双探针显色原位杂交技术检测HER2基因的应用

HER2是调节细胞生长过程中起关键作用的跨膜蛋白,编码基因定位于17q21.乳腺癌患者中,约20%～30%存在HER2基因或蛋白的过表达,导致肿瘤细胞过度增殖,使肿瘤的恶性程度远高于HER2未过度表达的患者.近年来,HER2的状态被公认为是判断乳腺癌预后风险和决策靶向治疗的重要指标.     

荧光原位杂交及其在肿瘤生物学中的应用

荧光原位杂交（ＦＩＳＨ）是一种应用荧光物质依靠核酸探针杂交原理在核中或染色体上显示ＤＮＡ序列位置的方法。ＦＩＳＨ具有快速、安全、经济、灵敏度高、特异性强等优点，在中期及间期细胞均可检测ＤＮＡ序列及其变化，广泛应用于细胞遗传学、产前诊断、肿瘤生物学、核组成、基因定位、基因扩增等领域。细胞内染色质含量的不平衡是引起肿瘤的根本原因，细胞遗传学上表现为染色体数目或结构异常，分子水平上表现为ＤＮＡ片段的扩增     

应用地高辛标记的cRNA探针原位杂交检测胰岛素mRNA

成年雄性Ｗｉｔａｒ大鼠，用地高辛标记的ｃＲＮＡ探针在胰腺切片上进行原位杂交，以检测胰岛Ｂ细胞胰岛素基因表达产物ｍＲＮＡ；同时并用免疫组织化学法显示相邻切片胰岛素免疫反应阳性细胞，进行比较，胰腺经４％多聚甲醋灌注固定，经Ｂｏｕｉｎ液再固定２０ｈ，常石蜡包埋和切片，经胰岛素ｃＲＮＡ探针杂交的胰腺切片中胰岛Ｂ细胞和核仁呈阳性反应，阳性信号为蓝色颗粒，胰岛其他细胞及胰腺泡细胞为阴性，方法照切片中均无阳性信     

荧光原位杂交检测乳腺癌HER2基因状态

目的 探讨荧光原位杂交（FISH）,与免疫组织化学（IHC）染色（3＋）和（2＋）检测HER2基因状态的结果的一致性、导致两者差异的可能原因及FISH检测HER2基因状态的必要性和可行性。方法 采用PathVysion^TM探针试剂盒,以FISH方法,对28例IHC EnVision法染色分别为（3＋）、（2＋）、（1＋）和阴性（-）的乳腺癌石蜡切片标本进行HER2基因状态的检测。结果 HER2表达（3＋）的12例标本中,10例HER2基L大1扩增,其中2例为17号染色体多体,另2例无扩增的病例均为多体;IHC为（2＋）的10例标本中,7例为HER2基因扩增,其中1例为多体,另3例无扩增病例中2例为多体;IHC为（1＋）的3例标本均无HER2基因扩增,其中1例为多体;IHC（-）的3例标本均无基因扩增,其中1例为多体。结论 IHC是初步筛查HER2状态的首选方法;因IHC（2＋）与FISH检测结果差异较大,所以这类患者应做FISH确诊;IHC（3＋）存在假阳性,主要原因可能是由于17号染色体非整倍体,必要时这类患者也应做FISH。     

双色荧光原位杂交检测c-erbB-2基因扩增及应用

ｃｅｒｂＢ2(ＨＥＲ2ｎｅｕ)癌基因编码一种跨膜酪氨酸激酶受体蛋白,该蛋白与表皮生长因子受体家族有广泛的同源性。有研究表明,ｃｅｒｂＢ2蛋白与某些实体肿瘤细胞间的信号传递有关。ｃｅｒｂＢ2基因的过度表达可影响细胞受体的激酶活性,从而影响或改变细胞的生长、增殖特性。经研究发现,ｃｅｒｂＢ2原癌基因的过度扩增或蛋白异常表达与乳腺癌、胃癌等实体瘤的发生、发展及预后密切相关,而且具有ｃｅｒｂＢ2基因过度表达的乳腺癌可对一种人源化的重组抗ｃｅｒｂＢ2抗体(Ｈｅｒｃｅｐｔｉｎ)有较好的治疗反应[1,2]。因此,ｃｅｒｂＢ2基因过…     

HER-2/neu基因探针的制备及特异性分析

目的 筛选、制备乳腺癌标志基因--HER-2/neu的标记探针,利用间接荧光原位杂交(FISH)技术,探索其临床应用价值.方法 通过文库构建、多级筛选、PCR鉴定、序列分析等技术获得HER-2/neu基因组片段,缺口转移法制备生物素标记探针,与生物素-亲和素放大系统和多种荧光显色系统组合,针对病理标本、细胞涂片进行间接FISH检测.结果 制备的HER-2/neu基因探针可以特异性地检测乳腺癌阳性、胃癌阳性病理片,也可以特异性检测阳性、阴性细胞标本,并与不同的荧光显色系统结合使杂交信号可以选择性地呈现绿色或红色荧光.结论 高特异性的HER-2/neu基因探针和灵活的显色系统为FISH的临床应用奠定了基础.     

荧光原位杂交的原理及在产前诊断中的应用

荧光原位杂交（fhorescenceinsituhybridization．FISH）是近几年来由细胞遗传学、分子生物学及免疫学技术相结合而产生的一种新技术。由于其兼备了细胞遗传学与分子生物学技术，克服了使用单一细胞遗传学技术的局限性，故在产前诊断、基因定位、肿瘤细胞遗传学、临床遗传病检测等研究领域显示出重要应用价值。下面就有关荧光原位杂交的原理及在产前诊断中的应用作一简单介绍。所谓FISH即利用生物素（biotin）等标记的已知外源核酸作为探针，通过特定的碱基互补序列与玻片（包括组织切片、细胞滴片、染色体制片）上待测核酸进行原位杂交…     

聚合酶链反应掺入生物素探针在细胞原位杂交中的应用

在PCR反应体系中，以Bio－11-dUTP部分代替dTTP，做人类白细胞抗原（HLA）－DQA、DQB、DRB及DPB等位点的聚合酶链反应（PCR）。扩增产物有较高的生物素掺入。以此为生物素标记探针应用于细胞原位杂交检查诸位点的mRNA表达水平，取得了满意的结果。此方法简化了基因探针的制备，可应用于任意目的mRNA和DNA的分子杂交。一、材料和方法1．模板DNA的制备：随机取正常人血标本sml，1／10体积的2％乙二胺四乙酸（EDTA）抗凝，经快速法分离染色体DNA「‘’ZHLA－DQA位点的PCR掺入探针的制备：依文献「Zj进行。同时以未掺入…     

小鼠Y染色体RNA探针的标记和应用

目的标记小鼠Y染色体RNA探针,检验该探针在检测外周血涂片和脑组织切片Y染色体阳性细胞的效率。方法以小鼠Y染色体重复序列pY353/B cDNA为模板标记RNA探针。以雌性小鼠为对照,用原位杂交法检测小鼠外周血涂片Y染色体阳性细胞;用荧光原位杂交法检测雄性小鼠脑组织切片Y染色体阳性细胞。结果Y染色体原位杂交法在外周血涂片Y染色体阳性的检测灵敏度和特异度均为100％,在脑切片检测Y染色体阳性细胞的灵敏度和特异度分别为85．67％和100％。结论小鼠Y染色体RNA探针的原位杂交和荧光原位杂交法检测Y染色体阳性细胞有较高的灵敏度和特异度。     

应用荧光原位杂交技术进行孕早期产前诊断的研究

目的探讨荧光原位杂交(FISH)技术用于产前诊断绒毛间期细胞染色体非整倍体异常的临床应用价值。方法采用FISH技术对我院计划生育门诊人工流产的53例40-84天的流产绒毛进行5条染色体(21、13、18、X和Y)的快速检测。同时,将绒毛接种、培养,进行常规细胞染色体核型分析,作为FISH检测结果的对照。结果被检测的53例样本中,用FISH检测,均获得诊断结果,检测成功率为100%,而常规细胞染色体核型分析,则只有51例获得诊断结果,有2例未培养成功,检测成功率为96.23%(51/53)。FISH检测结果与常规细胞染色体核型分析结果有2例不相符合,结果符合率为96.08%(49/51)。结论应用FISH技术检测未培养绒毛间期细胞染色体数目异常,具有快速,简便,使用样本量少等优势,具有一定的临床应用价值。     

应用荧光原位杂交技术进行早期难免流产胚胎绒毛细胞染色体检测的研究

目的探讨荧光原位杂交（FISH）技术用于早期难免流产绒毛间期细胞染色体非整倍体异常的临床应用价值。方法采用FISH技术对20例我院住院难免流产病人的流产绒毛进行5条染色体（21、13、18、X和Y）的快速检测。结果被检测的20例样本中,用FISH检测,均获得诊断结果,检测成功率为100%,FISH检测结果中有8例异常,异常率为40%,其中XO是难免流产中胚胎中最多的异常。结论应用FISH技术检测未培养绒毛间期细胞染色体数目异常,具有快速,简便,使用样本量少等优势,具有一定的临床应用价值。     

荧光原位杂交检测在产前诊断中的临床价值

目的探讨运用羊水间期细胞开展荧光原位杂交检测在产前诊断中的临床价值。方法运用FISH技术对110例孕16～24周孕妇的羊水间期细胞进行检测,每例均进行常规染色体核型分析。结果运用FISH法,所有样本均在19h内获得检测结果,除2例羊水培养失败外,所有样本均取得染色体核型分析报告结果。两种方法均检出特氏综合征、21-三体综合征各1例,所有样本的两种方法检测结果均互相符合。常规染色体核型分析有2例异常,因缺乏相应DNA探针。FISH法未能检出。结论运用羊水间期细胞开展荧光原位杂交检测,缩短了诊断时间,缓解了孕妇及家属因等待检测结果所产生的焦虑心情。检测程序简单,结果判定明确,在产前诊断实施过程中具有重要的临床价值。     

Chromosomal localization of the human myeloperoxidase gene by in situ hybridization using oligonucleotide probes

Oligonucleotide probes have been used to map the myeloperoxidase (MPO) gene locus to chromosome bands 17q21-22. This is in agreement with results reported using conventional cDNA probes. No evidence for the existence of a second MPO gene locus was obtained. Six synthetic 72-base oligonucleotides, corresponding to different exon regions of the MPO gene, were tritium-labeled and used as in situ hybridization probes. Synthetic oligonucleotide probes offer a useful alternative to conventional DNA probes for gene mapping.     

Identification of the origin of centromeres in whole-arm translocations using fluorescent in situ hybridization with alpha-satellite DNA probes

We detected 2 patients with whole-arm translocations resulting in a derivative chromosome consisting of 18q and 21q. Because the breakpoints were near the centromere, classical cytogenetic techniques could not determine the centromeric origin of the derivative chromosomes. Using nonradioactive in situ hybridization with a chromosome 18 alpha-satellite DNA probe (D18Z1), the centromeres in the abnormal chromosomes were determined to be from chromosome 18. The abnormality in one patient resulted in monosomy 18p with a karyotype 45,XX, -18, -21, + der(18)t(18;21) (p11;q11)mat complement. The second patient with a 46,XX, -21, + der(18)t(18;21)(p11;q11) de novo karyotype had complete trisomy of 18q. In both cases the appropriate phenotype was observed.     

Application of fluorescent in situ hybridization with X and Y chromosome specific probes to buccal smear analysis

Conventional X- and Y-chromatin and fluorescent in situ hybridization (FISH) analysis based on X- and Y-chromosome specific probes were conducted from buccal smear, on 15 normal males, 15 normal females, and 9 cases suspected of sex chromosome anomalies. The proportion of X- and Y-chromatin in normal females and males was 12% ± 3% and 51.5% ± 4.9%, respectively, by the conventional X- and Y-chromatin procedure. The CEP-X/Y analysis by FISH for the same specimens provided a proportion of 98.8% ± 0.7% cells with XX signals in the normal females and 99.8% ± 0.4% cells with XY signals in the normal males. The FISH method was superior to the conventional procedure in nine cases suspected of sex chromosome anomalies, including one case of mosaicism. The results of CEP-X/Y will sometimes be false; it will not detect structural anomalies of sex chromosomes, and it is not intended to detect low level mosaicism. However, the test is useful for rapid screening of sex chromosome aneuploidy at a fraction of the cost for chromosome analysis. The FISH test is also appropriate to detect tissue specific sex chromosome mosaicism, especially if it is relatively high. This FISH test is best used as an adjunct to chromosome analysis whenever possible. © 1996 Wiley-Liss, Inc.     

Differentiation of micronuclei in mouse bone marrow cells: a comparison between CREST staining and fluorescent in situ hybridization with centromeric and telomeric DNA probes

Immunofluorescent staining (CREST) of kinetochore proteins andin situ hybridization (FISH) with centromeric DNA probes areable to distinguish between micronuclei (MN) containing laggingchromosomes or acentric fragments. Different frequencies ofsignal-positive MN induced by mitomycin C (MMC) were obtainedby Miller et al. (Mutagenesis, 6, 297–302, 1991) betweenCREST labelling and FISH with the mouse major-     

Chromogenic and fluorescent in situ hybridization in breast cancer

Fluorescent (FISH) and chromogenic (CISH) in situ hybridization have recently become part of the diagnostic armamentarium of breast pathologists. HER2 gene testing by FISH and/or CISH has become an integral part of the diagnostic workup for patients with breast cancer. In this era of high throughput technologies, these techniques have proven instrumental for the validation of results from microarray-based comparative genomic hybridization and for the identification of novel oncogenes and tumor suppressor genes. Furthermore, FISH and CISH applied to tissue microarrays have expedited the characterization of genomic changes associated with specific breast cancer molecular subtypes and the identification of novel prognostic and predictive markers. In this review, we provide in this review a critical assessment of CISH and FISH and the impact of the analysis of amplification of specific oncogenes (eg, HER2, EGFR, MYC, CCND1, and FGFR1) and deletion of tumor suppressor genes (eg, BRCA1 and BRCA2) on our understanding of breast cancer.