双色FISH检测人精子染色体非整倍体方法的建立

目的　建立用双色FISH检测人精子染色体非整倍体的方法。方法　采用双色荧光原位杂交 (FISH)方法取适量精子标本用EDTA/PBS处理 ,然后用二硫苏糖醇 (DTT) ,使精子去凝集。固定后滴片 ,然后与双色荧光直接标记探针杂交。结果　在OLYMPUS荧光显微镜下可以清楚看到精子头部的蓝色杂交信号 ,头部有 1个绿色荧光杂交信号的精子为X染色体精子 (X精子 ) ,有 1个红色荧光杂交信号的精子为Y染色体精子 (Y精子 )。精子头部有 2个荧光杂交信号的精子为染色体数目异常精子。结论　双色荧光原位杂交 (FISH)方法可以用于测定人精子染色体非整倍体率的变化。     

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     

21号染色体特异性柯斯质粒DNA克隆区域定位

为提高检测２１号染色体数目和结构异常的精确性，应用ｄｉｇｏｘｉｇｅｎｉｎ－１１－ｄＵＴＰ通过缺口平移法标记Ｄ１３Ｚ１／Ｄ２１Ｚ１和６个柯斯质粒（Ｃｏｓｍｉｄ）ＤＮＡ克隆，将标记后的Ｄ１３Ｚ１／Ｄ２１Ｚ１和６个柯斯质粒ＤＮＡ克隆双探针与正常二倍体细胞系染色体进行荧光原位杂交（ＦＩＳＨ），结合Ｑ显带方法将６个柯斯质粒ＤＮＡ克隆定位于２１ｑ２２；用柯斯质粒ＤＮＡ克隆探针与两个已知的平衡易位细胞系ＧＭ９５４２和８３２７Ｌ的染色体进行荧光原位杂交，检测并分析杂交信号在两个平衡易位细胞系染色体上的分布，结果表明柯斯质粒ＤＮＡ克隆精确定位于２１ｑ２２．２，６个柯斯质粒ＤＮＡ克隆定位结果一致。     

荧光原位杂交（FISH）技术

荧光原位杂交是以荧光标记取代同位素标记而形成的一种新的原位杂交方法，探针首先与某种介导分子结合，杂交后再通过免疫细胞化学过程连接上荧光染料。ＦＩＳＨ具有敏感、快速、能同时显示多种颜色等优点，不但能显示于中期分裂相中，还能显示于间期核中。目前ＦＩＳＨ经不断丰富和完善，已衍生成一个技术系列，包括原位杂交显带（ＩＳＨＢ）；ＦＩＳＨ基固定位；染色体原位抑制性杂交（ＣＩＳＳ）；染色体涂色；全Ｃｏｓｍｉｄ和Ｙ     

荧光原位杂交（FISH）技术

荧光原位杂交（ｆｌｕｏｒｅｃｅｎｅｅｉｎｓｉｔｕｈｙｂｒｉｄｉｚａｔｉｏｎ，ＦＩＳＨ）是以荧光标记取代同位素标记而形成的一种新的原位杂交方法，探针首先与某种介导分子（ｒｅｐｏｒｔｅｒｍｏｌｅｃｕｌｅ）结合，杂交后再通过免疫细胞化学过程连接上荧光染料。ＦＩＳＨ具有敏感、快速、能同时显示多种颜色等优点，不但能显示于中期分裂相中，还能显示于间期核中。目前ＦＩＳＨ经不断丰富和完善。已衍生成一个技术系列，包括原位杂交显带（ＩＳＨＢ）；ＦＩＳＨ基因定位（ＦＩＳＨｍａｐｐｉｎｇ）；染色体原位抑制性杂交（ＣＩＳＳ）；染色体涂色（ｃｈｒｏｍｏｓｏｍｅｐａｉｎｔｉｎｇ）；全Ｃｏｓｍｉｄ和ＹＡＣ的ＣＩＳＳ；以及反向染色体涂色（ｒｅｖｅｒｓｅｃｈｒｏｍｏｓｏｍｅｐａｉｎｔｉｎｇ）等。本文着重叙述在这方面的进展，对ＦＩＳＨ在细胞遗传学，基因定位和基因诊断三个方面的应用情况也作了简要说明。     

应用荧光原位杂交技术检测骨髓增生异常综合征患者的染色体数目畸变

应用荧光原位杂交技术检测骨髓增生异常综合征患者的染色体数目畸变潘涌薛永权过宇谢新阮长耿我们应用７号和８号染色体着丝粒特异性α－卫星ＤＮＡ探针，对１３例骨髓增生异常综合征（ＭＤＳ）患者进行了荧光原位杂交（ＦＩＳＨ）研究，并将结果和细胞遗传学发现相比较，...     

荧光原位杂交新技术在恶性血液病听研究进展

荧光原位杂交（ＦＩＳＨ）是以非放射性物质标记核酸探针，根据核酸杂交原理，在间期核和分裂中期染色体上检测特异性ＤＮＡ序列的一种新技术，由于它克服了放射性原位杂交（ＲＩＳＨ）的一些弊端，具有快速，准确，灵敏，经济等优点，现已应用于肿瘤生物学，产前诊断和基因突变等诸多领域，因恶性血液病常规制备染色较大难度，成功率不高，检出较低等问题，而ＦＩＳＨ技术既可以分析中期核染色体，又能用于间期染色质，故该技术对恶     

染色体涂染技术及其在染色体病诊断中的应用

染色体涂染（ｃｈｒｏｍｏｓｏｍｅｐａｉｎｔｉｎｇ）技术是用染色体特异性或染色体区带特异性ＤＮＡ文库作为探针池，与中期分裂相染色体（或间期核）进行荧光原位杂交（ｆｌｕｏｒｅｓｃｅｎｃｅｉｎｓｉｔｕｈｙｂｒｉｄｉｚａｔｉｏｎ，ＦＩＳＨ）和染色体原位抑制（...     

用荧光原位杂交方法检测肺癌印片标本中性染色体数目的异常

目的 研究肺癌细胞中性染色体拷贝数的异常。方法 取新鲜的肺癌手术标本作印片,利用双色荧光原位杂交（ＦＩＳＨ）技术对１６例肺癌印片标本（其中男性病例１２例,女性病例４例）中Ｘ染色体和Ｙ染色体数目进行检测。结果 发现１２／１６（７５％）的标本中有Ｘ染色体数目增多,其中女性患者３例,男性患者９例;在１２例男性患者中,有１例发生Ｙ染色体数目增多,有５例（４１．７％）发生Ｙ染色体数目减少。总计出现性染色体数     

肺癌胸水间期细胞遗传学异常变化的研究

目的 探讨肺癌胸水肿瘤细胞的间期细胞遗传学改变,并将其结果与传统细胞学结果进行比较。方法 用７号、１１号、１７号、Ｘ染色体着丝粒特异的ＤＮＡ探针,以２６例肺癌胸水标本进行双色荧光原位杂交（ｆｌｕｏｒｅｓｃｅｎｃｅ ｉｎ ｓｉｔｕ ｈｙｂｒｉｄｉｚａｔｉｏｎ,ＦＩＳＨ）结果 在１９例肺癌阳性胸水标本中,７号、Ｘ、１７号、１１号染色体出现超二倍体的病例分别为１６例（８４．２％）、１４例（７３．７％）、     

Microdissection of the Y chromosome and fluorescencein situ hybridization analysis of the sex chromosomes of lake trout,Salvelinus namaycush

Lake trout,Salvelinus namaycush, is one of the few salmonids with morphologically differentiated sex chromosomes. Genetic analysis suggested that the sex-determining region of this species lies on the short arm of the Y chromosome. The differential arm of the Y chromosome was microdissected and the resulting DNA amplified in a sequence-independent manner. Amplified DNA was biotin labeled as a probe for fluorescencein situ hybridization (FISH). Strong hybridization signals were seen covering defined regions of both the Y and X chromosomes. Homeologous chromosomes of the ancestrally tetraploid genome were not identified by FISH with the Y probe, indicating diploidization of this region of the genome.     

Analysis of unfertilized oocytes subjected to intracytoplasmic sperm injection using two rounds of fluorescence in-situ hybridization and probes to five chromosomes

Chromosomal aberrations are the major cause of pre- and post- implantation embryo wastage and some studies suggest that half of all human conception have a chromosomal abnormality. Analysis of gametes provides information on the origin of these chromosomal aberrations. The purpose of this study was to develop a reliable multi-probe fluorescence in-situ hybridization (FISH) procedure that would enable us to investigate aneuploidy in unfertilized oocytes subjected to intracytoplasmic sperm injection (ICSI). Oocytes were spread with HCl and Tween 20 solution, and then two rounds of triple-probe FISH were performed on each oocyte using directly-labelled centromeric probes: chromosomes 1, 7, 15 (overnight hybridization); chromosomes 1, X, Y (2 h hybridization). After the first round, the slides were counterstained and evaluated, and the positions of FISH signals were recorded. For the second round, the counterstain was removed and the second probe cocktail was applied. The chromosome 1 probe was an internal control for the two hybridization procedures, while the Y chromosome probe was used to detect sperm DNA. To evaluate the method, a total of 79 oocytes from 27 patients were studied. Of these, 67 (84.8%) were successfully spread and 97% of these oocytes exhibited discernible FISH signals. Upon lysis, oocytes exhibited one or more DNA fragments (mean 1.9, range 1-3). Of the 65 analysable oocytes, 17 (26.2%) displayed a normal haploid chromosome constitution with paired spots for the two chromatids. A further 23 oocytes (35.4%) showed an ambiguous chromosome complement due to an abnormal number of DNA fragments which may have resulted from loss of DNA during spreading or to an abnormal oocyte, while 25 oocytes (38.4%) displayed aneuploidy for one or more of the chromosomes studied. In conclusion, this new approach is a quick and efficient method with which numerical chromosomal abnormalities in human oocytes can be studied; interpretation of the patterns of DNA fragments and FISH signals requires further clarification.      

Role of multicolor fluorescence in situ hybridization (FISH) in simultaneous detection of probe sets for chromosome 18, X and Y in uncultured amniotic fluid cells.

Major aneuploidies diagnosed prenatally involve the autosomes 13, 18, and 21, and sex chromosomes. Fluorescence in situ hybridization (FISH) allows rapid analysis of chromosome copy number in interphase cells. The purpose of this study was to evaluate the role of multicolor fluorescence in situ hybridization in simultaneous detection of probe sets for chromosome 18, X, and Y in uncultured amniotic fluid cells as a safer alternative method for aneuploidy detection prenatally. Fifty amniotic fluid samples were analyzed by FISH and standard cytogenetics. Mean time to obtain results was three days for fluorescence in situ hybridization and 20 days for karyotype. Fluorescence in situ hybridization was informative in 43 samples (86%), and within this group, two aneuploidies were correctly identified. This evaluation demonstrates that FISH with X, Y, and 18 alpha satellite DNA probes could accurately and rapidly detect aneuploidies involving these chromosomes and could be used in any prenatal clinical laboratory.     

间期FISH分析中细胞类型及探针的选择

荧光原位杂交已成为医学遗传学领域中一项极为重要的研究手段。为了摸索不同来源的产前诊断材料的间期核进行荧光原位杂交的可行性，选择了α重复序列ＤＮＡ及ＣｏｓｍｉｄＤＮＡ为探针，分别与未培养的绒毛间质细胞、羊水细胞及外周血白细胞的间期核杂交。结果表明：各种材料的间期核均见杂交信号，１３／２１α重复序列探针用于未培养的绒毛间质细胞及外周血白细胞的杂交信号最为特异，能有效的检出染色体非整倍体，羊水细胞则远不如绒毛细胞，绒毛组织是最理想的产前诊断材料。     

8例性发育异常患者SRY基因分析

目的对8例性发育异常患者进行细胞遗传学及分子遗传学检查以探讨性别发育异常与SRY基因关系.方法用PY3.4,X着丝粒,SRY特异探针进行荧光原位杂交,用于分析性发育异常病人Y染色体及SRY基因异位情况.聚合酶链反应(PCR)扩增SRY基因,直接测序检测SRY基因突变.结果 2例46,XX男性,1例46,XY女性,1例45,X/46,XY嵌合体及1例46,X,t(Y;Y)(p11;q11)男性患者SRY基因均为阳性,直接测序未发现SRY基因阳性患者该基因突变.剩余1例46,XX男性,1例46,XY男性及1例46,XY女性患者SRY基因为阴性.FISH技术证实2例46,XX且SRY基因阳性的男性患者SRY基因易位至X染色体短臂末端.结论 SRY基因是人类性别决定的主导基因,但尚有其他基因参与性别分化.     

Concurrent use of flow cytometry and fluorescence in-situ hybridization techniques for detecting faulty meiosis in a human sperm sample

Routine semen analysis in an infertile patient revealed severe teratospermia associated with malformation of head and tail in 100% of the sperm cells. Flow cytometry and fluorescence in-situ hybridization (FISH) were shown to supplement routine semen analysis by providing information on the sperm chromatin. Using flow cytometry, propidium iodide-stained spermatozoa from the same sperm sample were compared with a normal reference pool, and with human lymphocytes. The results point to a population of diploid sperm cells rather than to mature haploid spermatozoa. Numerical chromosomal abnormalities of the spermatozoa were subsequently evaluated using FISH. A total of 1000 sperm cells were scored for X and Y chromosomes, and an additional 1128 sperm cells for chromosome 18. Aneuploidy of chromosomes X and Y was revealed in 96.9% of the cells and of chromosome 18 in 90.3% of the cells. Non-disjunction of chromosome X and Y in meiosis I and II occurred in 54.8 and 2.7% of the sperm cells respectively. Non- disjunction in both meiosis I and II occurred in 39.4% of the sperm cells. A normal haploid pattern for chromosomes X and Y was observed in only 3.1%, and for chromosome 18 in 9.7%, of the cells. Using three colour FISH for the sex chromosomes and for chromosome 18, diploidy was demonstrated in 19.4% of 500 sperm cells and aneuploidy in virtually all sperm cells (99.2%). The use of flow cytometry and FISH in cases where genetic and developmental chromatin abnormalities are suspected is a valuable adjunct to other available techniques, and can guide the clinicians to decide which samples are unsuitable for intracytoplasmic injection.      

应用FISH和PCR技术鉴别Turner综合征患者标记染色体起源

目的　为指导遗传咨询,鉴别Turner 综合征患者微小标记染色体起源。方法　选择SRY 基因编码区1 对特异寡核苷酸引物、X 和Y 染色体特异探针,采用PCR 及荧光原位杂交方法,对8 例具有Turner 综合征体征的患者进行标记染色体分析。结果　5 例患者标记染色体起源于X染色体;3 例起源于Y 染色体,其中2 例SRY 基因序列扩增,可见男性特异扩增带,另1 例无男性特异扩增带。结论　FISH 与PCR 技术结合可准确鉴别标记染色体,对选择治疗方案及了解核型与表型关系有指导意义。     

荧光原位杂交同时检测精子X和Y染色体的方法

用实验手段分离X和Y染色体精子或富集特定性别染色体精子．对于预防性连锁遗传病具有潜在应用价值。建立精子X和Y染色体鉴定方法对于评估分离或富集特定性别染色体效果至关重要。本文介绍采用CY3TM和FlourXTM探针作荧光原位杂交（FISH），同时检测精子Y和X染色体。10份正常精液标本分析结果显示：X染色体精子为50．23％，Y染色体精子为49．77％；有效杂交率达91．99％。FISH方法比传统的精子染色体核型分析和奎纳克林染色检查Y荧光小体更具有简易、特异和快速的优点。     

Do morphological anomalies reflect chromosomal aneuploidies?: case report

In cases of severe teratozoospermia, the current morphological criteria used to assess chromosomal status is insufficient for the selection of spermatozoa for intracytoplasmic sperm injection (ICSI). Case histories are reported of four patients presenting 100% teratozoospermia, and the integrity of their individual chromosomal statuses is determined using a three-colour fluorescence in-situ hybridization (FISH) technique. Patient 1 presented shortened flagella syndrome, patient 2 globozoospermia, patient 3 spermatozoa with irregular acrosomes, and patient 4 macrocephalic spermatozoa with associated multiple flagella. Three-colour FISH analysis using chromosome X, Y and 1-specific probes showed that approximately 95% of the spermatozoa analysed from patients 1, 2 and 3 presented X,1 and Y,1 signals, X,Y ratios and aneuploidy/diploidy rates comparable with those observed in normal controls. In contrast, patient 4 showed a highly elevated Y to X sex ratio and a highly elevated aneuploidy/diploidy rate. Three-colour FISH analysis thus demonstrates an increased incidence of chromosomal abnormalities in association with macrocephalic spermatozoa. Moreover, the analysis shows that in patients affected with either globozoospermia, shortened flagella syndrome or a condition of abnormal acrosomal spermatozoa, no association exists between chromosomal status and phenotype. Since these patients display normal haploid, sex chromosome and aneuploidy status, ICSI can be conceivably offered as a treatment for their infertility.     

Use of fluorescence in situ hybridization for retrospective detection of aneuploidy in multiple myeloma

In malignancies with a low mitotic index such as multiple myeloma (MM), conventional cytogenetic studies may not be informative. This study's purpose was to assess specific numerical chromosomal aberrations in non-dividing MM cells by fluorescence in situ hybridization (FISH) of DNA chromosome probes on bone marrow smears. Old air-dried bone marrow smears from 18 MM patients were probed with alpha satellite DNA sequences for chromosomes 7, X, and Y, and a whole painting probe for chromosome 11. Plasma cells were identified by their morphologic characteristics so that counts of fluorescent signals in the nuclei of MM cells could be differentiated from those of normal marrow cells. Numerical chromosome aberrations were found in 66.7% of the cases (12 of 18), including 5 cases of trisomy 7, 2 cases of tetraploidy, 2 cases of monosomy X in females, 2 cases of disomy X in males, and 1 case of nullisomy Y. In addition, 2 of the 7 cases probed with chromosome 11 paint demonstrated 3 signals in about 15% of the cells. This study illustrates the advantages of FISH for interphase analysis of chromosome aberrations in slowly dividing cells, as well as the ability to use old slides for retrospective studies. © 1993 Wiley-Liss, Inc.