者均按指南所载方案进行化疗,并统计患者1年复发情况。ERCC1、TYMS、TUBB3表达阳性表达率分别为48.7%、70.5%、61.5%,ERCC1表达阳性率与年龄,TNM分期、组织分化程度等因素有关,TUBB3表达阳性率与性别有关,发病部位为食管上段的患者TYMS表达阳性率更高,差异均具有统计学差异。TYMS与TUBB3呈现出弱相关性,ERCC1表达与TYMS、TUBB3与均无相关性。2或3项基因阳性患者1年复发率显著高于0或1项基因阳性患者,分别为29.8%、9.7%。食管鳞癌患者ERCC1、TYMS、TUBB3表达具有特定的临床特征,高表达与复发率增加有关。     

高特异性HER2/CEP17双色探针的制备与临床应用

目的:探讨高特异性HER2/CEP17双色探针在浸润性乳腺癌中的诊断价值。方法:制备高特异性HER2/CEP17双色探针,通过外周血细胞核型滴片检测该探针的特异性,同时用传统探针和新制备探针对425例浸润性乳腺癌患者进行FISH检测,并对结果进行对比分析,评估高特异性HER2/CEP17探针在浸润性乳腺癌中的临床诊断价值。结果:染色体定位显示,传统的CEP17探针在17号染色体着丝粒处杂交信号明亮,在Y染色体着丝粒处可见一个较17号染色体略弱但肉眼可辨的杂交信号点;而新制备CEP17探针在17号染色体着丝粒处具有明亮的杂交信号,在其余染色体中未见明显的杂交信号点,表明该探针具有较高的特异性。临床样本检测中,新制备探针与传统探针对浸润性乳腺癌HER2基因扩增的检测无统计学差异(P=0.500,χ~2=0.5),然而,对于CEP17杂信号,统计结果表明新制备探针明显少于传统探针(P=0.013,χ~2=5.29)。结论:新方法制备HER2/CEP17探针具有较高的特异性,相比于传统探针更具有临床应用优势。     

性别决定和性染色体交换模型

人的性别究竟是由什么来决定?过去人们常认为:人的性别由性染色体决定,且主要由Y染色体所决定,即在人体的体细胞内,除了含有成对的常染色体以外,凡含有x和Y染色体的为男性,凡含有两个x染色体的为女性。但是也有性染色体组成为XX的男性。据估计,具有XX性染色体的男性约在两万个男性中就有一个。为什么体细胞含有XX染色体的个体是男     

利用染色体区段混合BAC探针鉴定食管癌细胞中的染色体畸变

染色体畸变是恶性肿瘤细胞的重要遗传学特征,文章旨在应用BAC DNA克隆鉴定食管癌细胞中的染色体臂和染色体区段的畸变。针对染色体各区段选取5~10个1 Mb BAC DNA,分别混合制备成特定染色体区段的BAC DNA混合克隆,然后将染色体臂上覆盖所有区段的上述混合克隆进一步混合制备成特定染色体臂BAC DNA混合克隆。利用简并寡核苷酸引物聚合酶链反应(Degenerate oligonucleotide primed PCR,DOP-PCR)标记染色体臂探针,利用切口平移法(Nick translation)标记染色体区段探针,并对食管癌细胞中期染色体进行荧光原位杂交(Fluorescence in situ hybridization,FISH)分析。正常人外周血淋巴细胞中期染色体FISH结果显示,上述方法标记的探针具有较高的特异性。进一步利用染色体臂混合探针,确定了多个食管癌细胞中的染色体重排所涉及的特定染色体臂;利用染色体区段混合探针,鉴定出KYSE140的t(1q;7q)衍生染色体中1q上的断点范围位于1q32-q41。文章成功建立了1 Mb BAC DNA混合克隆探针标记技术,并鉴定出多个食管癌细胞中的染色体臂和染色体区段畸变,不仅为利用M-FISH技术鉴定肿瘤细胞中的染色体畸变提供了更为准确的方法,而且还可能进一步将该法推广应用于恶性血液病的核型分析以及产前诊断。     

双向凝胶电泳-飞行时间质谱技术鉴定食管上皮细胞癌变时14-3-3 protein ε、S100A9的表达

[摘要] 目的：分析食管鳞癌和正常食管上皮细胞蛋白质的表达差异，获取鉴别两者的分子标志物。方法：通过激光捕获显微切割技术分离ESCC肿瘤细胞和癌旁上皮细胞，通过双向电泳和质谱技术鉴定表达异常的蛋白，并通过蛋白免疫印记证实部分差异蛋白的表达。结果：建立了食管癌组织和正常食管上皮蛋白的双向凝胶电泳图谱，通过质谱技术鉴定出14-3-3 protein ε、S100A9等蛋白在食管癌变时差异表达，蛋白印记结果证实14-3-3 protein ε、S100A9的表达量在食管癌变时分别上调和下调。结论：激光捕获显微切割是蛋白质组研究中的一个突破性的技术，可以有效地解决组织异质性的问题；本实验检测到的差异蛋白例如14-3-3 protein ε、S100A9可能成为鉴别食管癌组织和正常食管上皮特异性的分子标记物。     

Genomic imbalances in esophageal squamous cell carcinoma identified by molecular cytogenetic techniques

This review summarizes the chromosomal changes detected by molecular cytogenetic approaches in esophageal squamous cell carcinoma (ESCC), the ninth most common malignancy in the world. Whole genome analyses of ESCC cell lines and tumors indicated that the most frequent genomic gains occurred at 1, 2q, 3q, 5p, 6p, 7, 8q, 9q, 11q, 12p, 14q, 15q, 16, 17, 18p, 19q, 20q, 22q and X, with focal amplifications at 1q32, 2p16-22, 3q25-28, 5p13-15.3, 7p12-22, 7q21-22, 8q23-24.2, 9q34, 10q21, 11p11.2, 11q13, 13q32, 14q13-14, 14q21, 14q31-32, 15q22-26, 17p11.2, 18p11.2-11.3 and 20p11.2. Recurrent losses involved 3p, 4, 5q, 6q, 7q, 8p, 9, 10p, 12p, 13, 14p, 15p, 18, 19p, 20, 22, Xp and Y. Gains at 5p and 7q, and deletions at 4p, 9p, and 11q were significant prognostic factors for patients with ESCC. Gains at 6p and 20p, and losses at 10p and 10q were the most significant imbalances, both in primary carcinoma and in metastases, which suggested that these regions may harbor oncogenes and tumor suppressor genes. Gains at 12p and losses at 3p may be associated with poor relapse-free survival. The clinical applicability of these changes as markers for the diagnosis and prognosis of ESCC, or as molecular targets for personalized therapy should be evaluated.     

食管鳞状细胞癌中MMP-9、CD-147的表达及意义

目的：探讨基质金属蛋白酶9（MMP-9）和CD-147在食管鳞状细胞癌的表达及其与肿瘤浸润转移的关系。方法：应用免疫组化S-P法观察57例食管鳞癌组织中MMP-9、CD-147的表达,并探讨其与食管鳞癌临床病理资料的关系。结果：MMP-9、CD-147在癌组织中的阳性表达率分别为82．46％,64．91％;在癌旁组织中的阳性表达率分别是29．82％,8．77％。在食管鳞癌中,MMP-9及CD-147的表达均与食管鳞癌的浸润深度有关,与分化程度无明显关联;有淋巴结转移的病例阳性表达率明显高于无淋巴结转移组。结论：MMP-9及CD-147的表达均与食管鳞癌的浸润深度及淋巴结转移有关。     

Upregulation of Fucosyltransferase 3, 8 and protein O-Fucosyltransferase 1, 2 genes in esophageal cancer stem-like cells (CSLCs)

Recently, studies have shown that Fucosylation plays an important role in the invasion and metastatic process of CSLCs. Understanding the expression pattern of fucosyltransferase (FUT) genes may help to suggest better-targeted therapy strategies for esophageal squamous cell carcinoma (ESCC). The study aimed to address the expression pattern of FUT gene variants in esophageal CSLCs and parental adherent cells. Sphere formation method was used to enrich CSLCs. Expression of FUT genes was examined in tumor sphere and parental adherent cells using the RT-PCR method and then relative expression of detected variants was performed by the Real-Time PCR method in both groups. The detected FUTs, also, were assessed in fresh ESCC tumors and the matched healthy controls. Analysis of The cell surface carbohydrate Lewis x (LeX, CD15) was performed by flow cytometry. Molecular analysis showed that the expression of FUT 3, 8 and POFUT1, 2 genes in tumorsphere were significantly higher than parental adherent cells. Analysis of fresh ESCC tumor tissues and the matched healthy controls showed that FUT8 and POFUT1, 2 genes in contrast to FUT 3 have higher expression in tumor tissues than controls. Flow cytometric analyses revealed that tumorsphere and their parent cells do not differ significantly in Lewis x surface marker. The present study showed that FUT 3, 8 and POFUT1, 2 genes upregulated in esophageal CSLCs in comparison to adherent cells. Understanding the expression pattern of FUT gene variants may help to suggest better-targeted therapy strategies for ESCC.     

Abnormal chromosome 8 copy number in cytological smears from breast carcinomas detected by means of fluorescence in situ hybridization (FISH)

Numerical change in chromosome 8 is an acquired abnormality associated with high clinical stage and may be involved in the conversion of carcinoma in situ in the breast to invasive carcinoma. Fine needle aspiration smears from 53 cases of breast carcinoma were hybridized with centromeric probes for chromosome 8 and the X chromosome. Thirty-eight cases revealed chromosome 8 copy gain. Of the 45 grade II and III tumours, 28 showed polysomy (>3 signals) and six showed trisomy. Of the eight grade I tumours, four were trisomic, none were polysomic. There were only two cases of chromosome 8 copy loss (one each of grade I and III). X chromosome polysomy was also a frequent finding although the signal counts were similar to those for chromosome 8 in only a few cases. Chromosome 8 polysomy occurs frequently in breast carcinoma and high copy number (>3) is associated with high malignancy grade.     

Enhanced Expression of Fibroblast Growth Factor Receptor 3 IIIc Promotes Human Esophageal Carcinoma Cell Proliferation

Deregulated expression of fibroblast growth factor receptors (FGFRs) and their ligands plays critical roles in tumorigenesis. The gene expression of an alternatively spliced isoforms of FGFR3, FGFR3IIIc, was analyzed by RT-PCR in samples from patients with esophageal carcinoma (EC), including esophageal squamous cell carcinoma (ESCC) and adenocarcinoma (EAC). The incidence of FGFR3IIIc was higher in EC [12/16 (75%); p=0.073] than in non-cancerous mucosa (NCM) [6/16 (38%)]. Indeed, an immunohistochemical analysis of early-stage ESCC showed that carcinoma cells expressing FGFR3IIIc stained positively with SCC-112, a tumor marker, and Ki67, a cell proliferation marker, suggesting that the expression of FGFR3IIIc promotes cell proliferation. We used EC-GI-10 cells endogenously expressing FGFR3IIIc as a model of ESCC to provide mechanistic insight into the role of FGFR3IIIc in ESCC. The knockdown of endogenous FGFR3 using siRNA treatment significantly abrogated cell proliferation and the overexpression of FGFR3IIIc in cells with enhanced cell proliferation. EC-GI-10 cells and ESCC from patients with EC showed endogenous expression of FGF2, a specific ligand for FGFR3IIIc, suggesting that the upregulated expression of FGFR3IIIc may create autocrine FGF signaling in ESCC. Taken together, FGFR3IIIc may have the potential to be an early-stage tumor marker and a molecular target for ESCC therapy.     

NOB1在食管鳞状细胞癌中的表达及临床意义

目的研究NOB1基因在食管鳞状细胞癌（Esophageal squamous cell carcinoma,ESCC）中的表达及临床意义。方法利用免疫组织化学SP法检测59例ESCC及其相应（50例）的远端正常食管黏膜组织中NOB1的表达。结果 ESCC中NOB1的阳性率为71%,正常食管黏膜鳞状上皮中的阳性率为26%,两组比较,差异有统计学意义（P〈0.05）。NOB1的表达与ESCC的分化程度及淋巴结转移相关,与患者的性别,年龄以及肿瘤浸润深度无关。结论 NOB1在ESCC中表达升高,可能在ESCC发生发展过程中起重要作用。