应用双色荧光原位杂交检测慢性粒细胞白血病衍生9号染色体缺失的研究

目的 研究慢性粒细胞白血病(chronic myelogenous leukemia,CML)患者衍生9号染色体[derivative chromo-some 9,der(9)]部分序列缺失情况,探讨双色荧光原位杂交(FISH)技术检测der(9)部分缺失的应用价值.方法 对2002年3月至2005年12月中国协和医科大学血液学研究所150例CML患者采用不加任何刺激剂的骨髓24 h短期培养法制备染色体,R显带进行核型分析.应用bcr-abl双色双融合DNA探针对骨髓间期细胞行荧光原位杂交,检测der(9)部分序列缺失.结果 124例CML患者进行了染色体核型分析,其中3例分析失败,121例患者中典型Ph染色体易位97例,变异易位24例,同时伴附加染色体异常19例.150例CML患者均进行了双色荧光原位杂交检测,其中27例患者der(9)部分缺失,发生率为18.00%.9例为典型Ph染色体易位,占典型Ph染色体易位患者的9.27%;12例为变异易位,占变异易位患者的50.00%.变异易位患者中的der(9)缺失发生率明显高于典型易位患者,差异有统计学意义(P<0.01).结论 在CML患者中der(9)部分缺失的发生率约为18.00%,应用双色双融合FISH技术能够简便、快速、灵敏的检测出CML患者der(9)缺失.     

食管癌染色体畸变及其研究方法进展

食管癌是常见的消化道恶性肿瘤之一,主要有食管鳞癌和食管腺癌两种形式．细胞遗传学研究表明食管癌可出现多种多样的染色体异常,但迄今为止还未发现特征性的染色体改变．食管癌染色体异常包括数目异常和易位、扩增、缺失及环状染色体等结构异常．研究食管癌染色体畸变的方法主要有常规细胞遗传学、FISH、CGH等．现就食管癌的染色体畸变及其相关的研究方法予以综述．     

膀胱移行细胞癌术后复发与染色体畸变的相关性研究

目的探讨膀胱移行细胞癌(TCCB)术后复发与染色体畸变的相关性及其临床意义。方法 35例TCCB组织(复发19例、未复发16例)采用荧光原位杂交技术(FISH)检测,观察其3、7、17号染色体及9号染色体p16基因的畸变情况。另选10例癌旁正常组织为对照。结果 19例复发性TCCB组织中,3、7、17号染色体及9号染色体p16基因的畸变率分别为42.11%、57.89%、84.21%和89.47%;在16例未复发性TCCB中分别为31.25%、37.50%、43.75%和50.00%。17号染色体和9号染色体p16基因位点的畸变率TCCB复发者明显高于未复发者(P均<0.05),其畸变与TCCB复发呈正相关(r分别为0.391和0.399,P均<0.05);在复发性TCCB的G1级与G2～3级组织中7号染色体异常差异有统计学意义(P<0.05),其畸变与TCCB组织分级呈正相关(r为0.565,P<0.05);各染色体异常与TCCB病理分期、复发次数均无相关性(P均>0.05)。结论 TCCB术后复发与9号染色体p16基因及17号染色体畸变呈正相关,复发性TCCB中7号染色体畸变与TCCB组织分级呈正相关。TCCB患者术后定期随访并行FISH监测,对于及早发现病变并及时治疗具有重要临床意义。     

多发性骨髓瘤患者13号染色体长臂部分缺失及其临床意义

目的研究多发性骨髓瘤（MM）患者13号染色体长臂特定位点的缺失及与临床表现、预后的关系。方法采用荧光原位杂交技术检测68例MM患者骨髓标本中Rb-1基因和13q14．3位点的缺失,结合临床资料作统计分析。结果13号染色体部分缺失的总检出率为51％（35／68）,其中Rb-1基因缺失43％（29／68）,13q14．3位点缺失为52％（23／44）,两位点同时缺失者66％（29／44）。卡方检验分析显示13号染色体部分缺失与患者起病时多种临床特征及早期疗效、1年生存率有关。结论Rb-1基因和13q14．3位点的缺失在MM中均较为常见,13号染色体部分缺失对MM的生物学行为有一定影响。     

荧光原位杂交检测尿脱落细胞染色体异常对膀胱肿瘤诊断的意义

目的 探讨尿脱落细胞荧光原位杂交检测染色体异常在诊断膀胱肿瘤中的临床意义.方法 收集我院经治的35例膀胱肿瘤患者(试验组)和30例在我院进行健康体检的正常人(对照组)的尿液,采用3、7、17号染色体着丝粒探针进行荧光原位杂交,并观察两组对象细胞染色体畸变情况.结果 膀胱肿瘤患者的3、7、17号染色体畸变数与正常人的染色体均有显著性差异(P<0.05).3、7、17三种探针的阳性率分别为31.43%、45.71%、40.00%,而联合三种探针的阳性率为62.86%,与单独一种探针比较均具有显著性差异(P<0.05).结论 荧光原位杂交检测尿脱落细胞染色体异常可以作为膀胱肿瘤诊断的一项常规检查方法,无创、快速、有效,值得在临床推广应用.     

荧光原位杂交技术预测人大肠癌细胞放射敏感性的可行性研究

探讨荧光原位杂交（FISH）技术预测人大肠癌细胞放射敏感性的可行性.培养2种不同放射敏感性的人大肠癌细胞系Lovo和SW480,利用集落形成实验测得0、2、4、6 Gy X线照射后的细胞存活率,同时采用FISH技术和2号染色体涂染探针检测0、2、4、6 Gy X线照射后24 h细胞2号染色体的诱导易位畸变量,分析染色体诱导易位畸变量与细胞存活率的相关性.两种细胞的2号染色体诱导易位畸变量与细胞存活率显著相关（r=-0.89,P＜0.05）.FISH技术能快捷、可靠地预测人大肠癌细胞的放射敏感性.     

JAM3基因甲基化是食管鳞状细胞癌潜在的分子标志物

目的研究JAM3基因启动子区在食管癌中的甲基化情况及其表达调控机制,探讨JAM3基因启动子区异常甲基化作为食管鳞状细胞癌的潜在诊断标志物和治疗靶标。方法应用半定量RT-PCR、甲基化特异性PCR等技术对7个食管癌细胞系(KYSE140、KYSE150、KYSE410、KYSE450、COLO680N、KYSE520和TE13)、5例正常食管黏膜组织和83例原发性食管鳞状细胞癌组织进行分析。结果JAM3 mRNA在KYSE520、KYSE140、KYSE450细胞中高表达,这些细胞的JAM3基因启动子区呈非甲基化状态。JAM3 mRNA在KYSE410、COLO680N、TE13、KYSE150细胞中表达缺失,且其基因启动子区在这些细胞中呈完全甲基化。经过5-Aza-dc处理后,JAM3基因在KYSE410、COLO680N、TE13、KYSE150细胞中恢复表达。这些结果表明,JAM3在食管癌细胞中的表达受启动子区甲基化的调控。JAM3基因启动子区在5例正常食管黏膜组织中呈非甲基化状态(0/5),而在原发性食管鳞状细胞癌中其甲基化率为50.6%(42/83),且JAM3甲基化与肿瘤的位置相关(P<0.05)。结论JAM3在食管鳞状细胞癌中频繁发生甲基化,JAM3基因的表达受启动子区甲基化的调控,JAM3基因是潜在的食管癌诊断标志物和治疗靶标。     

荧光原位杂交技术检测慢性淋巴细胞白血病细胞遗传学的异常

目的:探讨荧光原位杂交(FISH)技术检测慢性淋巴细胞白血病(CLL)患者染色体异常的临床价值。方法:运用FISH技术,采用5种特异性DNA探针检测46例CLL患者的染色体异常,并结合患者的临床资料,分析各种染色体异常在临床诊断及预后判断中的价值。结果:46例CLL患者中,32例出现染色体异常,总异常检出率为69.6%,其中del(11q22.3)8例(17.4%),del(13q14.3)14例(30.4%),P53缺失7例(15.2%),del(13q14)5例(10.9%),CSP12阳性10例(21.7%)。结论:FISH技术检测染色体异常的敏感性和特异性较高。del(13q14)是CLL患者最常见的染色体异常。存在基因异常的患者预后较正常者差,尤其是存在P53基因缺失的患者。     

用FISH技术分析一例表型异常的染色体平衡易位

应用荧光原位杂交技术对一例染色体结构异常患者进行分析 ,阐明结构异常性质 ,并精细定位断点。对一先天性表型异常及染色体结构异常的病例 ,分别选用 5号染色体探针池以及用酵母人工染色体作为ＤＮＡ来源制备的断点区位点特异性探针 ,进行荧光染色体原位杂交。结果证实 ,患者染色体异常属平衡易位 ,并将 5号和 10号染色体的断点分别定位到 1.5Ｍｂ及约 3Ｍｂ的范围。患者的先天性表型异常可能由断点处染色体细微重排或致病基因断裂所致。用FISH技术分析一例表型异常的染色体平衡易位@朱冠山!200433$上海第二军医大学附属长海医院 @Oli…     

21例多发性骨髓瘤分子遗传学异常的FISH检测的意义

目的：为探讨间期荧光原位杂交（FISH）在检测多发性骨髓瘤（MM）间期细胞13q14缺失、1q21、p53缺失以及免疫球蛋白重链（IgH）基因重排中的意义。方法：采用组合探针（1q21/RB1、D13S319/p53、IgH）对21例MM患者骨髓进行FISH检测,分析其分子遗传学异常,比较其与常规染色体检查及临床指标的相关性。结果：21例MM患者中,19例（90.48%）检测出1种或1种以上的细胞遗传学异常,15例（71.43%）同时检测出2种及以上的异常。其异常比例从高到低分别为：＋1异常（66.67%）,IgH基因重排（57.14%）,13号染色体缺失（47.62%）和p53基因丢失（23.81%）。3例（14.29%）通过G-显带常规染色体检查发现异常,与FISH比较两者差异有统计学意义（P〈0.01）。结论：＋1、IgH基因重排及13q14缺失在MM中的发生率较高。FISH技术能提高MM分子遗传学异常的敏感性。     

An analysis of complex chromosomal aberrations in seven cases of myelodysplastic syndromes by M-FISH and whole chromosome painting

Complex chromosomal aberrations (CCAs) can be detected in a substantial proportion of myelodysplastic syndrome (MDS). Comprehensive analysis of the chromosomal rearrangements in these CCAs has been hampered by the limitations of conventional cytogenetics (CC). Multiplex fluorescence in situ hybridization (M-FISH) is a new generation FISH technique which allows simultaneous identification of all the 24 human chromosomes. So it is very useful in clarifing CCAs, identifing cryptic interchromosomal rearrangements and characterizing marker chromosomes. But it also has some limitations. We used M-FISH and whole chromosome painting (WCP) to accurately refine the CCAs revealed by R-banding CC in seven cases with MDS. The composition and origin of 6 kinds of marker chromosomes, nine kinds of chromosomes with additional material undetermined and five kinds of derivative chromosomes undefined by CC were defined after M-FISH analysis. Four kinds of cryptic translocations overlooked by CC were found on derivative chromosomes and previously normal appearing chromosomes. In addition, M-FISH revealed some nonrandom aberrations which most frequently involved chromosome 17 (5/7) and -5/5q-(4/7). Fluorescence flaring is a main factor leading to misinterpretations. Some misclassified and missed chromosomal aberrations by M-FISH were corrected by WCP. M-FISH is a powerful molecular cytogenetic tool in clarification of CCAs. Complementary WCP can further identify misclassified and missed chromosomal aberrations by M-FISH. CC in combination with molecular cytogenetic techniques including M-FISH and WCP can more precisely unravel CCAs.     

Expression of OCT4 in human esophageal squamous cell carcinoma is significantly associated with poorer prognosis

AIM: To explore the expression pattern of OCT4 in human esophageal squamous cell carcinoma and its significance in diagnosis and prognosis.METHODS: Using real-time polymerase chain reaction (PCR), Western blotting, immunocytochemistry and immunohistochemistry, the expression of OCT4 in three esophageal squamous cancer cell lines, KYSE70, KYSE140 and KYSE450, was characterized. OCT4 expression was investigated in a series of 153 esophageal squamous cell carcinoma samples using immunohistochemistry and explored its association with clinicopathological features.RESULTS: Immunohistochemically, OCT4 positive immunostaining was observed in cancer cell nuclei. OCT4 was variably expressed in three esophageal squamous cancer cell lines. Among 153 specimens, 105 (68.7%) were negative or weakly positive for OCT4 staining; 21 (13.7%) were moderately positive and 27 (17.6%) were strongly positive. Higher expression level of OCT4 was significantly associated with higher histological grade (P < 0.001) and poor clinic outcome (P < 0.001).CONCLUSION: The expression of OCT4 enables the tumor to have a higher degree of stemness, which in turn results in a poorer clinical outcome for patients with esophageal squamous cell carcinoma.     

Multicolor fluorescence in situ hybridization studies in multiple myeloma and monoclonal gammopathy of undetermined significance

The aim of the study was to test the multicolor fluorescence in situ hybridization technique (M-FISH) in seven multiple myeloma (MM) and eight monoclonal gammopathy (MGUS) patients. None of the eight MGUS patients had chromosomal abnormalities by conventional cytogenetics. In two of these patients structural abnormalities of chromosomes 2, 11 and 19 were found by M-FISH. However, these findings were not confirmed by conventional in situ hybridization. M-FISH only showed numerical chromosomal abnormalities in one out of the three MM cases with a normal karyotype. In the two MM cases with complex karyotype, M-FISH demonstrated the origin of the marker chromosomes. M-FISH is a useful technique to identify the origin of the marker chromosomes in MM. In contrast, MM or MGUS patients with normal karyotypes by conventional cytogenetics did not show structural abnormalities by M-FISH.     

Numerical Aberrations of Chromosomes 16, 17, and 18 in Hepatocellular Carcinoma (A FISH and FCM Analysis of 20 Cases)

Conventional cytogenetic studies havedemonstrated frequent abnormalities of specificchromosomes in hepatocellular carcinoma, although thereare few reports examining the relationship betweenchromosomal aberrations and clinicopathologic features. Inthis study, numerical aberrations of chromosomes 16, 17,and 18 were examined by fluorescence in situhybridization using pericentromeric DNA probes in 20 cases of surgically removed hepatocellularcarcinoma. DNA ploidy analysis was also performed byflow cytometry. Numerical abnormalities of chromosomes16, 17, and 18 were found in 7 of 19 cases, 15 of 20 cases, and 12 of 20 cases, respectively. Gainand/or loss of more than one chromosome was detected in16 of 19 cases. However, aneuploidy was seen in only 9of 20 tumors by flow cytometry. The incidence of aneusomy 17 and 18 increased with tumor sizeand stage progression. Fluorescence in situhybridization analysis demonstrated that numericalchromosomal aberrations accumulated with tumorprogression in hepatocellular carcinoma.     

Characterization of one newly established esophageal cancer cell line CSEC from a high-incidence area in China

SUMMARY.  The Chaoshan littoral is located in a high-incidence area of esophageal cancer in the south of China. In this study, a new esophageal cancer cell line CSEC was established from a 47-year-old female Chinese patient in this district. The biological characters of the cultured cells were investigated, including morphology, ultrastructure, growth kinetic features, tumorigenicity, expression of tumor-associated antigen and cytogenetic features. CSEC cell line grew continuously with a doubling time of 39.5 h and had been passaged over 80 times. The CSEC cells possessed features of squamous epithelial cells with cytokeratin indicated by immunohistochemical staining and tonofilaments and desmosomes revealed by electron microscopy. Tumorigenicity to severe combined immunodeficient mice was confirmed and the tumors developed revealed well-differentiated squamous cell carcinoma, similar to the origin tumor from which the cell line derived. The cytogenetic analysis demonstrated hypertetraploid karyotypes. Chromosome structure aberrations were common and complicated. Immunohistochemical staining showed that CSEC cells were infected with HPV and over-expressed p53. In summary, the CSEC cell line is a well-differentiated esophageal squamous cell carcinoma cell line from a high-incidence area in southern China. It may provide a useful model for the pathogenesis and therapeutic research of esophageal squamous cell carcinoma.     

Simian AIDS-related lymphoma growth in severe combined immunodeficiency mice is independent of karyotypic abnormalities or Bcl-6 mutations

Simian AIDS-related lymphomas (sARL) of cynomolgus monkeys infected with a simian immunodeficiency virus (SIVsm) were studied in relation to growth in severe combined immunodeficient (SCID) mice, karyotype abnormalities, and DNA sequence of the first noncoding region of the Bcl-6 gene. The tumors were diffuse large B cell lymphomas and expressed a simian homolog to Epstein-Barr virus (HVMF-1) in 12 of 13 primary tumors and corresponding cell lines. A tested cell line was tumorigenic in SCID mice. Tumors in the SCID mice showed cell growth features similar to those in the original lymphoma, suggesting that no subpopulation with growth advantage was selected for in the mice. Spectral karyotype analysis of sARL cell lines showed normal cytogenetic features except for a trisomy of monkey chromosome 2 (corresponding to human chromosomes 7 and 21) in two of five sARL lines, which was not recovered in SCID tumors established from the same cell line. Sequence analysis of a Bcl-6 gene fragment showed sequence variations indicative of population polymorphism(s) in 10 of 13 sARLs, and no evidence of Bcl-6 mutations. Thus Bcl-6 mutations in the first noncoding region are irrelevant for sARL development in cynomolgus monkeys and for tumorigenicity of sARL cell lines. We also demonstrate that no cytogenetic alterations are needed for the development of highly aggressive lymphomas in the SIV-immunosuppressed host.     

Establishment of two new myeloma cell lines from bilateral pleural effusions: evidence for sequential in vivo clonal change

Two new human myeloma cell lines have been established from a 36-year- old woman with refractory IgG kappa multiple myeloma in whom bilateral malignant pleural effusions developed. The malignant plasma cells from each effusion were set up in a liquid culture using an L-15 medium containing catalase, glutathione, selenous acid, ascorbic acid, insulin, transferrin, additional glutamine hydrocortisone, and 2- mercaptoethanol and designated as M-3 medium. Two IgG kappa cell lines, LB -831 and LB-832, were established and proved to be Epstein-Barr virus negative using the internal repeat sequence DNA probe. Characteristic plasma cell morphology was evident by light and electron microscopy. Immunotyping revealed an IgG kappa , B1+, B2-, Ia (HLA- DR)+, CALLA+ phenotype for each cell line as well as for the original pleural fluid and bone marrow myeloma cells. The supernatants also contained IgG kappa, beta 2 microglobulin, and large amounts of osteoclast-activating factor (indicating bone-resorbing activity). Cytogenetic analysis of the LB-831 cell line revealed a nearly triploid highly abnormal karyotype with numerous clonal chromosomal abnormalities involving chromosomes 1, 3, 5, 7, 13, and 15; several structurally abnormal marker chromosomes; and a putative homogeneously staining region on chromosome 7p at band p22. Analysis of the LB-832 cell line revealed several additional clonal abnormalities. These additional cytogenetic changes suggest that in vivo sequential clonal evolution occurred in this patient. Therefore, two new but related cell lines have been established, which should prove useful for further biological studies.