不同血清量标本外泌体提取及微小RNA的检测结果比较
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摘要
本文旨在探讨Qiagen exoRNeasy Serum/Plasma试剂盒提取血清标本中外泌体所需的最适血清量。采用Qiagen exoRNeasy Serum/Plasma试剂盒分别对250、500、1 000μL血清中的外泌体进行抽提,使用透射电子显微镜检测分离的外泌体大小和形态,蛋白质免疫印迹法检测外泌体蛋白标记CD63和TSG101的表达,实时荧光定量聚合酶链反应(polymerase chain reaction,PCR)检测外泌体中微小RNA-122(microRNA-122,miR-122)的表达。结果显示,透射电子显微镜下可见血清外泌体呈圆形或椭圆形,直径30~150nm,有完整的膜结构。蛋白免疫印迹法检测外泌体CD63和TSG101阳性。实时荧光定量PCR检测慢性乙型肝炎患者250、500、1 000μL血清外泌体中miR-122表达量,与正常人相比,分别上调22.44、21.48、20.69倍(P=0.42)。结果提示,在临床血清样本体积有限的情况下,采用Qiagen exoRNeasy Serum/Plasma试剂盒提取血清中外泌体,减少血清量至250μL也可达到所需实验目的。
The present paper aims to investigate the optimum amount of serum samples used to isolate exosomes by Qiagen exoRNeasy Serum/Plasma Midi Kit.Exosomes were isolated by Qiagen exoRNeasy Serum/Plasma Midi Kit from different gradient serum samples,including 250μL,500μL,1 000μL.The particle size and morphology of exosomes were measured by transmission electron microscopy.The expressions of exosomal surface protein markers CD63 and TSG101 were determined by Western blotting.The expression of microRNA-122(miR-122)in exosomes was detected by real-time fluorescent quantitative polymerase chain reaction(PCR).Serum exosomes were circular or elliptic under the transmission electron microscope with diameters of 30-150 nm and they had intact membrane structure.Western blotting results showed that CD63 and TSG101 were positive in exosomes.The real-time fluorescent quantitative PCR results showed that when the initial serum volume was 250μL,500μL and 1 000μL,the expression of miR-122 in chronic hepatitis B patients was up-regulated by 22.44 folds,21.48 folds and 20.69 folds,respectively compared with controls(P=0.42).It is suggested that a small amount of 250μL serum could be used for isolation of exosomes by Qiagen exoRNeasy Serum/Plasma Midi Kit,when the clinical serum sample is limited.
The present paper aims to investigate the optimum amount of serum samples used to isolate exosomes by Qiagen exoRNeasy Serum/Plasma Midi Kit.Exosomes were isolated by Qiagen exoRNeasy Serum/Plasma Midi Kit from different gradient serum samples,including 250μL,500μL,1 000μL.The particle size and morphology of exosomes were measured by transmission electron microscopy.The expressions of exosomal surface protein markers CD63 and TSG101 were determined by Western blotting.The expression of microRNA-122(miR-122)in exosomes was detected by real-time fluorescent quantitative polymerase chain reaction(PCR).Serum exosomes were circular or elliptic under the transmission electron microscope with diameters of 30-150 nm and they had intact membrane structure.Western blotting results showed that CD63 and TSG101 were positive in exosomes.The real-time fluorescent quantitative PCR results showed that when the initial serum volume was 250μL,500μL and 1 000μL,the expression of miR-122 in chronic hepatitis B patients was up-regulated by 22.44 folds,21.48 folds and 20.69 folds,respectively compared with controls(P=0.42).It is suggested that a small amount of 250μL serum could be used for isolation of exosomes by Qiagen exoRNeasy Serum/Plasma Midi Kit,when the clinical serum sample is limited.
引文
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[18]Arataki K,Hayes CN,Akamatsu S,Akiyama R,Abe H,Tsuge M,Miki D,Ochi H,Hiraga N,Imamura M,Takahashi S,Aikata H,Kawaoka T,Kawakami H,Ohishi W,Chayama K.Circulating microRNA-22correlates with microRNA-122and represents viral replication and liver injury in patients with chronic hepatitis B[J].J Med Virol,2013,85(5):789-798.
[19]王仁峰,耿振,李朝阳,鲍会静,李雪,周迪苏,宋士伟,刘运德.ExoQuick提取人血清外泌体方法改良及比较[J].实用医学杂志,2015,31(15):2458-2462.
[20]Widera C,Gupta SK,Lorenzen JM,Bang C,Bauersachs J,Bethmann K,Kempf T,Wollert KC,Thum T.Diagnostic and prognostic impact of six circulating microRNAs in acute coronary syndrome[J].J Mol Cell Cardiol,2011,51(5):872-875.
[21]Lugli G,Cohen AM,Bennett DA,Shah RC,Fields CJ,Hernandez AG,Smalheiser NR.Plasma exosomal miRNAs in persons with and without Alzheimer disease:altered expression and prospects for biomarkers[J].PLoS One,2015,10(10):e0139233.
[22]Madhavan B,Yue S,Galli U,Rana S,Gross W,Müller M,Giese NA,Kalthoff H,Becker T,Büchler MW,Z9ller M.Combined evaluation of a panel of protein and miRNA serum-exosome biomarkers for pancreatic cancer diagnosis increases sensitivity and specificity[J].Int J Cancer,2015,136(11):2616-2627.
[23]Sugimachi K,Matsumura T,Hirata H,Uchi R,Ueda M,Ueo H,Shinden Y,Iguchi T,Eguchi H,Shirabe K,Ochiya T,Maehara Y,Mimori K.Identification of a bona fide microRNA biomarker in serum exosomes that predicts hepatocellular carcinoma recurrence after liver transplantation[J].Br J Cancer,2015,112(3):532-538.
[24]孙一帆,唐石伏.外泌体应用于肝细胞癌诊疗的研究进展[J].肿瘤,2016,36(8):944-951.
[25]杨倩,刘兰心,黄荷凤.多囊卵巢综合征患者卵泡液外泌体的提取鉴定及其miRNAs的提取和检测[J].上海交通大学学报(医学版),2017,37(8):1085-1089.
[2]Chevillet JR,Kang Q,Ruf IK,Briggs HA,Vojtech LN,Hughes SM,Cheng HH,Arroyo JD,Meredith EK,Gallichotte EN,Pogosova-Agadjanyan EL,Morrissey C,Stirewalt DL,Hladik F,Yu EY,Higano CS,Tewari M.Quantitative and stoichiometric analysis of the microRNA content of exosomes[J].Proc Natl Acad Sci USA,2014,111(41):14888-14893.
[3]Keerthikumar S,Chisanga D,Ariyaratne D,Al Saffar H,Anand S,Zhao K,Samuel M,Pathan M,Jois M,Chilamkurti N,Gangoda L,Mathivanan S.ExoCarta:A web-based compendium of exosomal cargo[J].J Mol Biol,2016,428(4):688-692.
[4]Denzer K,Kleijmeer MJ,Heijnen HF,Stoorvogel W,Geuze HJ.Exosome:from internal vesicle of the multivesicular body to intercellular signaling device[J].J Cell Sci,2000,113(Pt 19):3365-3374.
[5]Michael A,Bajracharya SD,Yuen PS,Zhou H,Star RA,Illei GG,Alevizos I.Exosomes from human saliva as a source of microRNA biomarkers[J].Oral Dis,2010,16(1):34-38.
[6]黄依瑶,唐月汀,覃思华,徐咏,安泰学,郑磊.血清中外泌体及外泌体RNA提取方法的比较[J].中华检验医学杂志,2016,39(6):427-432.
[7]Zeringer E,Barta T,Li M,Vlassov AV.Strategies for isolation of exosomes[J].Cold Spring Harb Protoc,2015(4):319-323.
[8]Szabo G,Momen-Heravi F.Extracellular vesicles in liver disease and potential as biomarkers and therapeutic targets[J].Nat Rev Gastroenterol Hepatol,2017,14(8):455-466.
[9]王鑫伟,毛建文.试剂盒法与差速超速离心法所提血清外泌体的形态学比较[J].临床与病理杂志,2016,36(11):1837-1841.
[10]Tauro BJ,Greening DW,Mathias RA,Ji H,Mathivanan S,Scott AM,Simpson RJ.Comparison of ultracentrifugation,density gradient separation,and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes[J].Methods,2012,56:293-304.
[11]Théry C,Amigorena S,Raposo G,Clayton A.Isolation and characterization of exosomes from cell culture supernatants and biological fluids[J].Curr Protoc Cell Biol,2006.doi:10.1002/0471143030.cb0322s30.
[12]Enderle D,Spiel A,Coticchia CM,Berghoff E,Mueller R,Schlumpberger M,Sprenger-Haussels M,Shaffer JM,Lader E,Skog J,Noerholm M.Characterization of RNA from exosomes and other extracellular vesicles isolated by a novel spin column-based method[J].PLoS One,2015,10(8):e0136133.
[13]Tang YT,Huang YY,Zheng L,Qin SH,Xu XP,An TX,Xu Y,Wu YS,Hu XM,Ping BH,Wang Q.Comparison of isolation methods of exosomes and exosomal RNA from cell culture medium and serum[J].Int J Mol Med,2017,40(3):834-844.
[14]Li J,Zhang X,Chen L,Zhang Z,Zhang J,Wang W,Wu M,Shi B,Zhang X,Kozlowski M,Hu Y,Yuan Z.Circulating miR-210 and miR-22 combined with ALT predict the virological response to interferon-alpha therapy of CHB patients[J].Sci Rep,2017,7(1):15658.
[15]Lamontagne J,Steel LF,Bouchard MJ.Hepatitis B virus and microRNAs:Complex interactions affecting hepatitis B virus replication and hepatitis B virus-associated diseases[J].World J Gastroenterol,2015,21(24):7375-7399.
[16]Bala S,Petrasek J,Mundkur S,Catalano D,Levin I,Ward J,Alao H,Kodys K,Szabo G.Circulating microRNAs in exosomes indicate hepatocyte injury and inflammation in alcoholic,drug-induced,and inflammatory liver diseases[J].Hepatology,2012,56(5):1946-1957.
[17]Zhang X,Zhang Z,Dai F,Shi B,Chen L,Zhang X,Zang G,Zhang J,Chen X,Qian F,Hu Y,Yuan Z.Comparison of circulating,hepatocyte specific messenger RNA and microRNA as biomarkers for chronic hepatitis B and C[J].PLoS One,2014,9(3):e92112.
[18]Arataki K,Hayes CN,Akamatsu S,Akiyama R,Abe H,Tsuge M,Miki D,Ochi H,Hiraga N,Imamura M,Takahashi S,Aikata H,Kawaoka T,Kawakami H,Ohishi W,Chayama K.Circulating microRNA-22correlates with microRNA-122and represents viral replication and liver injury in patients with chronic hepatitis B[J].J Med Virol,2013,85(5):789-798.
[19]王仁峰,耿振,李朝阳,鲍会静,李雪,周迪苏,宋士伟,刘运德.ExoQuick提取人血清外泌体方法改良及比较[J].实用医学杂志,2015,31(15):2458-2462.
[20]Widera C,Gupta SK,Lorenzen JM,Bang C,Bauersachs J,Bethmann K,Kempf T,Wollert KC,Thum T.Diagnostic and prognostic impact of six circulating microRNAs in acute coronary syndrome[J].J Mol Cell Cardiol,2011,51(5):872-875.
[21]Lugli G,Cohen AM,Bennett DA,Shah RC,Fields CJ,Hernandez AG,Smalheiser NR.Plasma exosomal miRNAs in persons with and without Alzheimer disease:altered expression and prospects for biomarkers[J].PLoS One,2015,10(10):e0139233.
[22]Madhavan B,Yue S,Galli U,Rana S,Gross W,Müller M,Giese NA,Kalthoff H,Becker T,Büchler MW,Z9ller M.Combined evaluation of a panel of protein and miRNA serum-exosome biomarkers for pancreatic cancer diagnosis increases sensitivity and specificity[J].Int J Cancer,2015,136(11):2616-2627.
[23]Sugimachi K,Matsumura T,Hirata H,Uchi R,Ueda M,Ueo H,Shinden Y,Iguchi T,Eguchi H,Shirabe K,Ochiya T,Maehara Y,Mimori K.Identification of a bona fide microRNA biomarker in serum exosomes that predicts hepatocellular carcinoma recurrence after liver transplantation[J].Br J Cancer,2015,112(3):532-538.
[24]孙一帆,唐石伏.外泌体应用于肝细胞癌诊疗的研究进展[J].肿瘤,2016,36(8):944-951.
[25]杨倩,刘兰心,黄荷凤.多囊卵巢综合征患者卵泡液外泌体的提取鉴定及其miRNAs的提取和检测[J].上海交通大学学报(医学版),2017,37(8):1085-1089.