联合应用激光捕获显微切割与基因芯片技术筛选肝细胞癌差异表达基因

目的 联合应用激光捕获显微切割与基因芯片技术构建纯化肝细胞癌(HCC)组织和正常肝组织基因表达谱,筛选HCC发病相关基因.方法 4例HCC标本先行激光捕获显微切割,分别获取纯化肝癌细胞和正常肝细胞各4对样本.提取微量RNA,经线性扩增后再与全基因组基因芯片杂交,筛选差异表达基因.结果 4对样本间纯化肝癌细胞与正常肝细胞共同差异表达的基因有1361条,上调基因607条,下调基因754条.前10位显著上调和下调的差异表达基因(共20条)中,5条为HCC已知差异表达基因(信号比值为23.2529、27.372 9、0.014 8、0.019 7、0.019 8);11条表达变化与其他肿瘤研究报道一致(信号比值为32.694 9、28.480 9、23.703 0、21.342 3、19.109 3、17.291 9、0.009 8、0.012 5、0.015 8、0.016 1、0.016 5);4条为在HCC和(或)其他肿瘤中均无报道过的差异表达基因(信号比值为24.676 1、22.072 2、0.009 7、0.019 1).结论 联合应用激光捕获显微切割与基因芯片技术,可准确、高效地筛选出HCC差异表达基因.     

激光捕获显微切割联合基因芯片在肿瘤差异表达基因研究中的应用

 目的 为激光捕获显微切割（LCM）联合基因芯片在肿瘤差异表达基因研究中应用摸索可行的技术方法。方法 采用 LCM 技术分别自原发性膀胱移行癌患者手术切除的癌组织和癌旁正常组织冻存标本获取细胞，提取 RNA，用 Agilent 2100 Bioanalyzer 芯片分析系统进行 RNA 完整度（RIN）检测。取总 RNA 100 ng 进行线性扩增和荧光标记，获得 aRNA 探针。取等量的癌组织探针与癌旁正常组织探针，与 Agilent 人全基因组寡核苷酸基因表达谱芯片杂交。通过自身比较实验分析芯片的假阳性基因数，计算假阳性率（FPR）。通过数据分析寻找癌组织与癌旁正常组织差异表达基因。 结果 LCM 所获微量 RNA 的 RIN 都在 8.0 以上，表明LCM 后 RNA 完整度较高。100 ng RNA 经过线性扩增与荧光标记，获 aRNA 产量约 16 µg，片段大小 0.5 ~ 2.5 kb。芯片自身比较实验结果良好，FPR < 1%，验证了实验系统的可靠性。相对于癌旁正常组织，癌组织发生表达上调的基因有 286 条，下调的基因 112 条。 结论 以 LCM 技术获得的细胞提取 RNA 用于制备基因芯片探针，获得了可信的芯片杂交结果，为肿瘤基因差异表达研究提供了可行的技术方法。     

Analysis of odontoblast gene expression using a novel approach,laser capture microdissection

Studying the mechanisms of molecular interactions in developing tissues demands sensitive molecular biological in vivo and in vitro techniques. Laser capture microdissection (LCM) allows for the isolation of mRNA in histological sections even from single cells, thus enabling the identification of in vivo gene expression products in closely circumscribed tissue areas. The aims of this study were to assess the optimal fixation, processing, and staining conditions to retrieve RNA from microdissected odontoblasts. Fluorometric assays and RT-PCR analysis of alpha 1(I) collagen, dentin sialophosphoprotein (Dspp), and osteocalcin (OC) confirmed that the total RNA isolated from day 0 and day 3 captured odontoblasts was sufficient in quantity and quality. Our results indicate that individual odontoblasts obtained by LCM are morphologically intact and chemically unaltered, allowing accurate molecular and biochemical analyses.     

EGFR and KRAS mutation analysis in cytologic samples of lung adenocarcinoma enabled by laser capture microdissection

The discovery of activating mutations in EGFR and KRAS in a subset of lung adenocarcinomas was a major advance in our understanding of lung adenocarcinoma biology, and has led to groundbreaking studies that have demonstrated the efficacy of tyrosine kinase inhibitor therapy. Fine-needle aspirates and other cytologic procedures have become increasingly popular for obtaining diagnostic material in lung carcinomas. However, frequently the small amount of material or sparseness of tumor cells obtained from cytologic preparations limit the number of specialized studies, such as mutation analysis, that can be performed. In this study we used laser capture microdissection to isolate small numbers of tumor cells to assess for EGFR and KRAS mutations from cell block sections of 19 cytology samples from patients with known lung adenocarcinomas. We compared our results with previous molecular assays that had been performed on either surgical or cytology specimens as part of the patient's initial clinical work-up. Not only were we able to detect the identical EGFR or KRAS mutation that was present in the patient's prior molecular assay in every case, but we were also able to consistently detect the mutation from as few as 50 microdissected tumor cells. Furthermore, isolating a more pure population of tumor cells resulted in increased sensitivity of mutation detection as we were able to detect mutations from laser capture microdissection-enriched cases where the tumor load was low and traditional methods of whole slide scraping failed. Therefore, this method can not only significantly increase the number of lung adenocarcinoma patients that can be screened for EGFR and KRAS mutations, but can also facilitate the use of cytologic samples in the newly emerging field of molecular-based personalized therapies.     

Sensitive immunoassay of tissue cell proteins procured by laser capture microdissection

Coupling laser capture microdissection (LCM) with sensitive quantitative chemiluminescent immunoassays has broad applicability in the field of proteomics applied to normal, diseased, or genetically modified tissue. Quantitation of the number of prostate-specific antigen (PSA) molecules/cell was conducted on human prostate tissue cells procured by LCM from fixed and stained frozen sections. Under direct microscopic visualization, laser shots 30 microm in diameter captured specific cells from the heterogeneous tissue section onto a polymer transfer surface. The cellular macromolecules from the captured cells were solubilized in a microvolume of extraction buffer and directly assayed using an automated (1.5 hour) sandwich chemiluminescent immunoassay. Calibration of the chemiluminescent assay was conducted by developing a standard curve using known concentrations of PSA. After the sensitivity, precision, and linearity of the chemiluminescent assay was verified for known numbers of solubilized microdissected tissue cells, it was then possible to calculate the number of PSA molecules per microdissected tissue cell for case samples. In a study set of 20 cases, using 10 replicate samples of 100 laser shots per sample, the within-run (intraassay) SD was approximately 10% of the mean or less for all cases. In this series the number of PSA molecules per microdissected tissue cell ranged from 2 x 10(4) to 6. 3 x 10(6) in normal epithelium, prostate intraepithelial neoplasia (PIN), and invasive carcinoma. Immunohistochemical staining of human prostate for PSA was compared with the results of the soluble immunoassay for the same prostate tissue section. Independent qualitative scoring of anti-PSA immunohistochemical staining intensity paralleled the LCM quantitative immunoassay for each tissue subpopulation and verified the heterogeneity of PSA content between tissue subpopulations in the same case. Extraction buffers were successfully adapted for both secreted and membrane-bound proteins. This technology has broad applicability for the quantitation of protein molecules in pure populations of tissue cells.     

Clonality analysis of follicular lymphoma using laser capture microdissection method

Whether a common and a single clone present, or not, among follicles of follicular lymphoma (FL) was examined in 12 cases with FL. Histologic grade was I in 6 cases, II in 3, and III in 3. DNA was selectively extracted from the neoplastic follicles of paraffin-embedded samples with use of laser capture microdissection method, and used for PCR-based analysis of rearrangement of immunoglobulin heavy chain variable region gene. Three different follicles in each case of FL were microdissected. Semi-nested PCR was performed using two sets of primers (Fr2A and Fr3A). In PCR with Fr2A primers, nine of 12 cases showed a common band among neoplastic follicles. The remaining three cases showed no PCR products. With Fr3A primers, eight of 12 cases showed a common band among follicles of the same case. The other four cases showed oligoclonal bands, among them presence of a common band was difficult to assess. Oligoclonal bands were more frequently observed in PCR with Fr3A than that with Fr2A and in grade I or II than in grade III cases. In total, 11 of 12 cases showed a common band in PCR with either Fr2A or Fr3A primers. In two cases, DNA extracted from whole section was amplified with both Fr2A and Fr3A or only Fr3A primers, showing smear or oligoclonal bands. These results showed the presence of a single clone of cells in neoplastic follicles of FL and the usefulness of PCR-based rearrangement analysis of immunoglobulin heavy chain gene combined with microdissection methods for differential diagnosis of FL from follicular hyperplasia.     

Microarray analysis using amplified mRNA from laser capture microdissection of microscopic hepatocellular precancerous lesions and frozen hepatocellular carcinomas reveals unique and consistent gene expression profiles

The indirect labeling cDNA microarray technique was used to evaluate gene expression profiles of pure cell populations from frozen sections of carcinomas and adenomas harvested from precancerous hepatocellular lesions by using laser capture microdissection (LCM). The levels of differentially expressed genes were investigated using a cDNA microarray with 9,984 features with only 2 ug of two-round amplified aRNA, equivalent to 35 cells from LCM-adenomas and frozen samples of carcinomas from simian virus 40 (SV40) large T antigen transgenic rats. A total of 855 genes were identified as being 3-fold or more differentially expressed in carcinomas or adenomas as compared to normal tissue controls. Among these 855 genes, 71 genes were differentially expressed in both carcinomas and adenomas. Commonly up-regulated genes in both carcinoma and adenomas were 28 while 41 of the 71 genes were commonly down-regulated. Two genes, Igh1 (immunoglobulin heavy chain 1(Serum IgG2a), Image clone ID: 875880) and EST clone (AI893585, Image clone ID: 596604) were more than 7-fold up-regulated in carcinomas and 6-fold down-regulated in adenomas. In Cy5 and Cy3 reciprocal experiments for screening out false positive signals, the amplified carcinomas showed higher Pearson Correlation Coefficient values (-0.94 and -0.92) than the LCM-amplified adenoma samples (-0.79 and -0.84). LCM-amplified samples provided higher signal intensities over backgrounds and a greater average of Cy5:Cy3 ratios. Expression levels of mRNAs from selected genes, determined by using traditional dot blot analysis, revealed that 36 of 40 tested expression profiles were consistent with the microarray data. Thus, amplified aRNA harvested from homogeneous cell types using LCM can be applied to study gene expression profiles by use of microarray analysis.     

Application of laser capture microdissection and proteomics in colon cancer.

AIMS: Laser capture microdissection is a recent development that enables the isolation of specific cell types for subsequent molecular analysis. This study describes a method for obtaining proteome information from laser capture microdissected tissue using colon cancer as a model. METHODS: Laser capture microdissection was performed on toluidine blue stained frozen sections of colon cancer. Tumour cells were selectively microdissected. Conditions were established for solubilising proteins from laser microdissected samples and these proteins were separated by two dimensional gel electrophoresis. Individual protein spots were cut from the gel, characterised by mass spectrometry, and identified by database searching. These results were compared with protein expression patterns and mass spectroscopic data obtained from bulk tumour samples run in parallel. RESULTS: Proteins could be recovered from laser capture microdissected tissue in a form suitable for two dimensional gel electrophoresis. The solubilised proteins retained their expected electrophoretic mobility in two dimensional gels as compared with bulk samples, and mass spectrometric analysis was also unaffected. CONCLUSION: A method for performing two dimensional gel electrophoresis and mass spectrometry using laser capture microdissected tissue has been developed.     

Laser capture microdissection: methodical aspects and applications with emphasis on immuno-laser capture microdissection.

Laser capture microdissection (LCM) is an easy, extremely fast and versatile method for the isolation of morphologically defined cell populations from complex primary tissues for molecular analyses. However, the optical resolution is limited due to the use of dried sections without coverslip necessary for tissue capture, and routine stains such as hematoxylin and eosin are sometimes insufficient for precise microdissection, especially in tissues with diffuse intermingling of different cell types and lack of easily discernible architectural features. Therefore, several groups have adapted immunohistochemical staining techniques for LCM. In addition to providing high contrast targets for microdissection, immunostaining allows selection of cells not only according to morphological, but also phenotypical and functional criteria. In order to allow reliable tissue transfer on one hand and preserve the integrity of the target of analysis such as DNA, RNA and proteins on the other hand, immunostaining protocols have to be modified for the purposes of LCM. The following review gives an overview of immuno-LCM and describes some applications, e.g. in the field of hematopathology.     

Laser capture microdissection reveals dose-response of gene expression in situ consequent to asbestos exposure

The genes that mediate fibroproliferative lung disease remain to be defined. Prior studies from our laboratory showed by in situ hybridization and immunohistochemistry that the genes coding for tumour necrosis factor alpha, transforming growth factor beta, the platelet-derived growth factor A and B isoforms, and alpha-1 pro-collagen are expressed in fibroproliferative lesions that develop quickly after asbestos inhalation. These five genes, along with matrix metalloproteinase 9, a collagenase found to be increased in several lung diseases, are known to control matrix production and cell proliferation in humans and animals. Here we show by laser capture microdissection that (i) The six genes are expressed at significantly higher levels in the asbestos-exposed mice when comparing the same anatomic regions 'captured' in unexposed mice. (ii) The bronchiolar-alveolar duct (BAD) junctions, where the greatest number of fibres initially deposit, were always significantly higher than the other anatomic regions for each gene. The first alveolar duct bifurcation (ADB) generally was higher than the second ADB, the ADBs were always significantly higher than the airway walls and pleura, and the airway walls and pleura were generally higher than the unexposed tissues. (iii) Animals exposed for 3 days always exhibited significantly higher levels of gene expression at the BAD junctions and ADBs than animals exposed for 2 days. To our knowledge, this is the first demonstration of a dose-response to a toxic particle in situ, and this response appears to be dependent on the number of fibres that deposits at the individual anatomic site.     

激光捕获显微切割纯化的人正常支气管上皮与肺鳞癌组织的定量蛋白质组学研究

目的:建立肺鳞癌(human lung squamous carcinoma,HLSC)组织与正常支气管上皮(normal human bronchial epithelial,NHBE)组织的差异蛋白质表达谱.方法:运用激光捕获显微切割(laser capture microdissection,LCM)技术从HLS...     

胃癌hMSH2基因突变与HP感染的关系

目的 通过对胃癌组织中错配修复基因hMSH2突变与幽门螺旋杆菌(HP)感染关系的研究,探讨在胃癌中hMSH2基因突变与临床病理特征的关系,以及HP致癌的可能机制.方法 (1)应用Warthin-Starry银染法检测45例胃癌组织中HP感染的情况.(2)用激光捕获显微切割设备直接获取胃癌细胞.(3)应用聚合酶链反应-单链构象多态性分析(PCR-SSCP)-银染技术,分别检测45例胃癌组织中5个外显子hMSH2基因突变的情况.结果 (1)45例胃癌组织中,HP感染阳性26例(57.78%),HP感染阴性19例(42.22%).(2)45例胃癌组织中,hMSH2基因突变共4例(8.89%),hMSH2基因突变率与胃癌患者的年龄、性别、发生部位、有无淋巴结转移、组织学类型、分化程度及进展程度均无相关性(P>0.05).(3)45例胃癌组织中,19例HP感染阴性的胃癌中,无1例发生hMSH2基因突变;26例HP感染阳性的胃癌中,4例hMSH2基因突变(15.38%),HP感染阴性胃癌组与HP感染阳性组的hMSH2基因突变率经统计学分析,差异无显著性(P>0.05).结论 胃癌hMSH2基因突变与HP感染可能无关.     

Use of laser capture microdissection, cDNA microarrays, and tissue microarrays in advancing our understanding of prostate cancer

One difficulty in studying epithelial tumors has been the inability to isolate pure samples for DNA and RNA analysis. Prostate cancer, with its infiltrative nature, is particularly challenging. The Combination of several new technologies should help overcome these hurdles. Laser capture microdissection uses a laser beam to achieve transfer of pure cell populations for isolation of DNA, RNA, and protein. High-throughput analysis of these samples can be achieved by using cDNA expression microarrays. High-density tissue microarrays should allow for validation of differentially expressed genes. This review will concentrate on the application of laser capture microdissection, cDNA microarrays, and tissue microarrays in the area of prostate cancer research.     

Improved resolution by mounting of tissue sections for laser microdissection.

BACKGROUND: Laser microbeam microdissection has greatly facilitated the procurement of specific cell populations from tissue sections. However, the fact that a coverslip is not used means that the morphology of the tissue sections is often poor. AIMS: To develop a mounting method that greatly improves the morphological quality of tissue sections for laser microbeam microdissection purposes so that the identification of target cells can be facilitated. METHODS: Fresh frozen tissue and formalin fixed, paraffin wax embedded tissue specimens were used to test the morphological quality of mounted and unmounted tissue. The mounting solution consisted of an adhesive gum and blue ink diluted in water. Interference of the mounting solution with DNA quality was analysed by the polymerase chain reaction using 10-2000 cells isolated by microdissection from mounted and unmounted tissue. RESULTS: The mounting solution greatly improved the morphology of tissue sections for laser microdissection purposes and had no detrimental effects on the isolation and efficiency of amplification of DNA. One disadvantage was that the mounting solution reduced the cutting efficiency of the ultraviolet laser. To minimise this effect, the mounting solution should be diluted as much as possible. Furthermore, the addition of blue ink to the mounting medium restores the cutting efficiency of the laser. CONCLUSIONS: The mounting solution is easy to prepare and apply and can be combined with various staining methods without compromising the quality of the DNA extracted.