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。     

用气相色谱测定植物样品中的微量细胞分裂素

细胞分裂素广泛地存在于植物组织和微生物中,对植物的细胞分裂、分化、种子的萌发和休眠等生长发育过程有调节功能。因此,在组织培养、植物细胞工程和开花植物及微生物的生长发育理论和应用技术研究中,都需要对植物体内的细胞分裂素进行测定。随着生物科学的发展,在现有测定细胞分裂素含量的方法中,生物试法准确度差,已不适于研究工作的     

天祝白牦牛退行期毛囊细胞亚群鉴定以及特征基因生物信息学分析

从单细胞水平解析天祝白牦牛退行期毛囊发育过程主要细胞类型,旨在对主要细胞类群特异表达基因进行生物功能预测与生物信息学分析,探索退行期毛囊发育调控机制。采用单细胞转录组测序对退行期毛囊进行异质性分析,利用已知标记分子对细胞类群进行筛选鉴定,并对鉴定得到的主要细胞类群进行GO和KEGG分析,同时对毛囊形态发生过程中涉及的关键分子进行免疫组织分析。结果表明,天祝白牦牛退行期涉及IFE-DC细胞、表皮细胞系、黑色素细胞、INFU细胞等细胞类群。其特征基因主要参与表皮发育、上皮细胞分化、超纤维组织发育、组织形态发生以及细胞形态学发生等生物过程。KEGG富集分析发现,特征基因主要富集在黏附连接、细胞周期、RNA转运等与毛囊发育相关的通路中。涉及的不同细胞类型拥有GJA1、FKBP4、KRT1、KRT80、FGFR2等28个共同基因。该研究成功鉴定出天祝白牦牛退行期主要细胞类群,获得了特征基因富集通路,揭示了退行期毛囊发育机制。     

ProTracer——不间断记录体内细胞增殖的遗传示踪新技术

器官内细胞数量和组织稳态可以通过细胞增殖调节并维持.大多数用于鉴别具有显著增殖能力的细胞的相关研究,都是基于某一细胞亚群的谱系示踪,而这会导致潜在的选择性偏差.该团队利用双同源重组酶系统,开发了一种示踪增殖细胞的新技术—ProTracer,该技术能够实现在多个器官中长时间不间断地记录整个细胞类群的细胞增殖.在肝脏中,P...     

3D-SIM结构照明超分辨率显微镜实现蛋白质在植物亚细胞器内的定位

基因表达产物蛋白质的亚细胞定位是解析基因生物学功能的重要证据之一。近年来出现的超分辨率光学成像技术已成功应用于人类和动物细胞中,预示着显微成像技术继激光共聚焦技术后的又一重要进步。由于植物细胞的特殊性和成像技术的研发取向,超分辨率光学成像技术在植物细胞蛋白质亚细胞定位的应用尚未见报道。该研究利用Delta Vision OMX显微镜技术,克服了叶绿体基粒中叶绿素自发荧光与融合蛋白荧光不易区分的缺陷,解决了受分辨率局限无法将植物细胞中蛋白质在亚细胞器内可视化精确定位的技术难题,成功地将植物蔗糖合成酶Zm SUS-SH1定位在烟草表皮细胞叶绿体基粒周围。该研究同时建立了一套基于撕片制片法的简便OMX显微镜制片方法,并针对OMX显微成像技术在植物细胞中蛋白质亚细胞定位的应用进行了讨论。     

单细胞组学技术及其在植物保卫细胞研究中的应用

单细胞组学技术在动物研究中已经得到广泛应用,但在植物学领域尤其是保卫细胞研究中还处于起步阶段。由保卫细胞构成的气孔承担着植物生命过程中水分散发及气体交换大门的作用。将单细胞组学技术应用到保卫细胞功能解析中将有助于了解保卫细胞参与的基本生理过程。该文综述了植物单细胞组学技术的发展、保卫细胞研究现状及单细胞组学技术在植物保卫细胞研究中的初步应用,为借助该技术解决植物生物学中保卫细胞发育、代谢及对环境胁迫响应等基本问题提供研究思路和方法。     

高通量组织特异表达谱分析技术

测量基因表达谱是研究动物如何发育和应对刺激的重要途径。基因表达谱实验检测的RNA通常来自多种细胞的混合体。这样的数据具有较低的分辨率,不能区分动物体内不同细胞类型的转录组,而且会偏袒RNA含量占优势的组织以及上调的基因。这个问题可以通过获得特异组织的RNA进行表达谱分析来实现。这十几年来已有多种方法被开发出来获取特异的细胞类型或它们的RNA,并与高通量RNA分析技术结合起来生成各种特异组织或细胞的转录组。本综述将介绍这些方法的基本原理和在大尺度表达谱分析中的应用,并讨论它们各自的优点和局限性。     

基于电喷雾电离串联质谱技术的植物脂质组学研究进展

研究表明,脂质不但参与植物的信号转导、小泡运输、细胞骨架重组等多种细胞过程,而且在植物的生长发育和胁迫反应中具有重要作用．但是脂质本身的多样性、复杂性、以及分析手段的滞后限制了人们对脂质的深入认识．电喷雾电离串联质谱(ESI-MS/MS)技术作为一种直接进样的高通量分析技术,能够在短时间内对大多数脂质的不同分子种进行定量分析,极大地方便了人们了解植物因环境变化和生长发育引起的组织内脂质分子种的微量变化．近年来,该技术在植物上的成功应用,推动植物脂质组学研究取得了重要进展,揭示出脂质在植物的逆境胁迫反应、防御反应中的多种功能,促进了植物脂质代谢相关基因的鉴定．而且,该技术与其他脂质分析技术结合,促使人们在脂质的分布、运输、转化和新脂质种类的鉴定方面有新的进展．概要介绍了ESI-MS/MS技术的特点,重点综述了该技术在植物脂质组学研究中的应用进展,并展望了该技术今后的发展方向.     

沙棘属植物的分布格局及其成因

本文首先从植物类群、分布地域和垂直分布角度分别论述了沙棘属植物的地理分布概况,指出沙棘属在植物区系地理上,属旧世界温带分布类型;在区系成分上,是森林─—草原过渡带的成员;横断山及其毗邻的东喜马拉雅地区,是该属植物的类群分布中心、类群分化中心和原始类群中心。并对类群分布的地理替代现象、该属植物成林的丛片性特征以及果实中植化组成分与类群分布、类群进化间的相互关系进行了论述。文章最后着重对该属植物分布区格局的形成与该属植物起源的生态地理环境、散布的时间、路线以及繁育方式之间的关系进行了分析。     

利用激光显微切割与microarray 技术对恒河猴脑中白质与 灰质基因表达的差异性的研究

目的:利用激光显微切割技术和microarray技术比较恒河猴脑组织中前额叶皮质(prefontal cortex,PFC)与小脑皮质(cere-bellar cortex,CBC)的灰质与白质基因表达的差异。方法:利用激光显微切割技术(laser capture dissection,LCM)与microarray技术的有效结合,提取恒河猴PFC与CBC的白质与灰质,分别提取RNA,合成cDNA文库。最后利用GeneChip 1.0 ST芯片技术,分析得出大脑与小脑中灰质与白质的表达差异性。结果:无论是灰质还是白质,在PFC中的高表达基因都要远远多于在CBC中的高表达基因。结论:使用LCM可以提取单一的细胞群,从而用于要求更为精确的实验当中。     

Laser capture microdissection of cells from plant tissues

Laser capture microdissection (LCM) is a technique by which individual cells can be harvested from tissue sections while they are viewed under the microscope, by tacking selected cells to an adhesive film with a laser beam. Harvested cells can provide DNA, RNA, and protein for the profiling of genomic characteristics, gene expression, and protein spectra from individual cell types. We have optimized LCM for a variety of plant tissues and species, permitting the harvesting of cells from paraffin sections that maintain histological detail. We show that RNA can be extracted from LCM-harvested plant cells in amount and quality that are sufficient for the comparison of RNAs among individual cell types. The linear amplification of LCM-captured RNA should permit the expression profiling of plant cell types.     

Construction of a specialized cDNA library from plant cells isolated by laser capture microdissection: toward comprehensive analysis of the genes expressed in the rice phloem

Laser capture microdissection (LCM) is a powerful system which allows the isolation of selectively targeted cells from a tissue section for the analysis of gene-expression profiles of individual cells. The technique has been successfully used for the isolation of specific mammalian cells, mainly cancer cells. However, LCM has never been reported to be applied to the gene expression analysis of plant cells. We used a modified LCM system and successfully applied it to target and isolate phloem cells of rice leaf tissue whose morphology is apparently different from the surrounding cells. Total RNA was extracted from microdissected (approximately 150) phloem cells and the isolated RNA was used for the construction of a cDNA library following the T7 RNA polymerase amplification. Sequence analysis of 413 randomly chosen clones from the library revealed that there was a high level of redundancy in the population and the clones could be subclassified into 124 different groups that contained related sequences. Approximately 37% of both the redundant population and the non-redundant subgroups had novel components while approximately 63% were either homologues to the known genes reported to be localized in phloem of different plant species, or were homologues to other known genes. In situ hybridization revealed that putative amino acid permease, one of the non-redundant clones, was specifically expressed in the phloem. The results proved the effectiveness of construction of a specialized cDNA library from the specific plant cells.     

Laser-capture microdissection,a tool for the global analysis of gene expression in specific plant cell types: identification of genes expressed differentially in epidermal cells or vascular tissues of maize

Laser-capture microdissection (LCM) allows for the one-step procurement of large homogeneous populations of cells from tissue sections. In mammals, LCM has been used to conduct cDNA microarray and proteomics studies on specific cell types. However, LCM has not been applied to plant cells, most likely because plant cell walls make it difficult to separate target cells from surrounding cells and because ice crystals can form in the air spaces between cells when preparing frozen sections. By fixing tissues, using a cryoprotectant before freezing, and using an adhesive-coated slide system, it was possible to capture large numbers (>10,000) of epidermal cells and vascular tissues (vascular bundles and bundle sheath cells) from ethanol:acetic acid-fixed coleoptiles of maize. RNA extracted from these cells was amplified with T7 RNA polymerase and used to hybridize a microarray containing approximately 8800 maize cDNAs. Approximately 250 of these were expressed preferentially in epidermal cells or vascular tissues. These results demonstrate that the combination of LCM and microarrays makes it feasible to conduct high-resolution global gene expression analyses of plants. This approach has the potential to enhance our understanding of diverse plant cell type-specific biological processes.     

Optimized procedures for microarray analysis of histological specimens processed by laser capture microdissection

Analysis of cell-specific gene expression patterns using microarrays can reveal genes that are differentially expressed in diseased and normal tissue, as well as identify genes associated with specialized cellular functions. However, the cellular heterogeneity of the tissues precludes the resolution of expression profiles of specific cell types. While laser capture microdissection (LCM) can be used to obtain purified cell populations, the limited quantity of RNA isolated makes it necessary to perform an RNA amplification step prior to microarray analysis. The linearity and reproducibility of two RNA amplification protocols--the Baugh protocol (Baugh et al., 2001, Nucleic Acids Res 29:E29) and an in-house protocol have been assessed by conducting microarray analyses. Cy3-labeled total RNA from the colorectal cell line Colo-205 was compared to Cy5-labeled Colo-205 amplified RNA (aRNA) generated with each of the two protocols, using a human 10K cDNA array. The correlation of the gene intensities between amplified and total RNA measured in the two channels of each microarray was 0.72 and 0.61 for the Baugh protocol and the in-house protocol, respectively. The two protocols were further evaluated using aRNA obtained from normal colonic crypt cross-sections isolated via LCM. In both cases a microarray profile representative of colonic mucosa was obtained; statistically, the Baugh protocol was superior. Furthermore, a substantial overlap between highly expressed genes in the Colo-205 cells and colonic crypts underscores the reliability of the microarray analysis of LCM-derived material. Taken together, these results demonstrate that LCM-derived tissue from histological specimens can generate abundant amounts of high-quality aRNA for subsequent microarray analysis.     

High-quality RNA from cells isolated by laser capture microdissection

Laser capture microdissection (LCM) provides a rapid and simple method for procuring homogeneous populations of cells. However, reproducible isolation of intact RNAfrom these cells can be problematic; the sample may deteriorate before or during sectioning, RNA may degrade during slide staining and LCM, and inadequate extraction and isolation methods may lead to poor recovery. Our report describes an optimized protocol for preparation of frozen sections for LCM using the HistoGene Frozen Section Staining Kit. This slide preparation method is combined with the PicoPure RNA Isolation Kitfor extraction and isolation of RNA from low numbers of microdissected cells. The procedure is easy to perform, rapid, and reproducible. Our results show that the RNA isolated from the LCM samples prepared according to our protocol is of high quality. The RNA maintains its integrity as shown by RT-PCR detection of genes of different abundance levels and by electrophoretic analysis of ribosomal RNA. RNA obtained by this method has also been used to synthesize probes for interrogating cDNA microarray analyses to study expression levels of thousands of genes from LCM samples.     

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.