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.     

Gene expression profiling of embryo-derived stem cells reveals candidate genes associated with pluripotency and lineage specificity

Large-scale gene expression profiling was performed on embryo-derived stem cell lines to identify molecular signatures of pluripotency and lineage specificity. Analysis of pluripotent embryonic stem (ES) cells, extraembryonic-restricted trophoblast stem (TS) cells, and terminally-differentiated mouse embryo fibroblast (MEF) cells identified expression profiles unique to each cell type, as well as genes common only to ES and TS cells. Whereas most of the MEF-specific genes had been characterized previously, the majority (67%) of the ES-specific genes were novel and did not include known differentiated cell markers. Comparison with microarray data from embryonic material demonstrated that ES-specific genes were underrepresented in all stages sampled, whereas TS-specific genes included known placental markers. Investigation of four novel TS-specific genes showed trophoblast-restricted expression in cell lines and in vivo, whereas one uncharacterized ES-specific gene, Esg-1, was found to be exclusively associated with pluripotency. We suggest that pluripotency requires a set of genes not expressed in other cell types, whereas lineage-restricted stem cells, like TS cells, express genes predictive of their differentiated lineage.     

Gene expression profiling of oral squamous cell carcinoma using laser microdissection and cDNA microarray

Cancer diagnosis and therapy are performed on the basis of clinical stage and clinicopathological findings; however, sensitivity to therapy and prognosis may not always be the same even when considering similar cancers because it is difficult to recognize adequate biological characteristics of a cancer when determining cancer therapy. To enable personalized medicine for cancer diagnosis and therapy, which may solve this problem, we used laser microdissection and cDNA microarrays to study the gene expression profile of oral squamous cell carcinoma. Moreover, to establish an objective evaluation with this system, we examined which type of gene expression profile corresponded to the biological characteristics of this cancer. We identified several genes that were up- or downregulated in the majority of cases and clarified genes sharing common behavioral profiles between metastasis-positive and metastasis-negative cases. It was suspected that the genes that were commonly up- or downregulated in the majority of cases were important for histogenesis and acquisition of invasion and proliferation capability and that the genes sharing common behavioral profiles between metastasis-positive and metastasis-negative cases played a large role in cancer metastasis. Using the expression profile of these genes, it may be possible to evaluate cellular state and metastatic potential and use them as tumor markers. Alternately, we showed averaged gene expression profiles in cases with or without metastasis; this may reveal a profile that could evaluate metastatic potential, which is an important element in the biological characteristics of cancer. In conclusion, our system using laser microdissection and cDNA microarray may contribute to cancer diagnosis and therapy and improvement in the quality of life of cancer patients.     

Microbeam MOMeNT: non-contact laser microdissection of membrane-mounted native tissue.

The analysis of tissue-specific genetic alterations depends on the selective procurement of homogeneous cell populations. Microbeam microdissection of membrane-mounted native tissue (MOMeNT) permits the rapid, selective, and low-contamination procurement of tumor or other cells from histological sections by non-thermic non-contact laser microdissection. Tissue sections are mounted on a specifically designed ultrathin transparent supporter membrane. Tissue together with the membrane are then dissected with an ultraviolet (337-nm) pulsed laser microbeam coupled into a robot-stage microscope. The ultraviolet laser causes dissection by cold photolysis due to the high photon density of the microbeam rather than by local heating. The track of the laser microbeam can be preselected freely on a video screen, and the size and form of the dissectates can thus be adapted to the histological features of the section with a delineation accuracy in the micron range. Polymerase chain reaction amplification of DNA from the dissectates is not impaired, and tumor-specific loss of heterozygosity of the APC gene as well as homozygous deletion of the MTS1 gene are demonstrated in bladder carcinomas. Taken together, microbeam MOMeNT is a novel technique that utilizes membrane-based microdissection by an ultraviolet laser microbeam, thus providing a flexible, easy-to-use high-performance tool for the molecular pathologist.     

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.     

Embryoid body-mediated differentiation of mouse embryonic stem cells along a hepatocyte lineage: insights from gene expression profiles

Pluripotent embryonic stem (ES) cells represent a promising renewable cell source for the generation of functional differentiated cells. Previous studies incorporating embryoid body (EB)-mediated stem cell differentiation have, either spontaneously or after growth factor and extracellular matrix protein supplementation, yielded populations of hepatocyte lineage cells expressing mature hepatocyte markers such as albumin (ALB). In an effort to promote ES cell commitment to the hepatocyte lineage, we have evaluated the effects of four culture conditions on albumin and gene expression in differentiating ES cells. Quantitative in situ immunofluorescence and cDNA microarray analyses were used to describe not only lineage specificity but also to provide insights into the effects of disparate culture environments on the mechanisms of differentiation. The results of these studies suggest that spontaneous and collagen-mediated differentiation induce cells with the highest levels of ALB expression but mature liver specific genes were only expressed in the spontaneous condition. Further analysis of gene expression profiles indicated that two distinct mechanisms may govern spontaneous and collagen-mediated differentiation.     

Microdissection is essential for gene expression profiling of clinically resected cancer tissues.

The gene expression array method enables us to achieve expression profiling with thousands of genes. Clinically resected bulk cancer tissues, however, contain not only cancer cells but also stromal cells, which may affect gene expression profiling and hamper accurate analysis of the cancer cells per se. Therefore, a procedure for dissecting specific cells, such as laser capture microdissection, is neededfor the clinical application of a gene expression array. There has been no study actually comparing 2 gene expression profiles, one obtained using RNA extractedfrom cancer cells by laser capture microdissection and one obtained using RNA extractedfrom bulk cancer tissues. Wefirst demonstrated the difference in expression patterns between them, without any amplification procedures. In addition, differential expression analysis between tumor and nontumor tissue yielded quite different patterns between the 2 methods. We conclude that microdissection is essential for gene expression profiling of clinical specimens.     

Antigen-specific gene expression profiles of peripheral blood mononuclear cells do not reflect those of T-lymphocyte subsets

Advances in microarray technology have allowed for the monitoring of thousands of genes simultaneously. This technology is of particular interest to immunologists studying infectious diseases, because it provides tremendous potential for investigating host-pathogen interactions at the level of immune gene expression. To date, many studies have focused either on cell lines, where the physiological relevance is questionable, or on mixed cell populations, where the contributions of individual subpopulations are unknown. In the present study, we perform an intrasubject comparison of antigen-stimulated immune gene expression profiles between a mixed population of peripheral blood mononuclear cells (PBMC) and the two predominant cell types found in PBMC, CD4+ and CD8+ T lymphocytes. We show that the microarray profiles of CD4+ and CD8+ T lymphocytes differ from each other as well as from that of the mixed cell population. The independence of the gene expression profiles of different cell types is demonstrated with a ubiquitous antigen (Candida albicans) as well as with a disease-specific antigen (human immunodeficiency virus p24). This study has important implications for microarray studies of host immunity and underscores the importance of profiling the expression of specific cell types.     

Prospects for tissue-specific analysis of gene expression in Xenopus embryos through laser-mediated microdissection of histological sections

Analysis of spatiotemporal patterns of gene expression is an important prerequisite for understanding the molecular basis of embryogenesis. Tissue-specific resolution is desirable, but often not achieved owing to methodical limitations. We used a common model system for embryonic development--the South African clawed frog Xenopus laevis--to demonstrate that laser microdissection and laser-mediated catapulting of tissue samples from histologic sections are feasible even for yolk-rich, fragile embryonic tissue. A combination with RT-PCR provides the possibility of detecting tissue-specific gene expression with high resolution and fidelity. We show that specimens of various sizes and shapes can easily be procured by laser microdissection and pressure catapulting (LMPC). Subsequent RNA-isolation and nested RT-PCR for marker genes revealed that the combination of these methods allows for analysis of specific gene expression in micro-areas. We report on the efficiency and reliability of detection of marker genes in dissected tissue. We further discuss the question of whether such a combination can be applied to certain issues raised in developmental biology with regard to other techniques.     

Tumor–microenvironment interactions studied by zonal transcriptional profiling of squamous cell lung carcinoma

Invasion is a critical step in lung tumor progression. The interaction between tumor cells and their surroundings may play an important role in tumor invasion and metastasis. To better understand the mechanisms of tumor invasion and tumor–microenvironment interactions in lung tumors, total RNA was isolated from the inner tumor, tumor invasion front, adjacent lung, and distant normal lung tissue from 17 patients with primary squamous cell lung carcinoma using punch‐aided laser capture microdissection. Messenger RNA expression profiles were obtained by microarray analysis, and microRNA profiles were generated from eight of these samples using TaqMan Low Density Arrays. Statistical analysis of the expression data showed extensive changes in gene expression in the inner tumor and tumor front compared with the normal lung and adjacent lung tissue. Only a few genes were differentially expressed between tumor front and the inner tumor. Several genes were validated by immunohistochemistry. Evaluation of the microRNA data revealed zonal expression differences in nearly a fourth of the microRNAs analyzed. Validation of selected microRNAs by in situ hybridization demonstrated strong expression of hsa‐miR‐196a in the inner tumor; moderate expression of hsa‐miR‐224 in the inner tumor and tumor front, and strong expression of hsa‐miR‐650 in the adjacent lung tissue. Pathway analysis placed the majority of genes differentially expressed between tumor and nontumor cells in intrinsic processes associated with inflammation and extrinsic processes related to lymphocyte physiology. Genes differentially expressed between the inner tumor and the adjacent lung/normal lung tissue affected pathways of arachidonic acid metabolism and eicosanoid signaling. © 2012 Wiley Periodicals, Inc.     

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。     

Genome and transcriptome profiles of CD133-positive colorectal cancer cells

Colorectal carcinomas (CRC) might be organized hierarchically and contain a subpopulation of tumorigenic, putative cancer stem cells that are CD133 positive. We studied the biological and genetic characteristics of such cells in CRC cell lines and primary tumors. Three CRC cell lines were sorted in CD133 positive and negative fractions. The respective genetic aberration profiles were studied using array comparative genomic hybridization (aCGH) and expression profiling. Tumorigenicity for each cellular population was tested by injection into nude mice. Additionally, we compared CD133+ and CD133- cells of 12 primary colorectal tumors using laser capture microdissection and aCGH. Three of five CRC cell lines displayed both CD133+ and CD133- cells, but tumorigenicity of these subfractions did not differ significantly and aCGH revealed essentially identical genomic imbalances. However, 96 genes were differentially expressed between the two populations. Array comparative genomic hybridization analysis after laser capture microdissection of CD133+ and CD133- areas in primary colorectal tumors revealed genetic differences in 7 of 12 cases. The use of cell lines for studying genomic alterations that define cancer stem cell characteristics, therefore, seems questionable. In contrast, CD133+ cells in primary cancer samples showed a unique genomic aberration profile. In conclusion, our data suggest that CD133 positivity defines a genetically distinct cellular compartment in primary CRC, which potentially includes tumor initiating cells.     

Transcription profiling of platelet-derived growth factor-B-deficient mouse embryos identifies RGS5 as a novel marker for pericytes and vascular smooth muscle cells

All blood capillaries consist of endothelial tubes surrounded by mural cells referred to as pericytes. The origin, recruitment, and function of the pericytes is poorly understood, but the importance of these cells is underscored by the severe cardiovascular defects in mice genetically devoid of factors regulating pericyte recruitment to embryonic vessels, and by the association between pericyte loss and microangiopathy in diabetes mellitus. A general problem in the study of pericytes is the shortage of markers for these cells. To identify new markers for pericytes, we have taken advantage of the platelet-derived growth factor (PDGF)-B knockout mouse model, in which developing blood vessels in the central nervous system are almost completely devoid of pericytes. Using cDNA microarrays, we analyzed the gene expression in PDGF-B null embryos in comparison with corresponding wild-type embryos and searched for down-regulated genes. The most down-regulated gene present on our microarray was RGS5, a member of the RGS family of GTPase-activating proteins for G proteins. In situ hybridization identified RGS5 expression in brain pericytes, and in pericytes and vascular smooth muscle cells in certain other, but not all, locations. Absence of RGS5 expression in PDGF-B and PDGFR beta-null embryos correlated with pericyte loss in these mice. Residual RGS5 expression in rare pericytes suggested that RGS5 is a pericyte marker expressed independently of PDGF-B/R beta signaling. With RGS5 as a proof-of-principle, our data demonstrate the usefulness of microarray analysis of mouse models for abnormal pericyte development in the identification of new pericyte-specific markers.     

X chromosome-specific cDNA arrays: identification of genes that escape from X-inactivation and other applications

Mutant alleles are frequently characterized by low expression levels. Therefore, cDNA array-based gene expression profiling may be a promising strategy for identifying gene defects underlying monogenic disorders. To study the potential of this approach, we have generated an X chromosome-specific microarray carrying 2423 cloned cDNA fragments, which represent up to 1317 different X-chromosomal genes. As a prelude to testing cell lines from patients with X-linked disorders, this array was used as a hybridization probe to compare gene expression profiles in lymphoblastoid cell lines from normal males, females and individuals with supernumerary X chromosomes. Measurable hybridization signals were obtained for more than half of the genes represented on the chip. A total of 53 genes showed elevated expression levels in cells with multiple X chromosomes and many of these were found to escape X-inactivation. Moreover, the detection of a male-viable deletion encompassing three genes illustrates the utility of this array for the identification of small unbalanced chromosome rearrangements.