哺乳动物单细胞研究技术的现状与未来
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摘要
近年来,生命科学和医学的基础研究已深入到单细胞阶段。单细胞研究为揭示生命活动的基本规律、探索细胞异质性、提高对疾病发病机制的认识等提供了重要的线索和依据,同时,单细胞技术已被应用于日常实践中,如法医学和临床生殖医学。单细胞研究中使用的技术也在不断变化,并越来越复杂。文中主要介绍单细胞分离技术,包括手工挑取、激光捕获显微切割和微流控技术,以及单细胞中DNA、RNA和蛋白质分析方法的各种技术。此外,文中总结了近年来生命科学和医学领域的主要单细胞研究成果,讨论了单细胞相关技术和研究的不足,并介绍了其未来的发展方向。
Basic research in life science and medicine has dug into single cell level in recent years.Single-cell analysis offers to understand life from diverse perspectives and is used to profile cell heterogeneity to investigate mechanism of diseases.Single cell technologies have also found applications in forensic medicine and clinical reproductive medicine,while the techniques are rapidly evolving and have become more and more sophisticated.In this article,we reviewed various single cell isolation techniques and their pros and cons,including manual cell picking,laser capture microdissection andmicrofluidics,as well as analysis methods for DNA,RNA and protein in single cell.In addition,we summarized major up-to-date single cell research achievements and their potential applications.
Basic research in life science and medicine has dug into single cell level in recent years.Single-cell analysis offers to understand life from diverse perspectives and is used to profile cell heterogeneity to investigate mechanism of diseases.Single cell technologies have also found applications in forensic medicine and clinical reproductive medicine,while the techniques are rapidly evolving and have become more and more sophisticated.In this article,we reviewed various single cell isolation techniques and their pros and cons,including manual cell picking,laser capture microdissection andmicrofluidics,as well as analysis methods for DNA,RNA and protein in single cell.In addition,we summarized major up-to-date single cell research achievements and their potential applications.
引文
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[14]Alphonse RS,Vadivel A,Zhong S,et al.The isolation and culture of endothelial colony-forming cells from human and rat lungs.Nat Protoc,2015,10(11):1697-1708.
[15]Zhang LX,Chan C.Isolation and enrichment of rat mesenchymal stem cells(MSCs)and separation of single-colony derived MSCs.J Vis Exp,2010,(37):1852.
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[22]Schütze K,P?sl H,Lahr G.Laser micromanipulation systems as universal tools in cellular and molecular biology and in medicine.Cell Mol Biol(Noisy-le-Grand,France),1998,44(5):735-746.
[23]Shao YY,Wang L,Hicks DG,et al.Analysis of gene expression in mineralized skeletal tissues by laser capture microdissection and RT-PCR.Lab Invest,2006,86(10):1089-1095.
[24]Sun JX,Horst OV,Bumgarner R,et al.Laser capture microdissection enables cellular and molecular studies of tooth root development.Int J Oral Sci,2012,4(1):7-13.
[25]Kirschner J,Plaschke-Schluetter A.Advancing forensics with precise target excision:the CellCut Plus laser microdissection instrument.Nat Methods,2007,4(10):iii-iv.
[26]Simone NL,Paweletz CP,Charboneau L,et al.Laser capture microdissection:beyond functional genomics to proteomics.Mol Diagn,2000,5(4):301-307.
[27]Gawad C,Koh W,Quake SR.Single-cell genome sequencing:current state of the science.Nat Rev Genet,2016,17(3):175-188.
[28]Okumus B,Baker CJ,Arias-Castro JC,et al.Single-cell microscopy of suspension cultures using a microfluidics-assisted cell screening platform.Nat Protoc,2017,13(1):170-194.
[29]ddSEQ Single-Cell Isolator[EB/OL].[2018-03-08].www.bio-rad.com/zh-cn/product/ddseq-single-cell-isolat or?ID=OKNWBSE8Z.
[30]Wong-Staal F,Gillespie D,Gallo RC.Proviral sequences of baboon endogenous type C RNA virus in DNA of human leukaemic tissues.Nature,1976,262(5565):190-195.
[31]Fiers W,Contreras R,Haegeman G,et al.Complete nucleotide sequence of SV40 DNA.Nature,1978,273(5658):113-120.
[32]Zhang L,Cui X,Schmitt K,et al.Whole genome amplification from a single cell:implications for genetic analysis.Proc Natl Acad Sci USA,1992,89(13):5847-5851.
[33]Dean FB,Hosono S,Fang LH,et al.Comprehensive human genome amplification using multiple displacement amplification.Proc Natl Acad Sci USA,2002,99(8):5261-5266.
[34]Dean FB,Nelson JR,Giesler TL,et al.Rapid amplification of plasmid and phage DNA using Phi29DNA polymerase and multiply-primed rolling circle amplification.Genome Res,2001,11(6):1095-1099.
[35]Hou Y,Guo HH,Cao C,et al.Single-cell triple omics sequencing reveals genetic,epigenetic,and transcriptomic heterogeneity in hepatocellular carcinomas.Cell Res,2016,26(3):304-319.
[36]Marcy Y,Ouverney C,Bik EM,et al.Dissecting biological“dark matter”with single-cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth.Proc Natl Acad Sci USA,2007,104(29):11889-11894.
[37]Blainey PC.The future is now:single-cell genomics of bacteria and archaea.FEMS Microbiol Rev,2013,37(3):407-427.
[38]Telenius HK,Carter NP,Bebb CE,et al.Degenerate oligonucleotide-primed PCR:general amplification of target DNA by a single degenerate primer.Genomics,1992,13(3):718-725.
[39]Arriola E,Lambros MBK,Jones C,et al.Evaluation of Phi29-based whole-genome amplification for microarray-based comparative genomic hybridisation.Lab Invest,2007,87(1):75-83.
[40]de Bourcy CFA,de Vlaminck I,Kanbar JN,et al.Aquantitative comparison of single-cell whole genome amplification methods.PLoS ONE,2014,9(8):e105585.
[41]Gole J,Gore A,Richards A,et al.Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells.Nat Biotechnol,2013,31(12):1126-1132.
[42]Lu SJ,Zong CH,Fan W,et al.Probing meiotic recombination and aneuploidy of single sperm cells by whole-genome sequencing.Science,2012,338(6114):1627-1630.
[43]Zong CH,Lu SJ,Chapman AR,et al.Genome-wide detection of single-nucleotide and copy-number variations of a single human cell.Science,2012,338(6114):1622-1626.
[44]Chen CY,Xing D,Tan LZ,et al.Single-cell whole-genome analyses by Linear Amplification via Transposon Insertion(LIANTI).Science,2017,356(6334):189-194.
[45]QIAGEN单细胞扩增试剂盒[EB/OL].[2018-03-08].https://www.qiagen.com/cn/shop/sample-technologies/d na/genomic-dna/repli-g-single-cell-kit/#orderinginforma tion.
[46]亿康基因MALBAC微量基因组快速扩增试剂盒[EB/OL].[2018-03-08].www.yikongenomics.com/upload/2017/1124/KT1107003_MALBACweiliangjiyinz ukuaisukuozengshijihechanpinshuomingshu_zhongwenPM.pdf.
[47]SIGMA单细胞全基因组扩增试剂盒[EB/OL].[2018-03-08].https://www.sigmaaldrich.com/catalog/product/sigma/wga4?lang=en®ion=US.
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[49]Huang L,Ma F,Chapman A,et al.Single-cell whole-genome amplification and sequencing:methodology and applications.Annu Rev Genomics Hum Genet,2015,16(1):79-102.
[50]Shalek AK,Satija R,Adiconis X,et al.Single-cell transcriptomics reveals bimodality in expression and splicing in immune cells.Nature,2013,498(7453):236-240.
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