手工制作组织芯片在HPV原位杂交中的应用与体会

组织芯片亦称组织微阵列是近年发展起来的以形态学为基础的分子生物学新技术。其具有高通量、可比性强等优点．在实验研究过程中得到一定范围的应用。由于组织阵列仪价格昂贵，限制了组织芯片在临床广泛应用。在实践常规病理工作中。我们在无芯片制作仪情况下．利用组织芯片技术原理反复多次摸索．得到了一种简易可行的手工制作方法．并用于本科室开展最多的HPV原位杂交实验上取得了满意的效果，现介绍体会如下。     

组织芯片技术在免疫组化及原位杂交阳性对照中的应用

组织芯片亦称组织微阵列(tissue microaryyay,TMA)是将数十个、数百个组织标本整齐地排列在同一张载玻片上的微缩组织切片。组织芯片具有高通量、节约组织原材料和检测试剂、实验条件一致性及实验结果可比性好、实验误差小等优点，在病理学研究中有广阔的应用前景…。我们利用组织芯片技术制作免疫组化及原位杂交的阳性对照组织芯片，将阳性对照的组织芯片与待检测的组织裱在同一张玻璃切片上，进行免疫组化及原位杂交染色，取得了比较理想的结果，现介绍如下。     

组织芯片的基本应用范围

目的探索组织芯片的基本应用范围。方法将HE染色、组织化学、免疫组织化学、原位杂交和荧光原位杂交技术应用于组织芯片，了解这些技术应用于组织芯片的可行性和有效性。结果这些检测技术在组织芯片中均有清晰、定位明确的着色。结论HE染色、组织化学、免疫组化、原位杂交和荧光原位杂交技术可有效地用于组织芯片，因此，组织芯片可用于组织形态观察、组化特性分析以及蛋白和核酸(RNA和DNA)在组织细胞中的定位性研究。     

组织芯片技术在脑胶质瘤研究中的应用

目的 探讨组织芯片技术在脑胶质瘤研究中的应用价值.方法 构建包括50例人脑胶质瘤标本的组织芯片,运用免疫组织化学法检测细胞增殖核抗原Ki-67的表达,并用免疫组织化学法和原位杂交法检测突变型P53蛋白及野生型p53基因的表达.结果 Ki-67表达水平与肿瘤病理分级之间有关(P＜0.05).突变型P53蛋白与野生型p53 mRNA之间呈显著性负相关(P＜0.001).突变型P53蛋白与Ki-67的表达水平之间存在显著性正相关(P＜0.05).结论 应用组织芯片技术大规模高效检测胶质瘤临床组织样本是可行的,具有重要的实用价值和广阔的应用前景.     

组织芯片检测snail mRNA的表达及其与胃癌的关系

目的探讨转录因子snail mRNA在胃癌中的表达及其与临床病理特征和预后的关系。方法制作组织芯片，在组织芯片上通过原位杂交检测snail mRNA在25例非肿瘤胃黏膜组织和100例胃癌组织中的表达情况。结果100例胃癌中snail mRNA的阳性表达率46％，snail mRNA的阳性表达率与胃癌的TNM分期（r=0．176，P=0．027）、淋巴结转移（r=0．299，P=0．003）、脉管转移（r=0．208，P=0．038）、分化程度（r=0．203，P=0．042）呈正相关，与胃癌的病灶大小、浸润深度、远处转移无明显相关性。snail mRNA的阳性表达病例的平均生存时间和5年生存率明显低于阴性表达的病例。结论snail mRNA的表达可预测胃癌的浸润和转移，是指导临床治疗及估计预后的有意义指标。     

组织芯片应用的现状与前景

长期以来，各种传统的特殊染色技术、免疫组织化学、原位杂交等研究方法均建立在常规病理组织切片基础上．一张玻片上只能载有限的组织做一种测试，仅用于日常临床病理诊断尚可胜任．但要从事大规模、多样本的科研则费时、费力。自1998年由Kononen等构建了第一个组织微阵列后．组织芯片技术得到极大发展，     

组织芯片的制备技术

1998年Ｋｏｎｏｎｅｎ等〔1〕在Ｎａｔｕｒｅ《自然》杂志上发表了“组织芯片在肿瘤分子高通量分析中的应用” ,给病理学及生物分析技术开辟了新的领域。组织芯片 (ｔｉｓｓｕｅｍｉｃｒｏａｒｒａｙ ,ＴＭＡ)是将多个微小组织片汇集在一张固相载体上所形成的组织微阵列生物芯片 ,简称组织芯片〔1 ,2〕。ＴＭＡ克服了传统病理学技术步骤繁琐、试验速度慢和效率低的缺点 ,具有小体积、多样本、大信息、可同时分析数百种组织样品的优点。ＴＭＡ可根据实验要求和目的 ,设计出不同的排列组合 ,在疾病的分子诊断、流行病学调查、个体化治疗、预后…     

原位杂交技术在组织工程研究中的应用

背景:通过原位杂交技术对修复组织进行检测,是目前分析组织再生和修复状况的重要方法之一.目的:综述国内外关于原位杂交技术在组织工程应用中的研究概况.方法:应用计算机检索PubMed 1980-01/2009-07期间的相关文章,检索词为In situ hybridization,Tissue engineering,Insulin-like growthfactor-Ⅰ(IGF-Ⅰ),并限定文章语言种类为English.同时计算机检索中国期刊全文数据库1995-01/2009-07期间的相关文章,检索词为原位杂交,组织工程,胰岛素样生长因子1,并限定文章语言种类为中文.此外还手工查阅相关专著数部.纳入原位杂交技术基本原理及原位杂交技术在组织工程中相关应用的文章.结果与结论:原位杂交技术包括分子生物学、细胞遗传学和组织化学等方面的技术,是一种核酸定位和检测基因表达的有效方法.原位杂交技术尤其是荧光原位杂交技术,可对修复的组织进行准确有效的检测,在骨、软骨等组织工程实验研究中有广泛应用,已成为目前分析组织再生和修复状况的重要方法之一.近年来国内外对原位杂交技术在组织工程中的应用研究主要从生物医学和临床医学两方面着手,都获得了众多研究成果.此外,对与组织生长发育密切相关的生长因子胰岛素样生长因子1的原位检测也很有效,三维支架中胰岛素样生长因子1的原位检测是今后工作的重点.     

组织芯片技术在癌症研究中的应用

组织芯片技术已广泛应用于病理学各研究领域，现总结组织芯片技术在癌症研究中的应用现状，包括不同发展阶段相关基因的表达，以及组织芯片技术和DNA芯片技术的联合应用等方面的研究。     

应用原位杂交技术检测HPV的体会

宫颈癌是妇科常见的恶性肿瘤,全球每年约有20余万女性死于宫颈癌,我国每年新发病例有13万之多,严重的危害了女性的生命和健康。研究表明宫颈癌致病因素主要是人乳头瘤病毒(HPV),许多国家也已将HPV检测纳入筛查     

Localization of cystic fibrosis transmembrane conductance regulator mRNA in human fetal lung tissue by in situ hybridization.

The fetal pulmonary epithelium secretes fluid. Cl transport is presumed to provide the driving force for net fluid secretion, although the cellular mechanisms have not been well identified in the fetus. The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP- and nucleoside triphosphate-regulated Cl channel; mutations in CFTR cause cystic fibrosis. We hypothesized that if CFTR is involved in fetal lung fluid transport, the fetal pulmonary epithelium should express CFTR mRNA. We used the technique of in situ hybridization with 3H-anti-sense and, as a control, 3H-sense CFTR cRNA probes to localize CFTR mRNA in human fetal lung tissue and cultured lung explants and determine when in gestation it is expressed. Epithelial cells of both first and second trimester lung tissues expressed CFTR mRNA. A decreasing gradient of CFTR mRNA expression was present from the proximal to the distal pulmonary epithelium. Cultured second trimester lung tissue explants expressed more CFTR mRNA than the uncultured starting tissue, suggesting CFTR gene expression increased during the five days in culture. Furthermore, alveolar type II cells in cultured explants expressed CFTR mRNA, suggesting that these cells are Cl-secretory and may be involved in lung fluid transport. These data confirm that CFTR mRNA is expressed in the human fetal pulmonary epithelium, consistent with the Cl-secretory properties of the fetal lung.     

荧光原位杂交在细胞学诊断中的应用

荧光原位杂交(FISH)是一种可以识别染色体畸变的有效技术,近年来逐渐在细胞学诊断中得到应用.本文通过分别阐述FISH如何从尿液、胆道刷液、支气管刷液和痰液、食管刷液以及妇科脱落细胞学中检测肿瘤细胞,从而证明FISH能够提高细胞学诊断的敏感性,而且该方法 几乎适用于任何细胞学样本类型.但是因为FISH技术费时且昂贵,所以对于大多数细胞学样本来说,FISH技术是对传统细胞学的补充而不是替代.     

应用原位杂交技术比较肺腺癌和肺鳞癌的内在分子机制

目的分析肺腺癌及肺鳞癌肿瘤中染色体异常变化和基因组的改变。探讨肺腺癌及肺鳞癌细胞遗传物质的改变,揭示这两种NSCLC主要亚型发生发展的内在本质及其与临床特征之间的关系。方法应用M-FISH技术,检测肺腺癌及肺鳞癌细胞株各1株,观察其染色体数目及结构畸变情况,应用CGH技术对80例肺腺癌组织80例肺鳞癌组织中所提取的全基因组DNA进行检测,比较这两种类型的NSCLC全基因组的变化。结果M-FISH结果显示肺腺癌及肺鳞癌细胞中存在许多复杂的染色体重排,其中5、6、11、12、17号染色体最频繁参于染色体间的易位。CGH发现,在160例肺癌标本基因组中,最常见的扩增区域足1 q,2 p,3 q,5 p,5 q,7 p,8 q,11 q,12 q,14 q,16 p,17 p.19 p,20 q,21 q,22 q。最常见的缺失区域是2 q.3 p,4 p,5 q,7 q,8 p,9 p.13 q,14 q,17 p。在肺腺癌中最常见的扩增位点足16p13,阳性率达50%.而肺鳞癌中最常见的扩增位点是17q21(45%),并且这两个位点的变异在肺腺癌和肺鳞癌之间有着显著性的差异(P<0.05)。结论M- FISH和CGH技术是研究肺癌基因组变化的强有力的工具,本实验中发现的基因改变町能代表了与肺腺癌及肺鳞癌特有的病理,诊断相关和候选基因。     

Multicolor fluorescence in situ hybridization and comparative genomic hybridization reveal molecular events in lung adenocarcinomas and squamous cell lung carcinomas

We have used the molecular cytogenetic techniques of multicolor fluorescence in situ hybridization (M-FISH) and comparative genomic hybridization (CGH) to analyze two established lung cancer cell lines (A549, H520), 80 primary lung adenocarcinoma samples and 80 squamous cell lung carcinoma samples in order to identify common chromosomal aberrations. M-FISH revealed numerous complex chromosomal rearrangements. Chromosomes 5, 6, 11, 12, and 17 were most frequently involved in interchromosomal translocations. CGH revealed regions on 1q, 2p, 3q, 5p, 5q, 7p, 8q, 11q, 12q, 14q, 16p, 17p, 19q, 20q, 21q and 22q to be commonly over-represented and regions on 2q, 3p, 4p, 5q, 7q, 8p, 9p, 13q, 14q, and 17p to be under-represented. In lung adenocarcinomas the most common gains were found in 16p13 (50%); while in squamous cell lung carcinomas the common gains were found in 17q21 (45%) and these alterations were observed to be associated with their specific pathological subtype. In conclusion, the present study contributes to the molecular biological characterization in lung adenocarcinomas and squamous cell lung carcinomas and through evaluation of molecular events to the recently emergent focus on novel markers for lung cancer treatment.     

Comparative genomic hybridization of hepatocellular carcinoma: correlation with fluorescence in situ hybridization in paraffin-embedded tissue.

BACKGROUND: Proto-oncogene MYC, mapped to chromosomal band 8q24 and the genes for hepatocyte growth factor (HGF at 7q21) and its receptor, MET, at chromosomal band 7q31, have an important role in the biology and growth of normal and neoplastic liver. Comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) studies have reported frequent abnormalities of chromosomes 1 and 8 in hepatocellular carcinomas (HCCs) of various clinical and pathological stages. Chromosome 7 involvement is reported to be less frequent. MATERIALS AND METHODS: Frozen tissue from 17 HCCs was used for CGH analysis and sections of corresponding formalin-fixed, paraffin-embedded HCC tissue were used for dual-color FISH with locus-specific (LSI-cMYC for chromosome 8q24 and LSI D7S486 for chromosome 7q31) and centromeric probes, CEP8 (8p11.1-q11.2) and CEP7 (7p11.1-q11.2) (Vysis, Inc, Downers Grove, IL). This study intended to determine the pattern of chromosomal aberrations in early-stage (incidental) HCC and large surgically resected HCC, and also compared the efficiency and usefulness of the two cytogenetic methods. RESULTS: CGH showed abnormalities on chromosomes 1q, 5q, 7q, 8q, 9, 10, 13q, 15, 16, 17p, 18q, 19, 20, 21, 22, and X. Gains of 8q were noted in 50% of the HCCs, including five cases of incidental HCCs by CGH. Increase in copy numbers of MYC detected by FISH was noted in 25% of tumors that had shown 8q gains by CGH and in five cases with no chromosome abnormalities noted by CGH. Three cases with 7q31 copy number abnormalities were found by FISH in addition to those detected by CGH. CONCLUSION: Combined use of CGH and FISH may provide important information about early and/or primary genetic changes in the development of HCC.     

微组织芯片在免疫组化染色对照实验中的应用

免疫组化技术已经常规用于临床病理诊断。为了确保免疫组化结果的准确性 ,必须进行严格的质量控制 ,而免疫组化的内对照就是确保正常工作所必需的。在实际工作中 ,人们往往使用已知阳性切片作为对照 (外对照 ) ,这无疑增加了额外消耗 ,而且 ,还不能完全保证与患者组织染色的一致性。我们根据组织芯片的原理制作了多组织微芯片[1 ] ,用于免疫组化染色的内对照 ,取得了满意的效果。现报告如下。1　材料与方法1 1　材料　从解放军第 30 6医院病理科档案中选取 9种经4 %甲醛溶液固定、石蜡包埋的组织 ,这些组织均经免疫组化证实与不同抗体呈阳性…     

Quantitative fluorescence in situ hybridization in lung cancer as a diagnostic marker

The diagnosis of lung cancer is quite often hampered by the existence of various cell types within samples such as biopsies or pleural effusions. We have established a new marker for image cytometry of interphase tumor cells of the lung by using the most recurrent and early cytogenetic event in lung cancer, the loss of the short arm of chromosome 3. The method is based on the detection of the imbalance between the long and the short arms of chromosome 3 by performing two-color fluorescence in situ hybridization on both arms. Fourteen tumors were analyzed after short-term culture and compared with the corresponding cytogenetic data obtained from metaphase analysis. Results on interphase nuclei and control experiments on metaphases were the same, with imbalance ratios ranging from 1.0 to 2.0 (mean value 1.6, median 1.5). To assess the clinical significance of this approach, three pleural effusions were analyzed. Data showed that normal cells within the sample could have been distinguished from the tumor cells based on different imbalance values between the long and the short arms. Thus, our method allows refined detection of lung tumor cells within samples containing heterogeneous cell populations.