两种FISH试剂盒检测羊水染色体的比较

目的应用两种荧光原位杂交(FISH)试剂盒检测羊水染色体,比较两者在临床的应用效果。方法采用北京金菩嘉和美国雅培Vysis生产的FISH试剂盒对30例未培养的羊水进行染色体检测,比较两者在信号强度和杂交率的差异。结果 30例样本均获得FISH检测结果,且两种试剂临床诊断符合率达到100%,杂交率无统计学意义(P>0.05)。Vysis试剂21/13染色体荧光杂交信号较强,金菩嘉试剂18染色体信号清楚,不易融合。结论 Vysis与金菩嘉两种FISH检测试剂应用染色体异常产前诊断均可达到同样的临床诊断效果。     

Identification of autosomal supernumerary chromosome markers (SMCs) by fluorescent in situ hybridization (FISH)

Supernumerary marker chromosomes (SMCs) are rare chromosomal abnormalities resulting in partial trisomy of specific genomic regions with characteristic phenotypic effects. Twenty six cases with autosomal SMCs are reported. Four were identified prenatally and 22 postnatally in children, aged from 8 days to 15 years, who were referred for genetic evaluation because of various congenital anomalies and developmental delay. In 22 of the 26 cases, the SMCs were de novo, in two they were familial and in another two a 11;22 reciprocal translocation was revealed in the mothers. In only one patient was the SMC present in a mosaic form. Sequential fluorescent in situ hybridization studies (FISH) using Whole Chromosome Paint (WCP) probes were performed in order to determine the chromosomal origin of the SMCs. Sixteen of them originated from chromosome 15, five were shown to be an isochromosome 18p and one was derived from chromosome 22, but did not contain the DiGeorge/ VCFS critical region. In two instances, the SMCs were derivatives of chromosome 13 and in two the SMCs resulted from a 11;22 maternal translocation and contained material from both chromosomes 11 and 22. Molecular investigation of two of the patients with an SMC[15] revealed three copies of the SNRPN gene, but the diagnosis of PW/AS due to possible imprinting was excluded in both patients by a methylation-specific PCR. FISH and molecular studies have greatly facilitated the characterization of marker chromosomes. As more SMCs are classified, better genetic counseling and risk evaluation can be achieved.     

常规R显带和FISH技术检测慢性淋巴细胞白血病染色体异常的价值

目的 研究FISH技术检测慢性淋巴细胞性白血病(CLL)染色体异常的价值.方法 用3、12、18号标记有不同荧光素的染色体着丝粒探针,用荧光原位杂交技术(FISH)检测25例CLL,并和常规细胞遗传学(Conventional cytogenetics,CC)检测方法即R显带法进行比较,以明确何种方法对CLL染色体异常检出更敏感可靠.结果 25例CLL患者中,常规细胞遗传学检出 3, 12, 18 5例,检出率为20%;其中: 12 3例, 3、 12 1例, 3、 12、 18 1例; FISH方法检出8例异常(32%), 3 4例; 12 6例; 18 1例.CC可检出不确定异常:t(5;15) 1例;3q-,18p 1例;4q 13q-1例; 19,-22 1例.结论 FISH方法是检测CLL患者染色体异常的有效技术,可提高染色体异常检出率,明显优于常规显带法.     

Analysis of micronuclei induced under hyperthermic conditions in human lymphocyte culture by fluorescence in situ hybridization (FISH) and spectral karyotyping (SKY) methods]

The spectral karyotyping (SKY) method is a novel molecular cytogenetic technique which simultaneously discerns entire chromosomes. In order to elucidate the origins of micronuclei induced under hyperthermic conditions in human lymphocyte culture, peripheral blood cells were cultured at 40 degrees C or 42 degrees C for 3-24 h, using the cytokinesis-block method with cytochalasin B. The induced micronuclei were identified by the fluorescence in situ hybridization (FISH) and SKY methods. At 42 degrees C for more than 6 h, the frequency of occurrence of micronuclei in binucleated cells rose with increasing incubation time. By the FISH method, 83.3% of micronuclei induced in 24 h culture at 42 degrees C were shown to be positive for the human centromeric probes. By the SKY method, each micronucleus induced under the hyperthermic conditions was identified unequivocally and shown to contain a specific chromosome. These results suggest that the micronuclei induced under the hyperthermic conditions in human lymphocyte culture contain chromosomes which do not migrate to the poles at the anaphase of the cell cycle because of the breakdown of the spindle apparatus.     

Strategy of the scientific committee on occupational exposure limits (SCOEL) in the derivation of occupational exposure limits for carcinogens and mutagens

Setting standards, such as occupational exposure limits (OELs) for carcinogenic substances must consider modes of action. At the European Union level, the scientific committee on occupational exposure limits (SCOEL) has discussed a number of chemical carcinogens and has issued recommendations. For some carcinogens, health-based OELs were recommended, while quantitative assessments of carcinogenic risks were performed for others. For purposes of setting limits this led to the consideration of the following groups of carcinogens. (A) Non-threshold genotoxic carcinogens; for low-dose assessment of risk, the linear non-threshold (LNT) model appears appropriate. For these chemicals, regulations (risk management) may be based on the ALARA principle ("as low as reasonably achievable"), technical feasibility, and other socio-political considerations. (B) Genotoxic carcinogens, for which the existence of a threshold cannot be sufficiently supported at present. In these cases, the LNT model may be used as a default assumption, based on the scientific uncertainty. (C) Genotoxic carcinogens with a practical threshold, as supported by studies on mechanisms and/or toxicokinetics; health-based exposure limits may be based on an established NOAEL (no observed adverse effect level). (D) Non-genotoxic carcinogens and non-DNA-reactive carcinogens; for these compounds a true ("perfect") threshold is associated with a clearly founded NOAEL. The mechanisms shown by tumour promoters, spindle poisons, topoisomerase II poisons and hormones are typical examples of this category. Health-based OELs are derived for carcinogens of groups C and D, while a risk assessment is carried out for carcinogens of groups A and B. Substantial progress is currently being made in the incorporation of new types of mechanistic data into these regulatory procedures.     

Cytogenetic toxicity of cycloplatam in human lymphocytes: detection by the micronucleus test and fluorescence in situ hybridization

Cycloplatam has been shown to be effective in the treatment of pleural mesothelioma, myeloma and ovarian carcinoma. Cycloplatam is not nephrotoxic with respect to the platinum-based anti-tumor agents. We have investigated the mechanism underlying the induction of micronuclei (MN) in human lymphocytes by cycloplatam compared to that by its parent drugs cisplatin and carboplatin. The cytokinesis-block micronucleus assay in human lymphocytes was applied in combination with fluorescence in situ hybridization (FISH) with an all-chromosome centromeric probe allowing discrimination between MN due to chromosomal fragments (centromere negative, C) and those containing whole chromosomes (centromere positive, C). A statistically significant increase of MN frequency (P<0.001) was detected for cisplatin, carboplatin and cycloplatam. However, cycloplatam was active at a much lower dose (0.1 micromol/l) than cisplatin or carboplatin (1 micromol/l). No significant increase in the frequency of C or C MN was observed for cisplatin and carboplatin compared to the controls. A statistically significant (P<0.001) increase in the percentage of C MN was observed in cycloplatam-treated cells. The results obtained suggest different mechanisms for cytogenetic damage induced by platinum drugs. Cycloplatam induces one type of MN and it could be considered a clastogenic agent, whereas cisplatin and carboplatin appear to induce both chromosome breakage and numerical chromosomal abnormalities.     

Fluorescence in situ hybridization techniques (FISH) to detect changes in CYP19a gene expression of Japanese medaka (Oryzias latipes)

The aim of this study was to develop a sensitive in situ hybridization methodology using fluorescence-labeled riboprobes (FISH) that allows for the evaluation of gene expression profiles simultaneously in multiple target tissues of whole fish sections of Japanese medaka (Oryzias latipes). To date FISH methods have been limited in their application due to autofluorescence of tissues, fixatives or other components of the hybridization procedure. An optimized FISH method, based on confocal fluorescence microscopy was developed to reduce the autofluorescence signal. Because of its tissue- and gender-specific expression and relevance in studies of endocrine disruption, gonadal aromatase (CYP19a) was used as a model gene. The in situ hybridization (ISH) system was validated in a test exposure with the aromatase inhibitor fadrozole. The optimized FISH method revealed tissue-specific expression of the CYP19a gene. Furthermore, the assay could differentiate the abundance of CYP19a mRNA among cell types. Expression of CYP19a was primarily associated with early stage oocytes, and expression gradually decreased with increasing maturation. No expression of CYP19a mRNA was observed in other tissues such as brain, liver, or testes. Fadrozole (100 μg/L) caused up-regulation of CYP19a expression, a trend that was confirmed by RT-PCR analysis on excised tissues. In a combination approach with gonad histology, it could be shown that the increase in CYP19a expression as measured by RT-PCR on a whole tissue basis was due to a combination of both increases in numbers of CYP19a-containing cells and an increase in the amount of CYP19a mRNA present in the cells.     

应用荧光原位杂交对相互易位携带者进行植入前遗传学诊断

目的建立荧光原位杂交技术对平衡易位携带者的胚胎进行植入前遗传学诊断(FISH-PGD)。方法对1例因男方为t(1q42;4q21)易位携带而妊娠过染色体异常胎儿的不孕夫妇,经激素超促排卵和卵胞浆内单精子注射(ICSI)进行体外受精-胚胎移植(IVF-ET)。于受精后第3天进行胚胎活检及FISH分析,选择FISH结果为正常或平衡易位的囊胚予以冷冻,择期解冻移植。结果活检11个胚胎,均得到FISH结果。其中,对位(正常或平衡易位)胚胎2个(18.18%),邻位-1胚胎5个(45.45%),邻位-2胚胎为0,3∶1分离胚胎3个(27.27%),紊乱型胚胎1个(9.09%)。FISH诊断为正常或平衡易位的2个胚胎发育至第5天,1个形成囊胚冷冻备用移植,1个胚胎退化放弃冷冻。结论建立的FISH-PGD能够有效的选择正常或平衡易位的可移植胚胎,对解决相互易位携带者的生育障碍有重要价值。     

恶性淋巴瘤诊断中荧光原位杂交分析基因状态的研究

目的 探讨恶性淋巴瘤中基因重排的特点.方法 回顾性分析87例恶性淋巴瘤组织样本,利用荧光原位杂交技术,分别检测弥漫大B细胞淋巴瘤、滤泡性淋巴瘤、黏膜相关淋巴组织淋巴瘤、套细胞淋巴瘤、Burkitt淋巴瘤和间变性大细胞淋巴瘤中BCL-6、BCL-2、MALT1、CCND1、C-MYC和ALK基因重排.结果 弥漫大B细胞性淋巴瘤中BCL-6基因重排的阳性率为29.03％(9/31),滤泡性淋巴瘤中BCL-2基因重排的阳性率为61.11％(11/18),黏膜相关淋巴组织淋巴瘤中MALT1基因重排的阳性率为47.37％(9/19),套细胞淋巴瘤中CCND1基因重排的阳性率为88.88％(8/9),Burkitt淋巴瘤中C-MYC基因重排的阳性率为100％(2/2)和间变性大细胞淋巴瘤中ALK基因重排的阳性率为75.00％(6/8).结论 基因重排对恶性淋巴瘤的诊断具有重要的实际价值.     

Preimplantation genetic diagnosis for a patient with reciprocal translocation using fluorescence in situ hybridization

目的 建立荧光原位杂交技术对平衡易位携带者的胚胎进行植入前遗传学诊断(FISH-PGD).方法 对1例因男方为t(1q42; 4q21)易位携带而妊娠过染色体异常胎儿的不孕夫妇,经激素超促排卵和卵胞浆内单精子注射(ICSI)进行体外受精-胚胎移植(IVF-ET).于受精后第3天进行胚胎活检及FISH分析,选择FISH结果为正常或平衡易位的囊胚予以冷冻,择期解冻移植.结果 活检11个胚胎,均得到FISH结果.其中,对位(正常或平衡易位)胚胎2个(18.18％),邻位-1胚胎5个(45.45％),邻位-2胚胎为0,3:1分离胚胎3个(27.27％),紊乱型胚胎1个(9.09％).FISH诊断为正常或平衡易位的2个胚胎发育至第5天,1个形成囊胚冷冻备用移植,1个胚胎退化放弃冷冻.结论 建立的FISH-PGD能够有效的选择正常或平衡易位的可移植胚胎,对解决相互易位携带者的生育障碍有重要价值.     

荧光原位杂交技术在多发性骨髓瘤中的应用

目的 了解多发性骨髓瘤常见染色体异常情况,探讨其与临床分期及免疫学分型的关系。方法 对39例多发性骨髓瘤初诊患者进行1q21、RBl、D13S319、p53及IGH5种基因探针荧光原位杂交检测1q21扩增、13q14缺失、p53缺失以及IGH基因重排的发生率,统计分析染色体异常情况与临床分期及免疫学分型的关系。结果 1q21、RBl、D13S319、p53及IGH探针的阳性阈值分别为:7.2%、8.7%、7.9%、7.2%和9.8%。39例患者中,1q21扩增24例(61.5%);13q14缺失18例(46.2%),其中同时检出D13S319和RBl缺失15例;p53缺失8例(20.5%);IGH基因重排13例(33.3%)。除IGH重排与D-S分期之间(r=0.434,P=0.007)、p53缺失与IIS分期之间(r=-0.448,P=0.006)和D13S319缺失与免疫学分型之间(r=-0.335,P=0.040)呈显著相关性外,其余染色体异常与临床分期和免疫学分型均无相关性。结论 1q21扩增、13q14缺失、p53基因缺失及IGH重排是多发性骨髓瘤常见的核型异常,FISH检测技术有助于探索染色体异常与多发性骨髓瘤发展及预后的关系。     

应用荧光原位杂交技术进行产前诊断的可行性探讨

目的：探讨荧光原位杂交技术（FISH）应用于绒毛及羊水间期细胞进行产前诊断的可行性。方法 应用18、X号染色体着丝粒探针及21号染色体特定区域探针,分别对绒毛及羊水间期细胞进行荧光原位杂交,并与中期分裂相进行比较分析。结果 绒毛及羊水间期细胞均出现了杂交信号,万以绒毛细胞杂交比率高,信号强;常染色体探针出现3个及1个杂交点的比率约为1％和6％,X染色体探针出现3点的比例也在1％左右。结论 荧光原位杂交技术对检出染色体非整倍体有较强的特异性,同时也为染色体非整倍体的检出提供了诊断依据。FISH技术应用于产前诊断具有快速、简便、灵敏的优点,有较大的临床应用价值。     

荧光原位杂交检测垂体瘤染色体的异常

目的研究垂体瘤间期细胞遗传性畸变,探讨其异常与垂体瘤发生和发展的关系。方法应用荧光原位杂交技术研究了50例垂体瘤间期细胞核7、8、11染色体数目的异常。结果64％(32／50)的垂体瘤出现一个以上染色体数量的变异,其中以11染色体的异常最为常见28％(14／50),其余依次为7染色体22％(11／50)、8染色体20％(10／50)。7、8染色体的异常与垂体瘤的临床病理特征未见明显相关,但11号单体与垂体瘤的侵袭性密切相关。结论垂体瘤7、8、11染色体变异是频繁发生的。7、8染色体的获得可能参与了肿瘤形成的起始阶段,而11号染色体的丢失可能与肿瘤的演进及生物行为有关。     

Comet fluorescence in situ hybridization analysis for oxidative stress-induced DNA damage in colon cancer relevant genes.

Our objective was to study whether products of oxidative stress, such as hydrogen peroxide (H(2)O(2)), trans-2-hexenal, and 4-hydroxy-2-nonenal (HNE), cause DNA damage in genes, relevant for human colon cancer. For this, total DNA damage was measured in primary human colon cells and colon adenoma cells (LT97) using the single-cell gel electrophoresis assay, known as "Comet Assay." APC, KRAS, and TP53 were marked in the comet images using fluorescence in situ hybridization (Comet FISH). The migration of APC, KRAS, or TP53 signals into the comet tails was quantified and compared to total DNA damage. All three substances were clearly genotoxic for APC, KRAS, and TP53 genes and total DNA in both types of cells. In primary colon cells, TP53 gene was more sensitive toward H(2)O(2), trans-2-hexenal, and HNE than total DNA was. In LT97 cells, the TP53 gene was more sensitive only toward trans-2-hexenal and HNE. APC and KRAS genes were more susceptible than total DNA to both lipid peroxidation products but only in primary colon cells. This suggests genotoxic effects of lipid peroxidation products in APC, KRAS, and TP53 genes. In LT97 cells, TP53 was more susceptible than APC and KRAS toward HNE. Based on the reported gatekeeper properties of TP53, which in colon adenoma is frequently altered to yield carcinoma, this implies that HNE is likely to contribute to cancer progression. This new experimental approach facilitates studies on effects of nutrition-related carcinogens in relevant target genes.     

荧光原位杂交技术在绒膜绒毛间期细胞诊断中的应用

目的　探讨荧光原位杂交 (FISH)技术在绒毛间期细胞染色体分析中的应用价值。方法　应用地高辛标记的探针 p Y3.4以及生物素标记的探针 2 1q2 2 .3,对 10例流产绒毛进行荧光原位杂交 ,以绒毛染色体直接制备分析验证杂交结果。结果　 FISH杂交结果与绒毛染色体分析结果一致 ,诊断率达 95 %以上。结论　 FISH技术是一种非常有效的可用于产前诊断间期细胞染色体分析的手段     

多重荧光原位杂交检测多发性骨髓瘤复杂核型异常

目的探讨多重荧光原位杂交（M-FISH）技术在多发性骨髓瘤（MM）复杂核型异常（CCAs）检测中的价值。方法对5例常规R显带具有CCAs的MM患者应用MFISH确定复杂染色体的重排及标记染色体的组成。结粜明确了常规核型（CC）分析中的所有异常,共检出20种结构异常,其中缺失2种,易位18种,均为不平衡易位。最常累及的为14号染色体、1q的不平衡易位及6号染色体。结论对伴有CCAs的MM患者,M-FISH技术可以明确CC分析中复杂染色体异常,并发现和纠正CC分析中漏检及误检的异常,为MM的染色体异常研究提供了一种理想的方法。     

Evaluation of potential neurotoxic effects of occupational exposure to (L)-lactates

Organo psycho syndrome (OPS) or chronic toxic encephalopathy (CTE) is a neurotoxic condition reported following long-term exposure to paints containing organic solvent and to other solvents. Lactate esters are finding wider use as solvents. Lactate esters have been well studied in standard toxicity tests, but specific neurotoxicity studies have not been conducted. No clinical signs of chronic neurotoxicity have been observed in standard toxicity tests. Lactate esters are rapidly hydrolyzed in the body to lactic acid and the corresponding alcohol. Alcohols have been reported to have acute neurotoxic effects, usually following high levels of ingestion. The literature on alcohols was reviewed to establish the no-observed-adverse-effect level (NOAEL) for acute neurotoxicity and to look for any evidence of chronic neurotoxicity from the alcohols produced by hydrolysis of the lactate esters. The NOAELs were compared with the potential amounts of alcohol produced by hydrolysis of different lactate esters at 200 mg//m(3) (the NOAEL for most of the lactate esters). In all cases neither acute nor chronic neurotoxicity would be expected based on the amounts of alcohol produced by hydrolysis of the lactate esters at their NOAELs. L-Lactic acid is a normal metabolite in the body and is not considered neurotoxic. Based on this information there is no evidence to suggest that L-lactate esters can cause any chronic neurotoxicity, OPS, or CTE.     

荧光原位分子杂交技术检测乳腺癌组织中HER-2基因的表达

目的荧光原位杂交(FISH)方法检测乳腺癌组织中人表皮生长因子2(HER-2)的扩增情况,并与免疫组化(IHC)结果相比较,优化乳腺癌HER-2检测方法。方法采用相关质控措施,利用FISH法及IHC法检测乳腺癌HER-2的表达,分析相关性。结果 FISH检测Her-2基因扩增40例,阳性率30.5%(40/131),在IHC(-)标本中,Her-2基因未扩增19例,阴性符合率为95.0%;在IHC(+)标本中,Her-2基因扩增7例,阳性符合率为20.0%;在IHC(++)标本中,Her-2基因扩增15例,阳性符合率26.8%;在IHC(+++)标本中,Her-2基因扩增17例,阳性符合率89.5%。结论 FISH检测准确率较高,IHC阴性和(+++)时与FISH结果一致性较好,可作为治疗依据,IHC(+)和(++)时仅能作初步筛查,明确Her-2情况仍需结合FISH检测。     

免疫磁株法联合荧光原位杂交检测微小残留病

目的 探讨联合免疫磁珠分选和荧光原位杂交(FISH)技术检测慢性粒细胞白血病(CML)患者微小残留病的敏感性.方法 采集10例CML患者的骨髓,利用免疫磁珠法纯化CD34阳性细胞,对比分析纯化前后FISH检测结果.结果 CD34阳性细胞纯化前占细胞百分数为(0.7 ±0.2)%,纯化后为(93.4±2.5)%(P＜0.05).CD34阳性细胞纯化前FISH检测4例阳性,纯化后9例阳性(P＜0.05).结论 免疫磁珠技术联合FISH技术可提高检测CML患者微小残留病的敏感性.     

多色荧光原位杂交(M-FISH)在肺癌早期诊断中的应用

目的探讨多色荧光原位杂交(M-FISH)在肺癌早期诊断中的临床意义。方法通过苏北人民医院2007年1月至2011年1月89例肺癌患者支气管镜活检组织及刷检细胞标本作为观察组。结果 89份标本中61份标本中有X染色体数目的增多,其中男性45份,女性16份。具有异常X染色体数目的细胞比例为15.1%～93.2%。26例Y染色体异常,占29.21%,其中8例Y染色体增多的标本均为男性患者,占8.99%,另18例男性患者发生Y染色体数目减少,占20.22%。结论原位荧光杂交技术对肺癌组织细胞进行检测,有助于肺癌的早期诊断,同时对于判断治疗效果、预后及有无复发等均有辅助作用。