激光捕获显微切割技术在肿瘤研究中的进展

激光捕获显微切割技术(LCM,Laser Capture Microdissection)是目前最先进的组织纯化技术,LCM结合各种基于细胞、DNA、RNA、及蛋白质的分子生物学技术成功运用于肿瘤学研究的各个方面,通过对切割后细胞的基因组,转录组,蛋白组等分析研究后得到了大量有用的结论.本文就其在肿瘤学研究中的应用进行综述.     

激光捕获显微切割技术在植物基因组研究中的应用

植物的生长和发育在很大程度上取决于组织和(或)器官特异表达的基因, 但要获取某一发育阶段的特异细胞类群来进行基因表达分析又是相当困难的。近年发展起来的激光捕获显微切割技术可以在显微镜下快速准确地获取单一的细胞类群, 甚至单个细胞, 成功地解决了组织中细胞的异质性问题。介绍了该技术的原理, 并对其在植物中的应用进展情况做了综述, 同时指出了该技术在植物中应用的可能发展方向。     

Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) to provide an abundant supply of cells. NPCs were differentiated in the presence of a new combination of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), dibutyryl cAMP (DBC) and retinoic acid on dishes coated with poly-L-lysine and mouse laminin to obtain neuron-rich cultures. NPCs were also differentiated in the absence of neurotrophins, DBC and retinoic acid and in the presence of ciliary neurotrophic factor (CNTF) to yield astrocyte-rich cultures. Differentiated NPCs were characterized by immunofluorescence staining for a panel of neuronal markers including NeuN, synapsin, acetylcholinesterase, synaptophysin and GAP43. Glial fibrillary acidic protein (GFAP) and STAT3, astrocyte markers, were detected in 10-15% of differentiated NPCs. To facilitate cell-type specific molecular characterization, laser capture microdissection was performed to isolate neurons cultured on polyethylene naphthalate (PEN) membrane slides. The methods described in this study provide valuable tools to advance our understanding of the molecular mechanism of neurodegeneration.     

激光捕获显微切割在肿瘤多组学研究中的应用进展

当今社会,肿瘤因其高发病率和高死亡率成为威胁人类健康的重要疾病,研究者们对其发病机制及治疗手段的研究和探索也在不断深入。随着单细胞多组学测序技术的发展,肿瘤组织的异质性问题逐渐被研究人员所认识。为了解决这一问题,激光捕获显微切割（laser capture microdissection,LCM）技术应运而生。LCM技术是一种在显微镜直视下从器官或组织中准确获取某种特定的细胞群或单个细胞的样本收集技术。LCM技术结合多种分子生物学手段可以对异质性组织进行多组学研究,丰富了现有的肿瘤蛋白质组学、基因组学以及转录组学图谱,因此,LCM技术成为研究特异性表达及分子机制的有力工具,在肿瘤学领域得到广泛应用。基于此,对LCM的原理、优势及其在肿瘤多组学研究中的应用进行了综述,并对其未来可能的发展方向进行了展望,以期为肿瘤的研究和治疗提供新的思路。     

激光捕获显微切割技术及其在植物研究中的应用

若要获得植物特定类型细胞的准确信息,首要的是获得同质的目标细胞。近年发展起来的激光捕获显微切割技术能够在显微镜下准确、快捷地获得所需要的目标细胞群甚至是单个细胞,从而成功地解决了组织中细胞异质性问题。文章概述了激光捕获显微切割技术的原理、注意的问题以及在植物科学研究中的应用和前景。     

激光显微切割技术用于子宫内膜异位组织DNA的提取及其完整性分析

目的：探索一套激光显微切割（LCM）分离子宫内膜异位症腺体细胞后提取微量DNA并进行完整性分析的操作流程。方法：分别对20例石蜡标本及20例冰冻标本进行LCM,收集切割后的腺体细胞;2组标本各取10例提取微量DNA,检测DNA浓度并通过PCR扩增进行验证;余20例标本分别进行全基因组扩增,检测产物浓度并利用8种常见管家基因作为引物通过PCR扩增进行验证,对比分析其结果。结果：石蜡标本与冰冻标本在LCM获取腺体细胞及提取微量DNA两个环节中均可获得满意效果;但经全基因组扩增后,石蜡标本无法保留完整DNA信息。结论：LCM获取子宫内膜异位症腺体细胞提取微量DNA是一种操作简单、结果稳定的方法,可作为日后子宫内膜异位症基因组研究的常规方法;冰冻切片相对石蜡切片,更能保留完整的DNA信息。     

Editor's Choice: Mate Pair Sequencing of Whole-Genome-Amplified DNA Following Laser Capture Microdissection of Prostate Cancer

High-throughput next-generation sequencing provides a revolutionary platform to unravel the precise DNA aberrations concealed within subgroups of tumour cells. However, in many instances, the limited number of cells makes the application of this technology in tumour heterogeneity studies a challenge. In order to address these limitations, we present a novel methodology to partner laser capture microdissection (LCM) with sequencing platforms, through a whole-genome amplification (WGA) protocol performed in situ directly on LCM engrafted cells. We further adapted current Illumina mate pair (MP) sequencing protocols to the input of WGA DNA and used this technology to investigate large genomic rearrangements in adjacent Gleason Pattern 3 and 4 prostate tumours separately collected by LCM. Sequencing data predicted genome coverage and depths similar to unamplified genomic DNA, with limited repetition and bias predicted in WGA protocols. Mapping algorithms developed in our laboratory predicted high-confidence rearrangements and selected events each demonstrated the predicted fusion junctions upon validation. Rearrangements were additionally confirmed in unamplified tissue and evaluated in adjacent benign-appearing tissues. A detailed understanding of gene fusions that characterize cancer will be critical in the development of biomarkers to predict the clinical outcome. The described methodology provides a mechanism of efficiently defining these events in limited pure populations of tumour tissue, aiding in the derivation of genomic aberrations that initiate cancer and drive cancer progression.     

胶片吸附的激光显微切割技术及其在蛋白质组研究中的应用

组织显微切割在特定细胞亚群的比较研究中起着重要作用.新近发展的胶片吸附激光显微切割技术能从复杂的生物组织中快速准确地分离纯化出特定细胞亚群.在这项技术中,透明的乙烯乙酸乙烯基酯热塑性胶片覆盖于病理组织切片表面,CO₂激光束特异性作用于目的细胞群表面的胶片,胶片对细胞较强的吸附作用使得选择性自动移取目的细胞群成为可能.目前,这项技术已在蛋白质组分析中获得成功应用,并有望对其产生较深远的影响.     

Gene Expression Profiles of Beta-Cell Enriched Tissue Obtained by Laser Capture Microdissection from Subjects with Type 2 Diabetes

Background

Changes in gene expression in pancreatic beta-cells from type 2 diabetes (T2D) should provide insights into their abnormal insulin secretion and turnover.

Methodology/Principal Findings

Frozen sections were obtained from cadaver pancreases of 10 control and 10 T2D human subjects. Beta-cell enriched samples were obtained by laser capture microdissection (LCM). RNA was extracted, amplified and subjected to microarray analysis. Further analysis was performed with DNA-Chip Analyzer (dChip) and Gene Set Enrichment Analysis (GSEA) software. There were changes in expression of genes linked to glucotoxicity. Evidence of oxidative stress was provided by upregulation of several metallothionein genes. There were few changes in the major genes associated with cell cycle, apoptosis or endoplasmic reticulum stress. There was differential expression of genes associated with pancreatic regeneration, most notably upregulation of members of the regenerating islet gene (REG) family and metalloproteinase 7 (MMP7). Some of the genes found in GWAS studies to be related to T2D were also found to be differentially expressed. IGF2BP2, TSPAN8, and HNF1B (TCF2) were upregulated while JAZF1 and SLC30A8 were downregulated.

Conclusions/Significance

This study made possible by LCM has identified many novel changes in gene expression that enhance understanding of the pathogenesis of T2D.     

激光捕获显微切割技术在膀胱移行细胞癌相关基因研究中的应用

背景与目的:激光捕获显微切割技术 (LCM)是获取均一目的细胞的有效方法。利用LCM技术从膀胱粘膜中分离膀胱移行上皮细胞从肿瘤间质细胞中分离膀胱癌细胞,进行RNA提取、纯化、浓缩以备进一步研究。方法:采用LCM技术分别从正常膀胱粘膜及膀胱癌组织冰冻切片中获取膀胱移行上皮细胞及膀胱癌细胞,提取RNA,并对微量RNA进行纯化、浓缩。然后用RT PCR验证TotalRNA中β actin基因表达水平。结果:对照实验Ⅰ证实经LCM后RNA完整性较好;经对照实验Ⅱ初步确定设定条件下LCMshooting次数与可获得RNA量间对应关系。从膀胱粘膜中捕获膀胱移行上皮细胞 2 5万shootings;从膀胱癌组织中获取癌细胞 2 0万shootings。经RT PCR验证β actin基因表达表达完整。结论:使用LCM技术能成功地获取较为均一的研究目的细胞,RNA完整性较好,能用于进一步研究中。     

应用全外显子测序和显微切割技术识别1 例肺癌患者高频突变基因的异质性探索

目的:通过对一例肺鳞癌患者全外显子测序来识别这例肺癌的可能致病基因,并通过显微切割初步探索这例肺癌肿瘤细胞的起源与演化。方法:利用全外显子测序技术对肺癌肿瘤组织和相应癌旁组织测序;用COSMIC肿瘤数据库比较分析统计出肺癌可能致病基因;用激光显微切割技术提取五个不同部位肿瘤细胞;巢式PCR扩增,一代测序验证基因分型。结果:发现了这例肺癌病人的7个高频突变基因:LPHN2、TP53、MYH2、TGM2、C10orf137、MS4A3和EP300;这些基因在10×镜下和20×镜下经显微切割的肺癌组织的5个不同部位上的基因分型不同。结论:我们通过全外显子测序发现了这例肺癌的7个可能致病基因,并初步探索了这例肺癌肿瘤细胞是多克隆起源的。     

应用LCM和蛋白质组学技术筛选肺腺癌的差异表达蛋白质

为筛选肺腺癌(lung adenocarcinoma,AdC)发病相关蛋白质,首先采用激光捕获显微切割技术(laser capture microdissection,LCM)分别从AdC组织和正常支气管上皮(normal bronchial epithelium,NBE)组织中切割并收集AdC细胞和NBE细胞,再应用双向凝胶电泳技术(two-dimensional gel electrophoresis,2-DE)分离经LCM收集的细胞蛋白质,通过PDQuest软件分析差异表达的蛋白质点,基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)鉴定差异表达蛋白质,组织芯片免疫组化方法检测差异蛋白质膜联蛋白A4(annexin A4)在30例AdC组织、配对的癌旁组织和淋巴结转移癌组织中的表达水平。研究结果显示,通过蛋白质组学方法建立了LCM收集的AdC和NBE细胞的2-DE图谱,质谱鉴定得到了33个差异表达蛋白质,其中21个蛋白质在AdC细胞中表达上调,12个蛋白质在AdC细胞中表达下调。组织芯片免疫组化结果显示,与癌旁肺组织相比,annexin A4在AdC组织中的表达水平显著上调,且在AdC淋巴结转移癌组织中的表达明显高于其原发癌组织。上述结果提示annexin A4与AdC的发病及淋巴结转移相关,有望成为诊断AdC及预测AdC转移的分子标志物。     

Maintaining RNA integrity in a homogeneous population of mammary epithelial cells isolated by Laser Capture Microdissection

Background

We investigated the effects of the signaling molecules, cyclic AMP (cAMP) and protein-kinase C (PKC), on gap junctional intercellular communication (GJIC) between thymic epithelial cells (TEC).

Results

Treatment with 8-Br-cAMP, a cAMP analog; or forskolin, which stimulates cAMP production, resulted in an increase in dye transfer between adjacent TEC, inducing a three-fold enhancement in the mean fluorescence of coupled cells, ascertained by flow cytometry after calcein transfer. These treatments also increased Cx43 mRNA expression, and stimulated Cx43 protein accumulation in regions of intercellular contacts. VIP, adenosine, and epinephrine which may also signal through cyclic nucleotides were tested. The first two molecules did not mimic the effects of 8-Br-cAMP, however epinephrine was able to increase GJIC suggesting that this molecule functions as an endogenous inter-TEC GJIC modulators. Stimulation of PKC by phorbol-myristate-acetate inhibited inter-TEC GJIC. Importantly, both the enhancing and the decreasing effects, respectively induced by cAMP and PKC, were observed in both mouse and human TEC preparations. Lastly, experiments using mouse thymocyte/TEC heterocellular co-cultures suggested that the presence of thymocytes does not affect the degree of inter-TEC GJIC.

Conclusions

Overall, our data indicate that cAMP and PKC intracellular pathways are involved in the homeostatic control of the gap junction-mediated communication in the thymic epithelium, exerting respectively a positive and negative role upon cell coupling. This control is phylogenetically conserved in the thymus, since it was seen in both mouse and human TEC preparations. Lastly, our work provides new clues for a better understanding of how the thymic epithelial network can work as a physiological syncytium.     

激光微切割与定量PCR技术分析肾脏病理切片RNA

采用激光微切割与定量PCR技术,分析使用不同提取方法从不同固定方法固定的病理切片中提取的RNA.用70%乙醇、丙酮、甲醇、4%多聚甲醛固定肾脏冰冻切片,使用激光微切割技术切取肾小球,用硫氰酸胍方法(guanidinethiocyanatemethods,GTC)和Trizol试剂方法提取RNA,使用Taqman定量PCR方法分析比较各组RNA的量;选取丙酮固定的石蜡切片,使用激光微切割技术切取肾小球,采用RNA裂解液提取RNA,使用Taqman定量PCR方法,比较石蜡切片和冰冻切片中RNA含量.结果显示:提取沉淀性固定剂如乙醇、丙酮、甲醇固定的冰冻切片的RNA时,2种提取方法和3种固定方法对RNA含量的影响都无明显差异;但在提取4%多聚甲醛固定冰冻切片时,使用Trizol提取RNA含量明显高于使用GTC方法,且其含量与沉淀性固定剂固定的切片RNA含量无明显差异.石蜡切片中经激光微切割肾小球的RNA含量与冰冻切片经激光微切割肾小球的RNA含量无明显差异.结果提示:切片的固定方法和RNA的提取方法是影响切片RNA提取量的主要原因.     

Unambiguous Detection of Multiple TP53 Gene Mutations in AAN-Associated Urothelial Cancer in Belgium Using Laser Capture Microdissection

In the Balkan and Taiwan, the relationship between exposure to aristolochic acid and risk of urothelial neoplasms was inferred from the A>T genetic hallmark in TP53 gene from malignant cells. This study aimed to characterize the TP53 mutational spectrum in urothelial cancers consecutive to Aristolochic Acid Nephropathy in Belgium. Serial frozen tumor sections from female patients (n = 5) exposed to aristolochic acid during weight-loss regimen were alternatively used either for p53 immunostaining or laser microdissection. Tissue areas with at least 60% p53-positive nuclei were selected for microdissecting sections according to p53-positive matching areas. All areas appeared to be carcinoma in situ. After DNA extraction, mutations in the TP53 hot spot region (exons 5–8) were identified using nested-PCR and sequencing. False-negative controls consisted in microdissecting fresh-frozen tumor tissues both from a patient with a Li-Fraumeni syndrome who carried a p53 constitutional mutation, and from KRas mutated adenocarcinomas. To rule out false-positive results potentially generated by microdissection and nested-PCR, a phenacetin-associated urothelial carcinoma and normal fresh ureteral tissues (n = 4) were processed with high laser power. No unexpected results being identified, molecular analysis was pursued on malignant tissues, showing at least one mutation in all (six different mutations in two) patients, with 13/16 exonic (nonsense, 2; missense, 11) and 3/16 intronic (one splice site) mutations. They were distributed as transitions (n = 7) or transversions (n = 9), with an equal prevalence of A>T and G>T (3/16 each). While current results are in line with A>T prevalence previously reported in Balkan and Taiwan studies, they also demonstrate that multiple mutations in the TP53 hot spot region and a high frequency of G>T transversion appear as a complementary signature reflecting the toxicity of a cumulative dose of aristolochic acid ingested over a short period of time.     

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.