间期细胞遗传学及其在实体瘤分子诊断和发病机制研究中的应用

间期细胞遗传学即以标记的核酸探针用原位杂交的方法在完整细胞核中观察染色体的畸变。荧光原位杂交（ＦＩＳＨ）不仅在基因组物理制图上立下汗马功劳,随着比较基因组技术的进展,人类基因组全部基因的分离克隆,基因表达谱的发展,将在实体瘤的染色体畸变诊断以及分子机制研究方向发挥更大的用途。间期细胞遗传学也将连接分子遗传学和细胞病理学的桥梁学科。     

间期细胞遗传学及其在实体瘤分子诊断和发病机理研究中的应用

间期细胞遗传学即以标记的核酸探针用原位杂交的方法在完整细胞核中观察染色体的畸变。荧光原位杂交（ＦＩＳＨ）不仅在基因组物理制图上立下汗马功劳，随着比较基因组技术的进展，人类基因组全部基因的分离克隆，基因表达谱的发展，将在实体瘤的染色体畸变诊断以及分子机制研究方面发挥更大的用途。间期细胞遗传学也将成为连接分子遗传学和细胞病理学的桥梁学科。     

应用羊水细胞间期荧光原位杂交技术诊断胎儿Down综合征

目的 建立以未培养的羊水间期细胞直接制片进行荧光原位杂交(FISH)的简便方法并应用于Down综合征产前诊断。方法 Down综合征患者外周血染色体标本,以及10例产前诊断的孕妇,在妊娠16～20周行羊膜腔穿刺术,羊水细胞制片并以胃蛋白酶进行简单处理后,用位于染色体21q22.3区域的人类基因组BAC克隆探针进行荧光原位杂交。结果 全部杂交成功,信号强,背景低。讨论 该技术简便、稳定、易掌握,适用于检测胎儿Down综合征的快速产前诊断。     

荧光原位杂交技术与细胞遗传学分析在慢性淋巴细胞白血病检测中的评价应用

目的比较常规细胞遗传学分析（CCA）及荧光原位杂交（FISH）两种技术在慢性淋巴细胞白血病（CLL）染色体异常检测中的灵敏度和特异性。方法CCA法采用骨髓短期培养法,核型分析采用R带技术。FISH法采用间期FISH。结果采用CCA法,CLL患者的染色体异常检出率为35．71％,而采用FISH技术染色体异常检出率为64．2％。结论采用组合探针的FISH更为敏感和特异。     

线粒体DNA与人宫颈细胞癌变的关系

目的探讨线粒体DNA与人宫颈细胞癌变的关系.方法采用PCR方法制备地高辛标记的3条人mtDNA探针,对一株人宫颈癌Hela细胞系、1例原代培养人皮肤成纤维细胞和24例子宫颈癌患者的活检癌组织及癌旁宫颈组织的染色体或间期细胞核进行了荧光原位杂交分析.结果 3条mtDNA探针在原代培养人皮肤成纤维细胞染色体或间期核中均未出现阳性杂交信号;而在人宫颈癌Hela细胞和24例肿瘤组织标本的部分染色体或间期细胞核中均出现了阳性杂交信号.而在癌旁组织的部分染色体或间期细胞核中虽也出现了阳性杂交信号.但与肿瘤组织标本相比有显著性差异.结论表明癌细胞核基因组中存在mtDNA的同源序列,这些同源片段的出现可能与细胞癌变有关.     

荧光原位杂交技术产前诊断唐氏综合征

目的应用荧光原位杂交(fluorescence in situ hybridization,FISH)技术,简便、快速、准确产前诊断唐氏综合征.方法采用染色体特殊位点的特异性探针(LSI21)对30例羊水细胞和6例绒毛细胞标本进行未培养间期细胞荧光原位杂交.结果检测出21三体儿1例, 21三体和二倍体的嵌合体1例.检测结果与染色体核型分析及随访相符.结论 FISH技术应用于未培养间期细胞产前诊断唐氏综合征,可使诊断时间提早到孕50～70d,并且具有简便、快速、准确、灵敏度高、特异性强的独特优势.     

FISH在产前诊断及遗传咨询中的应用

荧光原位杂交(FISH)是近年发展起来的一种分子生物学、细胞遗传学及免疫学技术相结合的全新技术,在基础生物学领域和临床研究中得到广泛应用。FISH是一种非放射原性杂交法,它利用特殊的荧光素标记DNA探针,在染色体制片、细胞滴片或组织切片上进行DNA杂交,经免疫荧光显色,可以检测细胞内DNA或RNA存在与否,既可以显示染色体中期分裂相,又能显示未培养的间期核[1]。可用于性别鉴定、染色体非整倍体诊断、染色体结构异常诊断、基因定位、间期细胞遗传学及肿瘤遗传学方面的研究。一、FISH在产前诊断中的应用1.用于绒毛样本(CVS)的染色体分析绒毛细胞是胚胎外胚层细胞,其遗传信息与胎儿相同。因为绒毛膜绒毛取材获取的绒毛量相对较少,有时不能得到足够可够分析的染色体中期分裂相,给诊断带来困难,故同时应用FISH进行分析以提供更多的遗传信息,可及早做出诊断。孕早期的早检查、早诊断,可及时发现胎儿染色体异常,使孕妇在早期终止妊娠。当孕早期对绒毛检测无结果时,还可进一步于孕中期羊水细胞检测。Toth[2]等人为研究FISH技术用于诊断Down综合征,取331份标本,至少分析了50个细胞,无1例假阳性与假阴性。间期细胞FISH所分析...     

未培养外周血间期分子细胞遗传学研究

近年来，随着分子生物学技术的发展及制备探针技术的提高，荧光原位杂交（ｆｌｕｏｒｅｓｃｅｎ－ｃｅｉｎｓｉｔｕｈｙｂｒｉｄｉｚａｔｉｏｎ，ＦＩＳＨ）技术已被广泛应用于遗传学及其它相应学科中。本文选用Ｄ２１Ｚ１／Ｄ１３Ｚ１ＤｘＺ１探针与未培养的外周血间期白细胞进行荧光原位杂交来检测染色体非整倍体。结果表明：通过统计间期核中的杂交信号，能准确检出２１三体及Ｔｕｒｎｅｒ＇ｓ综合征，与常规细胞遗传学结果相符，该法省去了复杂的细胞培养过程和周期，不仅使常规的细胞遗传学得以简化，而且为产前诊断染色体非整倍体及肿瘤病因学的研究提供了捷径     

用荧光原位杂交技术产前诊断唐氏综合征

目的 用荧光原位杂交技术（Fluorescence in situ hybridization,FISH)快速产前诊断唐氏综合征。方法 采集23名孕妇14～24周的羊水标本，应用荧光标记的针对21号染色体特殊位点的探针（locus-sperifie probe,LSI）及X/Y染色体着丝粒探针（centromeric probe,CEP）对未培养的羊水间期细胞进行FISH；同步进行羊水细胞培养，行常规细胞遗传学染色体核型分析，以核型分析为标准，对FISH技术进行评价。结果 23份标本发生母血污染2例，培养失败1例，将其余20份羊水标本的FISH杂交结果与其染色体核型分析结果进行了比较。FISH分析羊水间期细胞性染色体数目正常者19例（XX11例，XY8例）与羊水中期细胞染色体核型分析结果一致，有1例羊水间期细胞FISH结果为X/XY，染色体核型分析结果为46，XY，因此，FISH与染色体核型分析结果的符合率为95%（19/20）；LSI21探针的FISH结果中21号染色体数目异常者1例，核型分析为典型的21三体，取脐血行G显带染色体核型分析得以验证为47，XY， 21。产前诊断染色体正常者追踪至分娩，新生儿行外周血染色体检查结果皆为正常核型。结论 荧光原位杂交技术可用于羊水间期细胞快速产前诊断唐氏综合征。     

荧光原位杂交技术研究在染色体异常中的应用

目的建立稳定的荧光原位杂交技术并用于染色体异常的研究.方法用地高辛标记的PY3.4探针、X-α着丝粒探针、生物素标记的特异区域单拷贝序列21q22.3探针对3例正常男性、3例正常女性及2例turner综合征、2例21三体综合征标本进行了原位杂交.结果间期细胞和分裂相中可见特异的荧光点.结论 FISH技术不仅可用于中期分裂相,还可在间期细胞显示杂交信号,是当今的一项分子细胞遗传学先进技术.     

应用荧光原位杂交检测人喉癌中EGFR基因扩增

目的 检测人喉癌Ｈｅｐ－２细胞系和５个喉癌组织中表皮生长因子受体（ｅｐｉｄｅｒｍａｌ ｇｒｏｗｔｈ ｆａｃｔｏｒ ｒｅｃｅｐｔｐｒ,ＥＧＦＲ）基因扩增。方法 荧光原位杂交技术。结果 在Ｈｅｐ－２细胞和２例喉癌组织中期染色体和间期细胞核中,检测到明显的集中成簇和多个斑点分散排布的杂效信号,另３例喉癌组织间期细胞核中杂交信号未见增强或数目增加。结论 以正常二倍休 色体和间期细胞核为对照,在喉癌Ｈｅｐ－     

Characterization of quantitative chromosomal abnormalities in renal cell carcinomas by interphase four-color fluorescence in situ hybridization

Renal cell carcinomas (RCC) in adults are histologically heterogeneous solid tumors with specific chromosomal abnormality patterns included in the World Health Organization (WHO) classification. To overcome some of the drawbacks of cytogenetic and comparative genomic hybridization (CGH) analyses, we designed a first-generation cytogenetic diagnostic test using four-color fluorescence in situ hybridization (FISH) on interphase nuclei. We selected 51 bacterial artificial chromosome and P1-derived artificial chromosome clones covering 17 chromosomal regions involved in the abnormalities of the adult RCC histologic subtypes. An initial set of probes allowed the identification of clear-cell RCC, papillary RCC, and other RCC on a single slide. A second test allowed the detection of additional chromosomal abnormalities or aberrations specific to chromophobic RCC and oncocytomas. We tested 25 cases of RCC, and the results were in agreement with those of cytogenetic techniques and/or CGH methods. The techniques appeared to be very sensitive, because small tumoral cell clones that were undetected by other cytogenetic methods were identified with this method. It was concluded that the multicolor FISH test was specific and sensitive, easy to perform, and could be part of the investigation process in RCC.     

High frequency of tissue-specific mosaicism in Turner syndrome patients

Interphase fluorescent studies of X chromosome aneuploidy in cultured and uncultured blood lymphocytes and oral mucosa epithelial cells using X centromere-specific DNA probe in addition to standard karyotype analysis were performed in 50 females with a clinical suspicion of Turner syndrome. All the patients were previously screened for the presence of 'hidden' Y chromosome mosaicism, using the primers DYZ3 and DYZ. The use of fluorescence in situ hybridization (FISH) analysis of interphase nuclei of tissues from different germ layers (lymphocytes from mesoderm and buccal epithelial cells from ectoderm) improves the accuracy of detection of low-level mosaicism. FISH studies on interphase nuclei revealed that 29% of patients with a pure form of monosomy X detected by metaphase analysis are, in fact, mosaics. The level of cells with the normal chromosomal constitution in lymphocytes of these cases as a rule was low, ranging from 3 to 18%, with an average of 7%. Two false-positive cases and one false-negative case of X monosomy mosaicism determined by standard cytogenetic approach were detected using FISH analysis. The majority of patients (92%) with mosaic form of Turner syndrome have considerable tissue-specific differences in levels of X aneuploidy. Our data indicate that in cases when mosaic aneuploidy with low-level frequency is questionable (approximately 10% and lower), the results of standard metaphase analysis should be supplemented with additional FISH studies of interphase nuclei. Tissue-specific differences in contents of different cell lines in the same patients point to the necessity of studying more than one tissue from each patient.     

应用荧光原位杂交产前诊断未培养羊水细胞非整倍体

目的探讨荧光原位杂交(fluorescenceinsituhybridization,FISH)诊断未培养羊水细胞非整倍体的临床应用价值。方法对55例孕16～32周未培养羊水细胞进行FISH快速产前诊断,应用多色FISH对另4条染色体(X、Y、13号和18号)进行检测。以经母腹穿刺取胎血常规核型分析作为FISH检测结果对照。结果被检55例羊水未培养细胞均获得诊断结果,发现两例异常胎儿。1例为标准型21三体;另1例为21三体嵌合体。FISH检测与常规核型分析结果一致。结论FISH检测未培养羊水细胞非整倍体具有快速、简便、所用样本量少的优势,结果准确可靠,可达到产前诊断要求,有较大临床应用价值。     

一种能精确检测人间期细胞核中21号染色体拷贝数的DNA探针

目的制备能精确检测人类间期细胞核中２１号染色体拷贝数的ＦＩＳＨ探针。方法利用万能引物ＰＣＲ法,从定位于人２１ｑ１１的ＹＡＣ克隆８８１Ｄ２分离制备ＤＮＡ探针,并用于与８例正常人和５例２１三体患者的外周血淋巴细胞中期相和间期核,及经细胞松弛素Ｂ（ｃｙｔｏｃｈａｌａｓｉｎＢ）诱导的双核细胞进行ＦＩＳＨ分析。结果该探针具有以下特点：（１）长度集中于３５０～７５０ｂｐ;（２）其靶序列位于２１号染色体长臂上且紧靠着丝粒;（３）特异性强;（４）杂交信号明亮,容易辨认;（５）对中期相及间期细胞核中２１号染色体的检出率分别高达９９．６０％和９８．４０％。结论制备的ＤＮＡ探针能精确检测人类间期细胞核中２１号染色体的拷贝数,且适用于细胞分裂时２１号染色体分离情况的研究     

Microfluidic channel‐assisted screening of hematopoietic malignancies

A simple microfluidic fluorescence in situ hybridization (FISH) device allowing accurate analysis of interphase nuclei in 1 hr in narrow channels is presented. Photolithography and fluorosilicic acid etching were used to fabricate microfluidic channels (referred to as FISHing lines) that allowed analysis of 10 samples on a glass microscope slide 0.2 µl of sample volume was used to fill a micro‐channel, which resembled a 250‐fold reduction compared to conventional FISH. FISH signals were comparable to conventional FISH, with 50‐fold less probe consumption and 10‐fold less time. Cells were immobilized in single file in channels just exceeding the diameter of the cells, and were used for minimal residual disease (MRD) analysis. To test the micro‐channels for application in FISH, MRD was simulated by mixing K562 cells (an established chronic myeloid leukemia cell line) carrying the BCR/ABL fusion gene across 1:1 to 1:1,000 Jurkat cells (an established acute lymphoblastic leukemia cell line). The limit of detection was seen to be 1:100 cells and 1:1,000 cells for FISHing lines and conventional FISH, respectively; however, the conventional method seemed to over‐score the presence of K562 cells. This may in part be attributed to FISHing lines practically eliminating the chance of duplicate screening of cells and hastened the time of screening, enhancing scoring of all cells within the channels. This was compared to 1 in 500 cells on the slide being analyzed with the conventional FISH. © 2013 Wiley Periodicals, Inc.     

Application of CARD-ISH for Assessment of Numerical Chromosome Aberrations in Interphase Nuclei of Human Tumor Cells

In this study based on the study of a centromeric DNA probe specific for chromosome 8 the authors standardized a method for the enzymatic detection of specific chromosome copy numbers on interphase nuclei from tumor tissue samples. Since the in situ hybridization (ISH) of chromosome 8 specific probe was revealed with a catalyzed reporter deposition (CARD), which allows a high amplification of the hybridization signal, the method was designated as CARD-ISH. This method has been standardized on interphase nuclei isolated from clinical samples of pituitary adenomas, as well as on human normal lymphocytes. On the same samples, they also evaluated chromosome 8 copy number distribution by FISH. Comparison between CARD-ISH and FISH results showed no significant differences between the two methods, proposing CARD-ISH as a reliable alternative to FISH for chromosome numerical aberration assessment in laboratories that do not have specific facilities for epifluorescence microscopy or cytogenetics and that need a long-term storage of slides that had been used for diagnostic purposes. Int J Surg Pathol 8(3):201-206, 2000     

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。     

Detection of chromosome aberrations in interphase nuclei using fluorescence in situ hybridization technique.

We report here several experiences of interphase cytogenetics, using fluorescence in situ hybridization (FISH) technique, for the detection of chromosome aberrations. FISH, using alpha satellite specific probes of 18, X, Y chromosomes, was done in interphase nuclei from peripheral blood of patients with Edwards'' syndrome, Klinefelter''s syndrome and Turner''s syndrome with healthy male and female controls, respectively. The distributions of fluorescent signals in 100 interphase nuclei were well correlated with metaphase findings. Nowadays FISH plays an increasingly important role in a variety of research areas, including cytogenetics, prenatal diagnosis, tumor biology, gene amplification and gene mapping.     

Formalin-fixed and paraffin-embedded nodal non-Hodgkin's lymphomas demonstrate the same chromosome changes as those found in frozen samples: a comparative study using interphase fluorescence in situ hybridization.

Cytogenetic studies in lymphomas classically require fresh or frozen tissue, whereas in many instances only paraffin-embedded biopsies are available. We applied an interphase FISH assay on nuclei extracted from thick paraffin sections to determine accuracy of molecular cytogenetics in such samples. Twenty-three lymphoma samples and 4 reactive lymph nodes were tested with various commercially available DNA probes, and hybridization patterns were compared with those obtained on frozen nuclei counterparts. Successful hybridization with all probes tested was observed for 23/27 (85%) paraffin-embedded tissues and for all (100%) frozen samples, and cut-off levels defining positivity were superimposable for both situations. Chromosome changes were detected in the same way, without any false-positive or false-negative cases. Hybridization signals observed on dewaxed samples were either those classically expected to define the relevant chromosome change or were atypical: all atypical changes could be demonstrated also into nuclei from the frozen counterpart. Moreover, all typical and atypical chromosome changes observed on frozen nuclei were also detected in paraffin-embedded tissues. Our study shows that our interphase FISH assay performed on paraffin-embedded samples is a valuable alternate to conventional methods to ascertain diagnosis of lymphomas as to include patients into therapeutic trials.