DETECTING EXPRESSION OF MRP-1/CD9 mRNA IN LUNG CANCERS USING TISSUE MICROARRAYS AND FLUORESCENCE IN SITU HYBRIDIZATION METHODS

Objective： The aim of this study was to investigate the MRP-1/CD9mRNA expression in lung cancer and normal lung tissues and the relationship between its expression and pathologic grades, clinical stages, metastasis and prognosis. Methods： To observe MRP-1/C9mRNA expression, tissue microarray （TMA） containing 54 lung cancers and 10 normal lung tissues was prepared and Fluorescence in situ hybridization was used. Results： The positive rate of MRP-1/CD9 expression was 48.1% in lung cancer, lower than that of normal lung tissues. The statistical difference was significant （P〈0.05）. Its protein expression had no relationship with the patients＇ ages, sex and the macroscopic type of tumor, but had relationships with the histological type, clinical stage, differentiated degree and metastasis. The expression in non-small cell lung cancer （NSCLC） was higher than that in small cell lung cancer （SCLC）; in well-moderately differentiated group was higher than that in poorly differentiated group; Earlier period group （I＋II） was higher than in later period group （Ⅲ＋Ⅳ）; and in group without lymphoid metastasis was higher than in patients with lymphoid metastasis. Conclusion： The progression of the lung cancer maybe related with the descended MRP-1/Cd9 expression, which may be useful in evaluating the prognosis of cancer patients.

CGH-based microarray detection of cryptic and novel copy number alterations and balanced translocations in cytogenetically abnormal cases of b-cell all

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children, with the majority of cases being of precursor B-cell phenoltype. Conventional cytogenetic analysis plays an important role in the diagnosis of B-cell ALL, identifying characteristic chromosomal abnormalities associated with a given prognosis therein facilitating optimized treatment. The more recent introduction of microarray technology to the analysis of B-cell ALL has afforded both higher resolution for the detection of known abnormalities and an ability to identify novel copy number abnormalities (CNAs) with potential clinical relevance. In the current study, microarray analysis was performed on 20 cytogenetically abnormal B-cell ALL cases (10 pediatric and 10 adult), while a novel microarray-based balanced-translocation detection methodology (translocation CGH or tCGH) was applied to that subset of cases with a known or suspected recurrent balanced translocation. Standard microarray analysis identified that CNAs was not detected by previous conventional cytogenetics in 75% (15/20) cases. tCGH identified 9/9 (100%) balanced translocations defining BCR/ABL1 (x4), ETV6/RUNX1 (x3), and MLL/AFF1 (x2) breakpoints with high resolution. The results illustrate the improved molecular detail afforded by these technologies and a comparison of translocation breakpoints, CNAs and patient age offers new insights into tumor biology with potential prognostic significance.

微阵列(microarrays)技术及其应用

微阵列分为ｃＤＮＡ微阵列和寡聚核苷酸微阵列。微阵列上“印”有大量已知部分序列的ＤＮＡ探针，微阵列技术就是利用分子杂交原理，使同时被比较的标本（用同位素或荧光素标记）与微阵列杂交，通过检测杂交信号强度及数据处理，把他们转化成不同标本中特异基因的丰度，从而全面比较不同标本的基因表达水平的差异。微阵列技术是一种探索基因组功能的有力手段。

特纳综合征患者额外小标记染色体的鉴定与分析

目的探讨染色体核型分析联合荧光原位杂交(FISH)和染色体微阵列分析(CMA)技术对特纳综合征(TS)患者额外小标记染色体(sSMC)的鉴定与分析,并预测sSMC的染色体核型。方法选取2020年1月至2022年12月经外周血染色体核型分析确诊为TS的5例患儿,使用FISH和CMA技术对sSMC的来源与结构组成进行分析。结果FISH结果提示5例TS患者的sSMC均被鉴定出来,其中2例sSMC来源于Y染色体,3例来源于X染色体,并且所有患儿均存在染色体嵌合现象。CMA结果提示5例患儿均存在异常的染色体拷贝数变异,其中2例患儿存在Y染色体片段的扩增,2例患儿存在X染色体的片段缺失,1例患儿存在整条X染色体缺失。结合染色体核型分析、FISH和CMA结果,除1例染色体低比例嵌合者外,其他4例患儿均可预测出该sSMC的染色体核型。结论染色体核型分析、FISH和CMA技术联合可以准确预测出sSMC的核型,但在染色体嵌合水平较低时无法预测出sSMC的核型。

应用aCGH技术建立胚胎植入前遗传学筛查

目的应用微阵列比较基因组杂交技术(aCGH)对移植前胚胎进行染色体遗传学筛查,建立胚胎染色体非整倍体检测方法。方法对冷冻囊胚、对照质控品先进行全基因组扩增,然后再继续aCGH检测。结果 4个冷冻囊胚中1个染色体正常,另外3个染色体异常,对照质控全部检测出。结论应用全基因组扩增以及aCGH技术,对囊胚成功进行了植入前遗传学筛查,能够全面评估胚胎染色体的非整倍体情况,为复发流产患者提高生殖成功率提供重要依据。

利用生物信息数据库筛选乳腺癌CGH微阵列BAC克隆

利用CGAP数据库和UCSC数据库检索出乳腺癌发生、发展过程有意义的BAC克隆,然后利用CGAP数据库设计更有意义的BAC克隆。结果:获得1286条BAC克隆,可用于打印CGH微阵列,进行乳腺癌的检测。

利用cDNA微阵列-CGH筛选支气管上皮细胞恶性转化中的扩增基因

目的:基因组不稳定是导致肺癌发生与发展的重要分子机理之一。本研究旨在筛选支气管上皮细胞恶性转化过程中拷贝数扩增的基因。方法:利用业已建立的支气管上皮细胞体外恶性转化模型,通过cDNA微阵列-CGH技术对支气管上皮来源的永生化细胞和癌变细胞的基因拷贝数进行了检测,并对部分结果进行了实时PCR验证。结果:永生化BEP2D细胞染色体中的某些区域存在不同程度的扩增,包括5q31.3、9q32-33.1、14q22.2-23.1、19p13.12-13.13、20q13.12-13.31;恶性转化BERP35T2细胞染色体中的扩增区域集中在1p12-13.1、5q33.1、5q31.3、9q32、19p13.12-13.13;5q31.3、9q32、19q13.12-13.13是以上2种细胞系中的共同扩增区域。共检测到201个基因的拷贝数发生扩增,其中PCNA、TP53及GADD45A基因的异常扩增已经实时PCR进一步验证。结论:在支气管上皮细胞恶性转化过程中,病毒与低剂量辐射的双重作用使得某些重要基因的拷贝数发生扩增,因基因剂量增加而导致某些癌基因高表达可能是细胞恶性转化的重要机制之一。

EXPRESSION AND SIGNIFICANCE OF SURVIVIN mRNA IN LUNG CANCER TISSUE MICROARRAY DETECTED BY FISH

Objective To investigate the expression of Survivin mRNA in lung cancer tissue microarray (TMA) by fluorescence in situ hybridization (FISH) method, and determine the role and significance of it in lung cancer genesis and progress. Methods The expression of Survivin mRNA was detected by FISH method and TMA technology. Fifty-four cases of lung cancer and 10 cases of normal lung tissue were examined. Results Survivin mRNA was expressed in 66.7% (36/54) of lung cancer; the positive ratio of lung cancer was significantly higher than that of normal lung tissue (0/10; χ2 = 15.238, P < 0.05). The positive ratio of Survivin mRNA was significantly higher in poor differentiated cancer (20/24, 83.3%) than moderate and well differentiated cancer (16/30, 53.3%; χ2 = 5.40, P < 0.05). The positive ratio of Survivin mRNA was significantly higher in group with lymph node metastasis (27/32, 84.4%) than without lymph node metastasis (9/22, 40.9%; χ2 = 11.084, P < 0.05). The positive ratio of Survivin mRNA was significantly higher in stage Ⅲ-Ⅳ(12/13, 92.3%) than stage Ⅰ-Ⅱ (24/41, 58.5%; χ2 = 5.066, P < 0.05). Conclusion Survivin mRNA highly expresses in lung cancer, which is related to the progress and malignant behavior. Survivin may play a promoting role in lung cancer genesis and progress and provide a basis for estimating prognosis and treatment.

Fiber-fluorescence in situ hybridization analyses as a diagnostic application for orientation of microduplications

Microduplications are normally invisible under microscopy and were not recognized before chromosomal microarray testing was available. Although it is difficult to confirm the orientation of duplicated segments by standard fluorescence in situ hybridization(FISH), our data indicates that fiber-FISH analysis has the potential to reveal the orientation of duplicated and triplicated segments of chromosomes. Recurrent microduplications reciprocal to microdeletions show tandem orientations of the duplicated segments, which is consistent with a non-allelic homologous recombination mechanism. Several random duplications showed tandem configurations and inverted duplications are rare. Further analysis is required to fully elucidate the basic mechanisms underlying such duplications/triplications.

Analysis of gene expression profile of aspermia using cDNA microarray

Objective: To identify the differential gene expression profiles between the normal and aspermia human testes utilizing cDNA microarray. Methods: cDNA probes were prepared by labeling mRNA of aspermia testes tissues with Cy5-dUTP and mRNA of normal testes tissues with Cy3-dUTP respectively through reverse transcription. The mixed cDNA probes were then hybridized with 4096 cDNA arrays (4096 unique human cDNA sequences), and the fluorescent signals were scanned by ScanArray 3000 scanner (General Scanning, Inc.). The values of Cy5-dUTP and Cy3-dUTP on each spot were analyzed and calculated by ImaGene 3.0 software (BioDiscovery, Inc.). Differentially expressed genes were screened according to the criterion that the absolute value of natural logarithm of the ratio of Cy5-dUTP to Cy3-dUTP was greater-than 2.0 or less-than 0.5. A randomly chosen gene RAP1A was studied by in situ hybridization to evaluate the accuracy of the results. Results: 623 differential expressed genes related to aspermia were found. There were 303 up-expressed genes and 320 down-expressed genes. A distinct up-expressed gene RAP1A was confirmed by in situ hybridization. Conclusions: Screening the differential gene expression profiles between the normal and aspermia human testis by cDNA microarray can be used in the study of aspermia-related genes and the further research due to its properties, RAP1A may play some roles in the development and progression of aspermia.

Identification of p63 expression in human lung cancer: analysis by complementary DNA and tissue microarray

Objective: To evaluate p63 expression at mRNA transcripts and protein levels in lung squamous cell cancer (SCC), adenocarcinoma, large cell lung cancer (LCLC) and small cell lung cancer (SCLC) and their matched metastatic tumors. The association between p63 expression and p63 locus at chromosomal 3q27 q29 was also investigated. Methods: p63 mRNA expression levels in a large series of lung cancers including SCC, adenocarcinoma, LCLC, SCLC and their matched metastatic tumors were analyzed by cDNA microarray technology. A tissue microarray from 150 primary lung cancer specimens was constructed and used for immunohistochemical detection of p63 protein expression. Chromosomal imbalances at the p63 locus in 70 primary lung cancers samples were studied by comparative genomic hybridization (CGH) technology. Results: mRNA levels were 10 fold in SCC compared to LCLC, SCLC, and adenocarcinoma. Interestingly, the mRNA expression of p63 in metastatic carcinomas was significantly higher than that in their matched primary tumors ( P <0 001). Immunohistochemistry demonstrated that p63 expression was 94.64% in SCC but only 1 79% in lung adenocarcinoma and 2 of 4 LCLC were positive staining. All the results in of SCLC were negative. There was a statistically significant difference for p63 positivity between pT1 tumors and those of higher stage ( P =0 035). The CGH results indicated that p63 locus at chromosomal 3q27 q29 was overrepresented in SCC. p63 immunopositivity correlated significantly with pronounced gains of the p63 locus at chromosomal 3q27 q29 (P=0.0001), indicating that strong expression of p63 in lung SCC correlated with increased gene amplification. Conclusion: p63 might play an important role not only in squamous differentiation of lung cancer but also in tumor development and progression.

SIMPLE METHOD OF PUNCHING AND RE-LOCATING TISSUES FOR MANUAL CONSTRUCTION OF TISSUE MICROARRAY

A series of human tissue samples and cultured cell lines were formalin-fixed and paraffin-embedded. Specimen cylinder (1.2-1.8mm) were punched by a modified bone marrow biopsy needle and arrayed on a recipient paraffin block. Microscopic analysis on the sections from this tissue microarray (TMA) block demonstrated that the spots of tissues and cells were well preserved, and the cultured cell samples were successfully embedded from 5×10 4 to 2×10 5 in number. These TMA sections were also suitable for immunohistochemistry and RNA in situ hybridization.

MALBAC-NGS和array CGH检测染色体异常囊胚的结果比较

目的比较基因组杂交微阵列芯片(array CGH)与多次退火环状循环扩增-二代测序(MALBAC-NGS)两种方法进行胚胎植入前染色体筛查的结果差异。方法收集西北妇女儿童医院生殖中心行胚胎植入前遗传学筛查/诊断(PGS/PGD),并经array CGH(BlueGnome 24SureV3软件)检测异常的囊胚(共32枚),复苏培养后再次活检,通过MALBAC-NGS的方法进行胚胎染色体非整倍体筛查,比较两种不同检测方法的结果差异。结果32枚囊胚中,内细胞团与外胚层分别活检的有16枚,通过MALBAC-NGS的方法检测,内细胞团与外胚层检测结果完全一致的为14枚,一致率87.5%(14/16);2枚囊胚内细胞团与外胚层部分不一致,不一致率12.5%(2/16)。32枚囊胚中,两种方法检测结果完全一致的有7枚,一致率21.87%(7/32);array CGH法检测为染色体异常的囊胚中,其中有15枚囊胚MALBAC-NGS法检测为正常(46XN),array CGH的假阳性率46.88%(15/32)。array CGH法检测染色体嵌合的胚胎数为0,MALBAC-NGS检测出染色体嵌合的胚胎数为3枚,嵌合率9.37%(3/32)。结论与array CGH法相比,MALBAC-NGS法检测能够降低假阳性率,嵌合检出率高。MALBAC-NGS法检测囊胚内细胞团与外胚层的整倍体一致性较高。

胎儿低比率嵌合体产前诊断方法的比较

目的:探讨染色体微阵列分析(CMA)、荧光原位杂交(FISH)和细胞核型分析在胎儿羊水低比率嵌合体中的诊断差异性。方法:回顾分析我院2019年1月-2020年12月无创DNA产前筛查提示21、18、13为高风险(Z值<5),补充报告为性染色体可能异常的病例,并进一步联合运用CMA、FISH与细胞核型分析确诊其是否为异常核型及嵌合比例。结果:收集30例,FISH检出率100%(95%CI:88.65%~100%),核型分析检出率70%(95%CI:52.12%~83.34%),CMA检出率13.33%(95%CI:5.31%~29.68%)。对于嵌合体的检测,核型分析与FISH方法有统计学差异(P=0.004),CMA与FISH检测也有统计学差异(P<0.001)。结论:对于无创DNA产前筛查提示21、18、13为高风险(Z值<5),或者性染色体数目存在异常的胎儿,FISH可以作为确定嵌合类型以及异常核型嵌合比例的首选检测方法。

1p36与12q24隐匿性易位致多发畸形胎儿的遗传学分析

目的明确超声异常的多发畸形胎儿的遗传学病因及其父母的细胞遗传学分析。方法家系中的父母及胎儿均行常规核型分析(CCA)、单核苷酸多态性微阵列(SNP)分析,并利用荧光原位杂交(FISH)进行验证。结果胎儿的CCA结果为46,XX,父亲和母亲均未见明显异常。SNP结果显示,胎儿存在1p36.33p36.13缺失(15.8 Mb)、12q24.31q24.33重复(10.8 Mb)和Xp22.12重复(506.1 kb);母亲未见异常;父亲存在Xp22.12重复(506.1 kb),但并未有临床疾病表型。FISH分析提示,胎儿ish der(1)t(1;12)(12q+,1q+),即存在1p部分单体和12q部分三体;父亲正常;母亲为ish t(1;12)(12q+,1q+;12p+,1p+),为染色体隐匿性易位携带者。结论1p36.33p36.13与12q24.31q24.33隐匿性易位会导致胎儿畸形,联合CCA、SNP和FISH技术可以更精确地对隐匿性易位患者进行遗传学分析。

染色体嵌合体在基因检测中的研究进展

染色体嵌合体是指个体中存在两个或多个染色体不同的细胞系,这些细胞系来源于单个受精卵。染色体嵌合体标本来源困难、临床结局多样导致临床结果解释和检测困难,是基因研究的难点之一。本文将对嵌合体的形成机制、致病性以及基因检测技术等进行综述,为临床治疗和产前遗传咨询提供更好的诊疗依据。

16例无创产前检测8号染色体异常病例的产前诊断结果及妊娠结局分析

目的探讨无创产前检测(non-invasive prenatal testing,NIPT)提示8号染色体异常病例的产前诊断、遗传学咨询和妊娠结局。方法2016年4月1日至2022年10月31日,在中国医学科学院北京协和医院进行NIPT检测的孕妇中,结果为8号染色体异常且接受产前诊断的孕妇共16例。所有病例均接受羊膜腔穿刺术,进行羊水细胞染色体核型分析及染色体微阵列分析(chromosomal microarray analysis,CMA),部分病例进行针对8号染色体的未培养细胞的荧光原位杂交(fluorescence in situ hybridization,FISH)。回顾性分析患者的临床资料、产前遗传学诊断结果和妊娠结局。结果①13例未提示其他染色体异常,为孤立性NIPT 8号染色体异常组。其中1例为胎儿8-三体嵌合体,经咨询后继续妊娠,后续超声检查未见异常,随访新生儿未见异常。其余12例产前遗传学检测均未见异常,除3例失访之外,所有病例均继续妊娠,随访新生儿均未见异常。②3例同时提示其他染色体异常,为非孤立性NIPT 8号染色体异常组。其中1例产前诊断结果为16-三体嵌合体,超声发现胎儿心脏异常,终止妊娠。其余2例产前遗传学检测未见异常,1例在孕29周发现孕妇罹患左侧乳腺癌,行剖宫产终止妊娠,随访新生儿未见异常;另1例失访。结论对于NIPT提示8号染色体异常的病例,应进一步行产前诊断,遗传学检测的方案建议包含染色体核型分析、CMA和未培养细胞的FISH分析。对于产前诊断胎儿8-三体嵌合体的病例,应进行详细的遗传咨询,如果超声检查未见异常,一般预后良好,可以考虑继续妊娠。

应用DNA微阵列芯片检测青年肺结核患者结核菌耐药性的研究

目的:以结核菌比例法药敏试验为标准,分析DNA微阵列芯片(以下简称DNA微阵列)在聊城市青年肺结核患者痰标本中进行结核分枝杆菌异烟肼和利福平耐药性检测的效能。方法:选取聊城市2019年10月-2021年9月确诊的18~45岁的青年肺结核患者,对其中痰涂片抗酸杆菌阳性的215例患者,通过DNA微阵列和比例法药敏对痰标本进行异烟肼和利福平的耐药性检测,分析DNA微阵列对异烟肼、利福平、耐多药结核诊断的敏感性、特异性、阳性预测值和阴性预测值,并与比例法进行一致性比较。结果:DNA微阵列检测耐多药结核的敏感度和特异度分别为71.4%和99.0%,阳性预测值和阴性预测值分别为83.3%和98.0%,Kappa=0.75,说明两种方法在耐多药结核检测中有高度一致性。DNA微阵列检测异烟肼耐药性的敏感度和特异度分别为76.5%和97.2%,阳性预测值和阴性预测值分别为83.9%和95.7%,Kappa=0.76,说明两种方法在异烟肼耐药性检测中有高度一致性。DNA微阵列检测利福平耐药性的敏感度和特异度为82.1%和98.9%,阳性预测值和阴性预测值分别为92.0%和97.4%,Kappa=0.85,说明两种方法在利福平耐药性检测中基本一致。结论:DNA微阵列适用于青年结核患者快速筛查和耐药性检测,为聊城青年结核病的防控能提供有力支持。

47例鼻咽癌遗传变异的研究

目的：分析鼻咽癌细胞的遗传变异特性。方法：①应用比较基因组杂交（CGH）技术检测47例NPC活检组织DNA拷贝数的改变；②应用微卫星序列分析16号染色体缺失情况。结果：发现与NPC相关的7个DNA拷贝高频增加区和7个高频缺失区：增加的染色体为1q、3q、4、6q、8q、12和18；缺失的染色体有1p、3p、gq、11q、14、16和19P。16号染色体进行了微卫星序列分析，检测出两个异常明显的区域：16p12．3和16q24．3。结论：鼻咽癌具有染色体变异，其中增加最多的是染色体1q和12，缺失最多的是染色体1p和16。