组织单腺体内单细胞的基因变异分析方法
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摘要
从单细胞尺度进行细胞异质性的分析是深度理解细胞群体关系的关键。组织中的单细胞由于细胞类型不同,尺寸往往相差很大,但是目前常用的基于微孔板和Fluidigm公司的微流控的单细胞组学研究方法,需要入口的单细胞大小相近。本研究以胃组织为例,建立了一种组织单细胞的基因变异分析方法,实现了尺寸差异较大的单细胞的基因变异分析。在该方法中,先将胃组织裂解获得单个腺体,再将单个腺体酶解得到不同大小的腺体内单细胞,然后把这些单细胞铺在聚乙烯萘膜载玻片上,进行激光显微切割分选、全基因组放大,最后测其微卫星的长度。利用该方法,成功在肠上皮化生腺体内部检测到微卫星长度的变化,并灵活地对尺寸差异大的组织细胞以及肠化生腺体细胞进行了精细分析。此外,这种单细胞分析方法还可以对带有不同标记的细胞进行低通量和高通量的基因组分析,为单细胞尺度上的组织异质性研究提供了一种高度灵活的分析方法。
Single-cell analysis of heterogeneity has become the cutting-edge technology for profound understandings of relationships between cell populations. At present, common methods used in single cellular genomic research are mainly microfluidic technologies(Fluidigm) or based on microwells, both requiring a uniform size of cells at the entrance. However, the size of cells in specific tissues can vary from type to type. To address this issue, we need to establish a method to identify genomic features of individual cells of different sizes. In this paper, we developed a robust method in the analysis of single cellular genomic mutations among gastric tissues. Briefly, the single gastric gland was isolated from the whole tissue, and further enzymatically digested into single cells of various sizes by trypsin. These single cells were then spread on the polyethylene naphthalene slides and selected by the laser microdissection method. Whole genome amplification(WGA) and capillary electrophoresis were performed subsequently to detect single cell microsatellite. This method enabled us to detect the existence of microsatellite instability(MSI) of each single cell within the intestinal metaplasia, and to carry out a flexible and fine analysis of single cells with different sizes in tissues and glands. This reliable and practical method is well performed in both low and high-throughput genome analysis when combined with cell labeling methods, thus providing a novel and highly flexible way to study tissue heterogeneity on the single cell scale.
Single-cell analysis of heterogeneity has become the cutting-edge technology for profound understandings of relationships between cell populations. At present, common methods used in single cellular genomic research are mainly microfluidic technologies(Fluidigm) or based on microwells, both requiring a uniform size of cells at the entrance. However, the size of cells in specific tissues can vary from type to type. To address this issue, we need to establish a method to identify genomic features of individual cells of different sizes. In this paper, we developed a robust method in the analysis of single cellular genomic mutations among gastric tissues. Briefly, the single gastric gland was isolated from the whole tissue, and further enzymatically digested into single cells of various sizes by trypsin. These single cells were then spread on the polyethylene naphthalene slides and selected by the laser microdissection method. Whole genome amplification(WGA) and capillary electrophoresis were performed subsequently to detect single cell microsatellite. This method enabled us to detect the existence of microsatellite instability(MSI) of each single cell within the intestinal metaplasia, and to carry out a flexible and fine analysis of single cells with different sizes in tissues and glands. This reliable and practical method is well performed in both low and high-throughput genome analysis when combined with cell labeling methods, thus providing a novel and highly flexible way to study tissue heterogeneity on the single cell scale.
引文
[1]Hu P,Zhang WH,Xin HB,Deng G.Single cell isolation and analysis.Front Cell Dev Biol,2016,4:116.
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[10]Kimmerling RJ,Lee Szeto G,Li JW,Genshaft AS,Kazer SW,Payer KR,de Riba Borrajo J,Blainey PC,Irvine DJ,Shalek AK,Manalis SR.A microfluidic platform enabling single-cell RNA-seq of multigenerational lineages.Nat Commun,2016,7:10220.
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[13]Ren KN,Chen Y,Wu HK.New materials for microfluidics in biology.Curr Opin Biotechnol,2014,25:78–85.
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[17]Choi E,Roland JT,Barlow BJ,O'Neal R,Rich AE,Nam KT,Shi CJ,Goldenring JR.Cell lineage distribution atlas of the human stomach reveals heterogeneous gland populations in the gastric antrum.Gut,2014,63(11):1711–1720.
[18]Hunt RH,Camilleri M,Crowe SE,El-Omar EM,Fox JG,Kuipers EJ,Malfertheiner P,Mc Coll KEL,Pritchard DM,Rugge M,Sonnenberg A,Sugano K,Tack J.The stomach in health and disease.Gut,2015,64(10):1650–1668.
[19]El-Zaatari M,Kao JY,Tessier A,Bai LC,Hayes MM,Fontaine C,Eaton KA,Merchant JL.Gli1 deletion prevents Helicobacter-induced gastric metaplasia and expansion of myeloid cell subsets.PLo S One,2013,8(3):e58935.
[20]Serizawa T,Hirata Y,Hayakawa Y,Suzuki N,Sakitani K,Hikiba Y,Ihara S,Kinoshita H,Nakagawa H,Tateishi K,Koike K.Gastric metaplasia induced by Helicobacter pylori is associated with enhanced SOX9 expression via Interleukin-1 signaling.Infect Immun,2015,84(2):562–572.
[21]Geigl JB,Speicher MR.Single-cell isolation from cell suspensions and whole genome amplification from single cells to provide templates for CGH analysis.Nat Protoc,2007,2(12):3173–3184.
[22]Shirai M,Arikawa K,Taniguchi K,Tanabe M,Sakai T.Vertical flow array chips reliably identify cell types from single-cell m RNA sequencing experiments.Sci Rep,2016,6:36014.
[23]Huang AY,Zhou Y,Cheng M,Zhang XD,Li JF,Liu BY,Guo Y.Precise analysis of microsatellite length in small amounts of tissue cells.Chin J Biochem Mol Biol,2016,32(6):714–720.黄阿源,周彦,程酩,张小丹,李建芳,刘炳亚,郭妍.组织中少量细胞的微卫星长度精细分析.中国生物化学与分子生物学报,2016,32(6):714–720.
[24]Luo WY,Hu J,Li XF.The evolution and application of microsatellites.Hereditas(Beijing),2003,25(5):615–619.罗文永,胡骏,李晓方.微卫星序列及其应用.遗传,2003,25(5):615–619.
[25]Guo Y,Zhou J,Huang AY,Li JF,Yan M,Zhu ZG,Zhao XD,Gu JR,Liu BY,Shao ZF.Spatially defined microsatellite analysis reveals extensive genetic mosaicism and clonal complexity in intestinal metaplastic glands.Int J Cancer,2015,136(12):2973–2979.
[26]Guo Y,Huang A,Hu CS,Zhou Y,Zhang XD,Czajkowsky DM,Li JF,Cheng SD,Shen RZ,Gu JR,Liu BY,Shao ZF.Complex clonal mosaicism within microdissected intestinal metaplastic glands without concurrent gastric cancer.J Med Genet,2016,53(9):643–646.
[27]Sun S,Deng YL.Single-cell detection of EGFR gene mutation in circulating tumor cells in lung cancer.Hereditas(Beijing),2015,37(12):1251–1257.孙帅,邓宇亮.肺癌循环肿瘤细胞的单细胞EGFR基因突变检测.遗传,2015,37(12):1251–1257.
[28]Lee WC,Bhagat AA,Huang S,Van Viet KJ,Han J,Lim CT.High-throughput cell cycle synchronization using inertial forces in spiral microchannels.Lab Chip,2011,11(7):1359–1367.
[29]Wang JB,Fan HC,Behr B,Quake SR.Genome-wide single-cell analysis of recombination activity and de novo mutation rates in human sperm.Cell,2012,150(2):402–412.
[30]Wu WJ,Wang ZH,Wang Z,Deng YL,Shi QH.Fast isolation and ex vivo culture of circulating tumor cells from the peripheral blood of lung cancer patients.Hereditas(Beijing),2017,39(1):66–74.吴文君,王智华,王卓,邓宇亮,施奇惠.肺癌患者外周血中循环肿瘤细胞的快速分离与体外培养.遗传,2017,39(1):66–74.
[31]Swennenhuis JF,Tibbe AGJ,Stevens M,Katika MR,van Dalum J,Tong HD,van Rijn CJM,Terstappen LWMM.Self-seeding microwell chip for the isolation and characterization of single cells.Lab Chip,2015,15(14):3039–3046.
[32]Xu JJ,Jiang DP,Qin YL,Xia J,Jiang DC,Chen HY.C3N4nanosheet modified microwell array with enhanced electrochemiluminescence for total analysis of cholesterol at single cells.Anal Chem,2017,89(4):2216–2220.
[2]Lo SJ,Yao DJ.Get to understand more from single-cells:current studies of microfluidic-based techniques for single-cell analysis.Int J Mol Sci,2015,16(8):16763–16777.
[3]Frieda KL,Linton JM,Hormoz S,Choi J,Chow KK,Singer ZS,Budde MW,Elowitz MB,Cai L.Synthetic recording and in situ readout of lineage information in single cells.Nature,2016,541(7635):107–111.
[4]Kanter I,Kalisky T.Single cell transcriptomics:methods and applications.Front Oncol,2015,5:53.
[5]Zong CH,Lu SJ,Chapman AR,Xie XS.Genome-wide detection of single-nucleotide and copy-number variations of a single human cell.Science,2012,338(6114):1622–1626.
[6]Junker JP,van Oudenaarden A.Every cell is special:genome-wide studies add a new dimension to single-cell biology.Cell,2014,157(1):8–11.
[7]Wu JC,Tzanakakis ES.Deconstructing stem cell population heterogeneity:single-cell analysis and modeling approaches.Biotechnol Adv,2013,31(7):1047–1062.
[8]De Laughter DM,Bick AG,Wakimoto H,Mc Kean D,Gorham JM,Kathiriya IS,Hinson JT,Homsy J,Gray J,Pu W,Bruneau BG,Seidman JG,Seidman CE.Single-cell resolution of temporal gene expression during heart development.Dev Cell,2016,39(4):480–490.
[9]Khalili AA,Ahmad MR.Numerical analysis of hydrodynamic flow in microfluidic biochip for single-cell trapping application.Int J Mol Sci,2015,16(11):26770–26785.
[10]Kimmerling RJ,Lee Szeto G,Li JW,Genshaft AS,Kazer SW,Payer KR,de Riba Borrajo J,Blainey PC,Irvine DJ,Shalek AK,Manalis SR.A microfluidic platform enabling single-cell RNA-seq of multigenerational lineages.Nat Commun,2016,7:10220.
[11]Gross A,Schoendube J,Zimmermann S,Steeb M,Zengerle R,Koltay P.Technologies for single-cell isolation.Int J Mol Sci,2015,16(8):16897–16919.
[12]Li P,Gao Y,Pappas D.Multiparameter cell affinity chromatography:separation and analysis in a single microfluidic channel.Anal Chem,2012,84(19):8140–8148.
[13]Ren KN,Chen Y,Wu HK.New materials for microfluidics in biology.Curr Opin Biotechnol,2014,25:78–85.
[14]Zhang K,Gao M,Chong ZC,Li Y,Han X,Chen R,Qin LD.Single-cell isolation by a modular single-cell pipette for RNA-sequencing.Lab Chip,2016,16(24):4742–4748.
[15]Liu X,Barizuddin S,Shin W,Mathai CJ,Gangopadhyay S,Gillis KD.Microwell device for targeting single cells to electrochemical microelectrodes for high-throughput amperometric detection of quantal exocytosis.Anal Chem,2011,83(7):2445–2451.
[16]Rettig JR,Folch A.Large-scale single-cell trapping and imaging using microwell arrays.Anal Chem,2005,77(17):5628–5634.
[17]Choi E,Roland JT,Barlow BJ,O'Neal R,Rich AE,Nam KT,Shi CJ,Goldenring JR.Cell lineage distribution atlas of the human stomach reveals heterogeneous gland populations in the gastric antrum.Gut,2014,63(11):1711–1720.
[18]Hunt RH,Camilleri M,Crowe SE,El-Omar EM,Fox JG,Kuipers EJ,Malfertheiner P,Mc Coll KEL,Pritchard DM,Rugge M,Sonnenberg A,Sugano K,Tack J.The stomach in health and disease.Gut,2015,64(10):1650–1668.
[19]El-Zaatari M,Kao JY,Tessier A,Bai LC,Hayes MM,Fontaine C,Eaton KA,Merchant JL.Gli1 deletion prevents Helicobacter-induced gastric metaplasia and expansion of myeloid cell subsets.PLo S One,2013,8(3):e58935.
[20]Serizawa T,Hirata Y,Hayakawa Y,Suzuki N,Sakitani K,Hikiba Y,Ihara S,Kinoshita H,Nakagawa H,Tateishi K,Koike K.Gastric metaplasia induced by Helicobacter pylori is associated with enhanced SOX9 expression via Interleukin-1 signaling.Infect Immun,2015,84(2):562–572.
[21]Geigl JB,Speicher MR.Single-cell isolation from cell suspensions and whole genome amplification from single cells to provide templates for CGH analysis.Nat Protoc,2007,2(12):3173–3184.
[22]Shirai M,Arikawa K,Taniguchi K,Tanabe M,Sakai T.Vertical flow array chips reliably identify cell types from single-cell m RNA sequencing experiments.Sci Rep,2016,6:36014.
[23]Huang AY,Zhou Y,Cheng M,Zhang XD,Li JF,Liu BY,Guo Y.Precise analysis of microsatellite length in small amounts of tissue cells.Chin J Biochem Mol Biol,2016,32(6):714–720.黄阿源,周彦,程酩,张小丹,李建芳,刘炳亚,郭妍.组织中少量细胞的微卫星长度精细分析.中国生物化学与分子生物学报,2016,32(6):714–720.
[24]Luo WY,Hu J,Li XF.The evolution and application of microsatellites.Hereditas(Beijing),2003,25(5):615–619.罗文永,胡骏,李晓方.微卫星序列及其应用.遗传,2003,25(5):615–619.
[25]Guo Y,Zhou J,Huang AY,Li JF,Yan M,Zhu ZG,Zhao XD,Gu JR,Liu BY,Shao ZF.Spatially defined microsatellite analysis reveals extensive genetic mosaicism and clonal complexity in intestinal metaplastic glands.Int J Cancer,2015,136(12):2973–2979.
[26]Guo Y,Huang A,Hu CS,Zhou Y,Zhang XD,Czajkowsky DM,Li JF,Cheng SD,Shen RZ,Gu JR,Liu BY,Shao ZF.Complex clonal mosaicism within microdissected intestinal metaplastic glands without concurrent gastric cancer.J Med Genet,2016,53(9):643–646.
[27]Sun S,Deng YL.Single-cell detection of EGFR gene mutation in circulating tumor cells in lung cancer.Hereditas(Beijing),2015,37(12):1251–1257.孙帅,邓宇亮.肺癌循环肿瘤细胞的单细胞EGFR基因突变检测.遗传,2015,37(12):1251–1257.
[28]Lee WC,Bhagat AA,Huang S,Van Viet KJ,Han J,Lim CT.High-throughput cell cycle synchronization using inertial forces in spiral microchannels.Lab Chip,2011,11(7):1359–1367.
[29]Wang JB,Fan HC,Behr B,Quake SR.Genome-wide single-cell analysis of recombination activity and de novo mutation rates in human sperm.Cell,2012,150(2):402–412.
[30]Wu WJ,Wang ZH,Wang Z,Deng YL,Shi QH.Fast isolation and ex vivo culture of circulating tumor cells from the peripheral blood of lung cancer patients.Hereditas(Beijing),2017,39(1):66–74.吴文君,王智华,王卓,邓宇亮,施奇惠.肺癌患者外周血中循环肿瘤细胞的快速分离与体外培养.遗传,2017,39(1):66–74.
[31]Swennenhuis JF,Tibbe AGJ,Stevens M,Katika MR,van Dalum J,Tong HD,van Rijn CJM,Terstappen LWMM.Self-seeding microwell chip for the isolation and characterization of single cells.Lab Chip,2015,15(14):3039–3046.
[32]Xu JJ,Jiang DP,Qin YL,Xia J,Jiang DC,Chen HY.C3N4nanosheet modified microwell array with enhanced electrochemiluminescence for total analysis of cholesterol at single cells.Anal Chem,2017,89(4):2216–2220.