应用荧光原位杂交技术检测84例慢性淋巴细胞白血病遗传学异常及预后分析

 摘要 目的：探讨荧光原位杂交 (fluorescence in situ hybridization,FISH) 技术检测慢性淋巴细胞白血病(chronic lymphocytic leukemia,CLL)患者的遗传学异常,并分析与其他预后指标的相关性及预后价值。方法：回顾性分析采用FISH技术进行着丝粒探针CSP12(12p11.1～12q11.1)和序列特异性探针D13s25(13q14.3)、ATM (11q22.3)、RB1(13q14)、p53(17p13.1)检测过的84例CLL患者的临床以及实验室资料。分析FISH技术检测CLL患者中遗传学异常的发生率,并分析遗传学异常与CLL患者年龄、性别、Binet分期、血清乳酸脱氢酶(lactate dehydrogenase,LDH)、CD38表达、生存期之间的相关性。结果：(1)84例CLL患者中,62例有至少有一种遗传学异常。遗传学异常的检出率为73.8%。(2)共发现5种遗传学异常,依次为13q14缺失（56%）、P53缺失（28.6%）、-12（16.7%）、ATM缺失（13.1%）、+12（13.1%）。其中-12为CLL病人中新发现遗传学异常。(3)分析上述5种遗传学异常与年龄、性别、Binet分期、LDH水平、CD38表达的关系,在>60岁年龄病人里,+12阳性率显著高于<60岁病人(24%VS3%, P=0.042),与性别、Bient分期、LDH水平、CD38表达无关。(4)在LDH增高组中,CD38高表达的病例比例增高（67%VS20%, P=0.015）。(5) 伴有del(17p13)或del(11q22)或复杂异常中位生存时间为75个月,单纯具有del(13q14)异常组的中位生存时间为150个月,其他中位生存时间为120月,但两两比较没有达到统计学差异。结论：(1)FISH技术是一种在分析CLL患者遗传学异常方面较为快速、准确和敏感的方法; (2)CLL病人中除了常见13q14缺失、P53缺失、ATM缺失、+12之外,还可以出现-12异常。     

Detection of specific genetic abnormalities by fluorescence in situ hybridization in soft tissue tumors

For the detection of chromosome translocations/chimeric genes and specific genetic abnormalities in soft tissue tumors, we conducted fluorescence in situ hybridization (FISH) analysis on 280 cases of soft tissue and other tumors using formalin-fixed paraffin-embedded tissue sections. The detection rate of the FISH split-signal was 84% (129/154 cases) for the translocation-associated soft tissue tumors, such as Ewing's sarcoma/primitive neuroectodermal tumor, synovial sarcoma, alveolar rhabdomyosarcoma, myxoid liposarcoma, clear cell sarcoma and so forth. Positive split-signals from EWSR1, SS18 and FOXO1A probes were detected in 3% (2/64) of various histological types of carcinoma, lymphoma, melanoma, meningioma and soft tissue tumors. In FISH using the INI1/CEP22 probe, the INI1 deletion signal was detected in 100% (9/9) of epithelioid sarcoma. In well-differentiated and dedifferentiated liposarcomas, detection of MDM2 amplification signals in FISH using the MDM2/CEP12 probe were both as high as 85% (11/13) and 100% (13/13), respectively. In other adipocytic and non-adipocytic tumors requiring differentiation from these types, detection was only 13% (5/39), and CEP12 polysomy was frequently detected. As these results demonstrate the high sensitivity and specificity of FISH, we concluded FISH to be a useful pathological diagnostic adjunct for definite and differential diagnosis of soft tissue tumors.     

Comparison of HER2 immunohistochemical results using a monoclonal antibody (SV2-61γ) and a polyclonal antibody (for Dako HercepTest) in advanced gastric cancer

We compared a monoclonal antibody (SV2-61γ) and a polyclonal antibody (Dako HercepTest) in immunohistochemical assessments of human epidermal growth factor receptor 2 (HER2) expression in 73 samples of advanced gastric cancer. Results were scored as 0 to 3+, and equivocal or discordant (SV2-61γ/Dako HercepTest = 0/2+, 0/3+, 1+/3+ or 2+/3+) cases were subjected to fluorescence in situ hybridization (FISH) analysis. The frequencies of HER2 scores of 2+ or 3+ were 15.1% (11/73) using SV2-61γ and 38.4% (28/73) using Dako HercepTest. All of the equivocal or discordant cases with a HER2 score of 3+ using Dako HercepTest exhibited amplification of the HER2 gene regardless of the HER2 score determined with SV2-61γ. The results of the HER2 tests differed according to the antibodies used for immunohistochemistry that preceded FISH analysis, being 15.1% (11/73) using SV2-61γ and 23.3% (17/73) using Dako HercepTest. Thus, therapeutic decisions might be markedly influenced by the selection of antibody used in the HER2 test.     

Examination of the stimulatory signaling potential of a channel catfish leukocyte immune-type receptor and associated adaptor

Expressed by various subsets of myeloid and lymphoid immune cells, channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs) are predicted to play a key role in the initiation and termination of teleost cellular effector responses. These type I transmembrane proteins belong to the immunoglobulin superfamily and display features of immunoregulatory receptors with inhibitory and/or stimulatory signaling potential. Expanding on our previous work, which demonstrated that putative stimulatory IpLITR-types associated with the catfish adaptor proteins IpFcRγ and FcRγ-L, this study focuses on the functional significance of this immune receptor-adaptor signaling complex. Specifically, we generated an epitope-tagged chimeric receptor construct by fusing the extracellular domain of IpLITR 2.6b with the transmembrane region and cytoplasmic tail of IpFcRγ-L. This chimera was stably expressed in a rat basophilic leukemia (RBL) cell line, RBL-2H3, and following cross-linking of the surface receptor with an anti-hemagglutinin monoclonal antibody or opsonized microspheres, the chimeric teleost receptor induced cellular degranulation and phagocytic responses, respectively. Site-directed mutagenesis of the immunoreceptor tyrosine-based activation motif encoded within the cytoplasmic tail of the chimera confirmed that these functional responses were dependent on the phosphorylated tyrosines within this motif. Using a combination of phospho-specific antibodies and pharmacological inhibitors, we also demonstrate that the IpLITR/IpFcRγ-L-induced degranulation response requires the activity of Src homology 2 domain containing protein tyrosine phosphatases, phosphatidylinositol 3-kinase, protein kinase C, and mitogen-activated protein kinases but appears independent of the c-Jun N-terminal kinase and p38 MAP kinase pathways. In addition to this first look at stimulatory IpLITR-mediated signaling and its influence on cellular effector responses, the advantage of generating RBL-2H3 cells stably expressing a functional IpLITR-adaptor chimera will be discussed.     

The expression and role of c-Myc in mouse hair follicle morphogenesis and cycling

Although the function of c-Myc has been clarified in many tissues, until now its expression and role in hair follicle morphogenesis and the hair cycle remains unknown. In this study we detected c-Myc expression pattern in the process of mouse hair follicle development and normal cycle. We found that during hair follicle morphogenesis, the stage-specific expression of c-Myc was detected in mouse skin and was predominantly localized to the hair follicle epithelium. c-Myc expression was also consistently found in mouse skin throughout the hair follicle cycle. Through the in vivo injection of c-Myc inhibitory peptide and c-Myc expression plasmid, we also investigated the direct effects of c-Myc on the hair follicle structures during the hair follicle cycle. Our results showed that c-Myc inhibitory peptide significantly restrained the development of anagen hair follicles, while the injection of plasmid DNA encoding c-Myc in vivo clearly promoted anagen development. Our data indicate that c-Myc may play an important role in the proliferation and differentiation of the hair follicle keratinocytes during hair follicle development. c-Myc also was shown to participate in the regulation of the mouse hair growth cycle and could promote the proliferation of the hair matrix keratinocytes as well as the differentiation of the inner root sheath.     

HBV DNA免提取核酸荧光定量PCR检测法应用评价

目的评价乙型肝炎病毒核酸免提取荧光定量PCR方法(一步法)对不同病毒载量时检测性能。方法评价试剂为圣湘公司HBV DNA荧光定量PCR检测免提取试剂盒,对照试剂为匹基公司、科发公司、达安公司3家煮沸核酸提取法试剂盒。将4种试剂盒按各自要求对配套的不同浓度标准品、阴性、临界值的反应曲线考核其线性和灵敏度。另检测1份已知浓度质控品、5份未知浓度样本血清考核其准确性、可比较性。检测仪器为瑞士罗氏公司Light Cycler荧光定量仪。结果一步法、匹基公司、科发公司、达安公司4家标准品浓度相关性R2分别为0.992、0.998、0.999及0.963,线性均满意。4个不同浓度标准品扩增曲线图形:一步法曲线比其他3家更为光滑,呈间距均匀的S形抛物线,2次结果重复曲线更趋重迭。对已知定值为(1.5E+06)IU/mL的质控品检测:一步法、匹基公司、科发公司、达安公司相应结果为(1.16E+06)、(5.27E+05)、(4.32E+05)及(4.90E+06)IU/mL,一步法最接近靶值。5份血清结果显示:4种试剂盒除P公司对2号样本结果为低于检出下限外,其他对1号、2号、3号、5号检出结果均与其他3家接近,可为临床接受。4号样本为一个低值临界范围,出现低于报告限、低值及临界追踪值3种结果现象。结论 4家HBV DNA荧光定量PCR检测检测试剂线性结果均满意,对中、高浓度样本检测结果较接近,但在低浓度临界范围检测报告可能会对临床诊断带来误导,需追踪复查。HBV PCR一步法检测技术免除了核酸高温裂解提取步骤,操作简便,人为误差减少,性能稳定,有利提高检测灵敏度、结果准确性。     

SISH检测乳腺癌HER2基因及17号染色体倍体分析

目的:评价银增强原位杂交(SISH)用于乳腺癌患者人表皮生长因子受体2(HER2)基因扩增状态检测的可行性和17号染色体多体的发生情况及其意义。方法:对90例手术后乳腺浸润性导管癌患者标本行全自动免疫组织化学(IHC)染色检测HER2蛋白表达,荧光原位杂交(FISH)和SISH检测HER2基因状态及17号染色体多体的发生率。结果:在IHC(3+)、(2+)、(1+/0)3组中,SISH检测与FISH检测在检测HER2基因扩增方面的总体符合率为96%。17号染色体多体的发生率在IHC(3+)、(2+)、(1+/0)3组中分别为44%,26%和0.02%,其总的发生率为18%。结论:SISH技术用于乳腺癌HER2基因状态检测结果与FISH相当,具有可行性和实际使用价值;17号染色体可能与乳腺癌患者的预后有关。     

宫颈上皮内瘤变与正常宫颈组织差异表达蛋白分析

目的探讨蛋白质组学方法筛查正常宫颈与宫颈上皮内瘤变(cervical intraepithelial neoplasia,CIN)组织中的差异表达蛋白质标志物,为研究CIN发生的分子机制及临床诊治工作提供依据。方法收集2008年8月至2009年9月间首都医科大学附属北京妇产医院妇瘤科收治的正常子宫颈组织因子宫肌瘤手术行全子宫切除术患者9例为对照组、CIN组织23例(其中CINⅠ7例、CINⅡ8例、CINⅢ8例)。应用二维荧光差异凝胶电泳(two-dimensional fluorescence difference in gel electrophoresis,2-DDIGE)及DeCyder软件寻找差异表达蛋白质点,基质辅助激光解析飞行时间串联质谱(MALDI-TOF/TOF MS)分析差异蛋白质点,数据库搜索鉴定差异表达最明显的S100蛋白家族A9(S100 calcium-binding protein A9,S100A9),真核延长因子1α1(eukaryoticelongation factor 1-alpha-1,eEF1A1)及丙酮酸激酶M2(pyruvate kinase M2,PKM2)3种蛋白质;应用免疫组织化学法(immunohistochemistry,IHC)(其中正常宫颈组织10例、CINⅠ10例、CINⅡ10例、CINⅢ10例)及蛋白印记法(Western blotting)(其中正常宫颈组织12例,CINⅠ～Ⅱ12例,CINⅢ12例)进一步验证上述3种蛋白在CIN组织和正常宫颈组织的表达差异。结果获得CIN组织与正常宫颈组织2-D DIGE图,成功鉴定25个蛋白;免疫组化及蛋白印迹验证结果提示:S100A9蛋白表达于细胞质中,在CIN中表达水平高于正常组,差异有统计学意义(P=0.010);eEF1A1蛋白表达于细胞质中、PKM2蛋白表达于细胞核中,2者在CIN中表达水平均低于正常组,差异有统计学意义(P分别为0.352,0.000)。结论在正常宫颈组织与CIN组织之间存在差异蛋白质表达,S100A9蛋白可能成为辅助诊断CIN的相关标志物,PKM2蛋白可能成为抑制CIN发生的蛋白质,并可根据2种差异蛋白表达预测CIN的发生发展。     

荧光定量RT-PCR检测沙门氏菌方法的建立

目的建立沙门氏菌实时荧光定量PCR的快速检测方法 ,探讨该方法的可行性和应用价值。方法根据沙门氏菌fimY基因序列设计引物和探针,采用基因重组技术构建用于沙门氏菌检测的定量标准品,建立实时荧光定量PCR检测沙门氏菌的方法。结果成功构建了沙门氏菌重组质粒标准品和沙门氏菌实时荧光定量PCR方法 ;通过特异性、敏感性、稳定性和重复性验证,结果表明具有较好的特异性、敏感性、稳定性和重复性;对模拟标本与分离培养对比,二者符合率为100%。结论沙门氏菌实时荧光定量PCR检测方法的建立,为食源性沙门氏菌污染的快速检测提供依据,可应用于食品卫生监管、商品检验检疫以及临床诊断等。