用双色荧光原位杂交技术检测罗伯逊易位携带者的精子染色体

目的 :探讨用双色荧光原位杂交技术 (D FISH)检测罗伯逊易位携带者的精子染色体的实验方法和应用价值。　方法 :采用荧光原位杂交 (FISH)技术 ,以Biotin标记的 13q 14 .3特异性探针和以Digoxigenin标记的 14 q11.1特异性探针对 2例罗伯逊易位携带者精子标本进行原位杂交 ,并统计精子间期核 13、14号染色体的杂交信号颗粒数量。　结果 :在显微镜下可见精子头部有以Biotin标记的 13q 14 .3特异性探针显示 1个绿色杂交信号 ,以Digoxi genin标记的 14 q11.1特异性探针显示 1个红色杂交信号 ,间期核背景经DAPI复染显示蓝色 ;共统计 30 0 0个精子间期核 ,显示 1个绿色 1个红色杂交信号的精子为 13q/ 14q ,占39.33% ;显示 1个绿色 2个红色杂交信号为 13q/ 14q ,14q ,占 11.5 7% ;仅显示 1个绿色杂交信号为 13q/ ,占 9.2 7% ;显示 2个绿色 1个红色杂交信号为 13q ,13q/ 14 q ,占12 .87% ;仅显示 1个红色杂交信号为 / 14 q ,占9.87% ;显示 2个绿色 2个红色杂交信号为 13q ,13q/ 14q ,14 q ,占12 .2 6 %。　结论 :用双色荧光原位杂交技术检测染色体结构异常患者的精子 ,可以分析其减数分裂过程中染色体分离规律 ,在人类生殖如显微授精和植入前胚胎遗传学诊断等方面具有非常广泛的应用价值     

介绍一种应用双色荧光复位杂交检测人精子染色体数目异常的技术

研究人类精子染色体常用的方法有两种,一种是用人类精子体外穿透金黄地鼠卵[1],这个方法虽然可以观察到精子的全部染色体组成,但由于这个方法操作复杂,十分耗时,且只适合于可以穿透金黄地鼠卵的精子,因而在临床上受到很大限制,推广起来也很困难.     

人类精子间期核荧光原位杂交

为研究人类精子染色体的畸变，用３号染色体涂染探针对人类精子间核进行荧光原位杂交，检测了１０名正常健康男子和２名涉及３号染色体平均易位携带者ｔ（３；７）、ｔ（３；９）的精液。结果表明，正常男性精子间期核中３号染色体数目和（或）结构畸变率〈２％，涉及３号染色体平衡易位携带者的畸变率〉１０％。     

人类精子间期核荧光原位杂交

为研究人类精子染色体的畸变，用3号染色体涂染探针对人类精子间期核进行荧光原位杂交（简称FISH），检测了10名正常健康男子和2名涉及3号染色体平衡易位携带者t（3；7）、t（3；9）的精液。结果表明，正常男性精子间期核中3号染色体数目和（或）结构畸变率＜2％，涉及3号染色体平衡易位携带者的畸变率＞10％。     

多色荧光原位杂交技术在精子染色体研究中的应用

随着胞质内精子显微注射 (ICSI)的应用 ,严重少、弱、畸精子症病人也能获得治疗。现有许多研究发现这部分病人精子染色体非整倍率增加。如非整倍体的精子受精 ,产生的子代将导致流产、畸胎、死胎 ,因此对精子染色体数目的研究已成为男性不育精子检测的一项重要内容。近年来 ,多色荧光原位杂交 (FISH)技术在检测精子染色体非整倍体方面取得了一些进展 ,本文对此作一综述     

应用荧光原位杂交技术检测人精子染色体(摘要)

介绍一种简便而快速的检测精子染色体数目异常的方法，以Ｄ２１Ｚ１／Ｄ１３Ｚ１、ＤＸＺ１探针与精子间期核进行荧光原位杂交（ＦＩＳＨ），能准确计数精子核中染色体数目。报告如下。实验资料４例新鲜精液由北京协和医院泌尿外科提供。主要试剂：二硫苏糖醇（ＤＴＴ）购...     

荧光原位杂交技术检测不育男性精子染色体非整倍体

目的 :检测不育男性与正常男性的精子染色体非整倍体之间的差异 ,为宫腔内人工授精提供指导。方法 :1 2例不育患者 ,其中 1 0例为少、弱、畸精症患者 ( A组 ) ,2例精子数接近正常 ( B组 ) ,2例正常健康的献精者作为对照 ( C组 ) ,选用 X、Y和 1 8号染色体探针进行荧光原位杂交 ( FISH)检测精子的非整倍体。结果 :B组精子 X、Y、和 1 8号染色体的二体频率分别为 0 .3 0 %、0 .3 0 % ,与 C组 ( 0 .1 5%、0 % )相比差别无显著性 ;A组的X、Y和 1 8号染色体二体率分别为 1 .1 3 %、0 .96 % ,缺体率为 1 .1 3 %和 1 .6 % ,与其他两组相比 ,差别有显著性 ( P<0 .0 5)。估算 A组总的非整倍体率为 42 .44 % ,与 B组 6 .0 5%及 C组的 2 .59%比较 ,差别有显著性 ( P<0 .0 5)。结论 :不育男性精子的染色体非整倍体显著高于正常 ,对少、弱、畸精症患者的精液标本可进行洗涤优化处理后行宫腔内人工授精 ,达到助孕、优生的目的     

评估人类精子中原位非整倍体:引物原位杂交和缩氨酸核酸荧光原位杂交技术的使用(英文)

引物原位杂交(PRINS)和缩氨酸核酸荧光原位杂交(PNA-FISH)技术都可替代传统的荧光原位杂交(FISH)技术,用于染色体的研究。PRINS 反应的基本物是 DNA 聚合酶和引物原位扩展反应中的标引核苷。缩氨酸核酸探针是合成的不带电聚酰胺主链的 DNA 类似物。这两种技术特异、快速、识别能力强的优点使它们在细胞遗传学研究中很受欢迎。它们在人类精子研究中的应用使男性配子的染色体筛选过程得到新的快速发展,成为男性配子非整倍体原位评估中 FISH 技术的有效补充。     

染色体平衡易位患者精子染色体荧光原位杂交分析

目的:探讨外周血染色体平衡易位患者在精子发生过程中染色体分离模式,了解各分离模式产生精子所占比例,预测染色体平衡易位携带者在胚胎移植前遗传学诊断(PGD)中得到正常表型胚胎的概率,评估其进行PGD的风险。方法:应用三色荧光原位杂交(FISH)对4例染色体平衡易位患者46,XY,t(9;11)(q22;q21)、46,XY,t(11;22)(q23;q11)、45,XY,t(13q;15q)、45,XY,t(13q;14q)进行精子相关染色体分析,计算出各染色体分离模式产生精子所占比例,同时以染色体正常男性的正常精液作为对照。结果:上述4例异常精子所占比例分别为50.86%、58.33%、13.00%和22.82%,均明显高于对照组(0.85%、1.63%、1.60%和1.37%)。结论:通过FISH检测染色体平衡易位患者精子染色体,有助于预测染色体平衡易位携带者在PGD过程中的风险,从而选择应用PGD。     

介绍一种应用双色荧光复位杂交检测人精子染色体数目异常的 …

研究人类精子染色体常用的方法有两种 ,一种是用人类精子体外穿透金黄地鼠卵[1] ,这个方法虽然可以观察到精子的全部染色体组成 ,但由于这个方法操作复杂 ,十分耗时 ,且只适合于可以穿透金黄地鼠卵的精子 ,因而在临床上受到很大限制 ,推广起来也很困难。另一种是应用ＦＩＳＨ技术直接检查精子间期核的染色体组成[2 ] 。这个方法克服了前一个方法的缺点 ,而且由于它的高度敏感性和特异性 ,一次杂交就可检测成千上万个精子非常适合于精子染色体数目异常的研究。为了研究男性不育症患者和染色体平衡易位携带者中精子染色体的异常 ,我室建立了一…     

染色体荧光原位杂交技术研究小片段和复杂的染色体易位

染色体之间的平衡易位，当片段很小或涉及染色体数目多时，显微镜下容易漏诊，或难以准确判定断裂点。本文报告了用染色体荧光原位杂交（ＦＩＳＨ）的实验技术结合常规Ｇ显带、高分辨Ｇ显带技术诊断了２例难辨认的染色体易位。本研究表明，ＦＩＳＨ技术结合细胞遗传学核型分析，对于检测小片段难辨认或复杂的染色体易位是非常有帮助的。     

Analysis by fluorescence in situ hybridization (FISH) of the relationship between gonosomic aneuploidy and the results of assisted reproduction in men with severe oligozoospermia

The aim of this study was to analyse the frequency of sex-chromosomal aneuploidy in human spermatozoa of severe oligozoospermic men undergoing intracytoplasmic sperm injection (ICSI), to evaluate the impact of these chromosomal anomalies on the results of the ICSI. Fluorescence in situ hybridization (FISH) with direct label fluorescence DNA probes specific for chromosome X, Y and 18 was performed on decondensed spermatozoa from fresh ejaculates of 12 patients with severe oligozoospermia undergoing ICSI. A total of 500 spermatozoa were analysed per donor. The rate of gonosomal aneuploidy was significantly increased in the patients compared with normal donors (3.5% and 0.8% respectively). The sex-chromosomal anomalies due to meiosis I (XY) are less important than the anomalies caused by meiosis II (XX or XY), but the difference was not statistically significant. There was a negative correlation between the rate of aneuploidy and the percentage of spermatozoa with normal morphology (r = -0.71; P < 0.05). The correlation was negative between the percentage of gonosomic aneuploidy and the rate of fertilization (r = -0.7; P < 0.001). Our results suggest an increased rate of gonosomic aneuploidy in the patients with oligozoospermia compared with the normal population. This aneuploidy, although it decreases the rate of fertilization, does not seem to affect the rate of cleavage, nor the embryonic quality.     

双色间期荧光原位杂交技术在异性间造血干细胞移植中的应用

目的 探讨双色间期荧光原位杂交（FISH）法在检测异性异基因造血干细胞移植后植活标志及微量残留病灶（MRD）中的作用。方法 采用X、Y染色体探针和间期双色间期FISH法检测35例异性异基因造血干细胞移植受者移植后不同时期的性染色体荧光杂交信号。并设对照组，分析检测的有效率。结果 35例受者移植后的细胞遗传学及双色间期FISH性染色体分析均可见植入证据。当受者出现原病复发时，双色间期FISH性染色体可检测出受者源克隆的增加；其再次缓解后受者源克隆也相应减少。在移植后不同时间，当染色体分析为100％XX或100％XY结果时，双色间期FISH可显示不同比例的受者源染色体。结论 双色间期FISH应用于异性异基因造血干细胞移植后植活标志及MRD检测，操作简单快速，是细胞形态学及细胞遗传学检查很好的补充。     

Rapid detection of human HLA transgenes in pigs by fluorescence in situ hybridization (FISH) for adjuvant study of human xenotransplantation

OBJECTIVES: We have recently generated several lines of transgenic pigs for HLA-DP and -DQ to elucidate the role of HLA-II antigens in the modulation of cell-mediated rejection of xenotransplantation. Using fluorescence in situ hybridization (FISH) analysis, the aim of this study was to determine integration sites and to test zygosity of these transgenes in the piglets after cross mating. METHODS: Blood lymphocytes of transgenic pigs for HLA-DP and -DQ were collected and cultured. Chromosome spreads were prepared by standard methodology. Gene constructs of HLA-DP A1+B1, -DQ A1 & B1 were labeled with fluorescein isothiocyanate or Texas Red by nick-translation. Hybridization was based on a standard FISH protocol. RESULTS: FISH analysis revealed that the HLA-DP probe hybridized to porcine chromosome 6, while both HLA-DQ A1 and B1 probes hybridized to porcine chromosome 11 at the same site. There was no cross-hybridization of HLA transgenes to the swine leukocyte antigen complex. Mosaic integration of HLA-DQ transgenes in the genome of F0, but full penetrance in F1 after selective breeding was observed. Both HLA-DP and HLA-DQ lines were determined to be heterozygous at the integration site. CONCLUSION: By FISH, we have detected specific integration sites of the HLA-DP and -DQ transgenes in pig genome and determined mosaic levels and zygosity types of these transgenes. We conclude that FISH is both sensitive and labor-efficient in confirming and differentiating transgenic pigs for multiple rejection-regulatory genes by visualizing individual integration sites in chromosomes or interphase nuclei.     

荧光原位杂交技术在膀胱尿路上皮癌诊断中的应用价值

目的:评估荧光原位杂交技术(FISH)在膀胱尿路上皮癌诊断中的应用价值。方法:收集60例疑似膀胱癌的血尿患者的尿液标本,分别作尿细胞学检测和荧光原位杂交分析。20例正常人尿液标本,用于建立FISH阀值,作为阳性判断的标准。结果:细胞学和FISH的总敏感性分别为42.0%、82.2%,特异性分别为:93.3%、86.7%。细胞学和FISH在低级别及非肌层浸润性肿瘤等敏感性的均差异有统计学意义(P<0.05)。结论:FISH技术能明显提高膀胱尿路上皮癌的检出率,尤其是早期和低级别病变,可以成为筛查膀胱尿路上皮癌的一种新的无创性检查方法。     

Application of dual color fluorescence in situ hybridization （D—FISH） to the diagnosis of a 49,XXXXY chromosomal abnormality

To study the technique of D-FISH and its application in the diagnosis of a 49, XXXXY chromosomal abnormality. Methods: Biotin-labeled alpha satellite X chromosome DNA (pBamX7) probe and digoxi-genin-labeled Y chromosome long arm terminal repetitive sequence (pY3.4) probe in situ hybridized with pre-treated slides of peripheral blood chromosome and interphase nucleus. After washing, the slides were treated with avidin-FITC, rhodamine-FITC and anti-avidin, amplified with an additional layer and counter-stained with DAPI in an antifade solution. The hy bridization signals and chromosomal or interphase nucleus settings were observed respectively with WIB, WIG and WU filters under fluorescent microscope (Olympus AX-70) and the number of metaphase chromosome and interphase nucleus in the peripheral blood was counted. Results: The biotin-labeled pBamX7 probe showed 4 green hybridization signal and the digoxigenin-labeled pY3.4 probe showed 1 red hybridization signal. The chromosome or cytoplasm counter-stained     

Urinary fluorescence in situ hybridization assay for detecting urothelial carcinoma in Taiwanese patients

Study Type – Diagnosis (case series)
Level of Evidence 4

OBJECTIVES

To investigate the cytogenetic marker detected by fluorescence in situ hybridization (FISH; UroVysion^TM, Vysis, Inc., Abbott Laboratories, Des Plaines, IL, USA) in the diagnosis of bladder cancer and upper tract urothelial carcinoma (UC) in Taiwanese patients, as FISH has been used in Western countries for detecting UC, but there are limited results in Asian populations.

PATIENTS AND METHODS

We analysed polyploidy of chromosome 3, 7, 17 and aneuploidy of chromosome 21, using uroepithelial cells collected at the first void or by instrumental extraction of urine, for bladder cancer, and shedding cells from the upper tract flushed by normal saline via ureteric catheterization or ureterorenoscopy. The criteria for positive tumour cells included three or more positive staining in two or more chromosomes showing polyploidy or <50% staining of the chromosome 9p21.

RESULTS

In all, 32 patients with bladder UC and 12 with upper tract UC were assessed. The overall sensitivity for bladder cancer by UroVysion was 96.8%. The sensitivity of the cytology test was 36% for UC of the bladder. The sensitivity for UroVysion in upper tract UC was 12/12 but the specificity was three of nine.

CONCLUSIONS

This preliminary report shows that UroVysion was a sensitive screening method for UC of the bladder and upper urinary tract.