技术与方法

流式细胞术检测组蛋白乙酰化水平方法的建立与应用

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  • 第四军医大学附属唐都医院传染科,西安 710038
李建辉,硕士研究生,专业方向:艾滋病功能性治愈。E-mail: lijianhui0403@outlook.com

收稿日期: 2016-02-24

  修回日期: 2016-05-13

  网络出版日期: 2016-05-25

基金资助

国家科技重大专项 -艾滋病和病毒性肝炎等重大传染病防治(编号:2014ZX10001002)

Establishment and application of a flow cytometry-based method for detecting histone acetylation levels

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  • Department of Infectious Diseases, Tang-du Hospital affiliated to Fourth Military Medical University, Xi’an 710038, China

Received date: 2016-02-24

  Revised date: 2016-05-13

  Online published: 2016-05-25

Supported by

Supported by the National Science and Technology Major Project (No; 2014ZX10001002)

摘要

组蛋白去乙酰化酶抑制剂是近年来出现的一类新的抗肿瘤药物,在艾滋病等其他疾病中同样也受到关注。但是在基础和临床研究中,目前还缺乏统一可靠的组蛋白乙酰化水平的检测手段。本文利用全血和外周血单个核细胞,通过一系列的对比实验,比较了不同样品处理温度(冰上和室温)、破膜方法(细胞内因子染色破膜和核内因子染色破膜)、抗体剂量(抗体滴定)和抗体孵育时间(时间梯度)等实验条件对流式细胞术检测的影响,最终建立了一套基于流式细胞术的组蛋白乙酰化水平检测手段。同时,将优化后的流式细胞检测技术应用于西达本胺(目前国内唯一上市的组蛋白去乙酰化酶抑制剂)的体外实验和临床试验,结果均证明本文建立的组蛋白乙酰化流式细胞检测方法可以作为基础和临床研究中一个可靠、快速、便捷的检测手段。

本文引用格式

李建辉, 汪春付, 白帆, 庄严, 毛卓君, 孙永涛 . 流式细胞术检测组蛋白乙酰化水平方法的建立与应用[J]. 遗传, 2016 , 38(6) : 581 -587 . DOI: 10.16288/j.yczz.16-065

Abstract

Histone deacetylase inhibitors, which have also received attention in AIDS and other diseases, are a new class of anticancer drugs developed in recent years. However, there is still a lack of a unified and reliable method for detecting histone acetylation levels in basic and clinical research. In this study, we developed a flow cytometry-based method to detect histone acetylation levels by comparing different sample processing temperature (on ice vs. room temperature), permeabilization method (intracellular vs. nuclear), antibody dose (antibody titration) and antibody incubation time (time gradient) using whole blood and peripheral blood mononuclear cells. In addition, we applied this optimized method in in vitro experiment and clinical trial of Chidamide (the only China FDA approved HDACi), the result of which confirmed that the flow cytometry-based method for detecting histone acetylation levels is a reliable, fast and convenient method which can be used in basic and clinical research.

参考文献

[1] Li XX, Lu J, Luo DH, Huang BQ. The histone deacetylase activity of SIR2 and chromatin silencing. Hereditas (Beijing) , 2003, 25(4): 484-488. 李晓雪, 陆军, 罗巅辉, 黄百渠. 组蛋白去乙酰化酶SIR2与染色质沉默. 遗传, 2003, 25(4): 484-488.
[2] Yang XJ, Seto E. HATs and HDACs: from structure, function and regulation to novel strategies for therapy and prevention. Oncogene , 2007, 26(37): 5310-5318.
[3] Lakshmaiah KC, Jacob LA, Aparna S, Lokanatha D, Saldanha SC. Epigenetic therapy of cancer with histone deacetylase inhibitors. J Cancer Res Ther , 2014, 10(3): 469- 478.
[4] Das GK, Shakespear MR, Iyer A, Fairlie DP, Sweet MJ. Histone deacetylases in monocyte/macrophage development, activation and metabolism: refining HDAC targets for inflammatory and infectious diseases. Clin Transl Immunol , 2016, 5(1): e62.
[5] Dong M, Ning ZQ, Xing PY, Xu JL, Cao HX, Dou GF, Meng ZY, Shi YK, Lu XP, Feng FY. Phase I study of chidamide (CS055/HBI-8000), a new histone deacetylase inhibitor, in patients with advanced solid tumors and lymphomas. Cancer Chemother Pharmacol , 2012, 69(6): 1413-1422.
[6] Rigby L, Muscat A, Ashley D, Algar E. Methods for the analysis of histone H3 and H4 acetylation in blood. Epigenetics , 2012, 7(8): 875-882.
[7] Kaypee S, Sudarshan D, Shanmugam MK, Mukherjee D, Sethi G, Kundu TK. Aberrant lysine acetylation in tumorigenesis: Implications in the development of therapeutics. Pharmacol Ther , 2016, doi:10.1016/j.pharmthera.2016.01.011.
[8] Archin NM, Liberty AL, Kashuba AD, Choudhary, SK, Kuruc JD, Crooks AM, Parker DC, Anderson EM, Kearney MF, Strain MC, Richman DD, Hudgens MG, Bosch RJ, Coffin JM, Eron JJ, Hazuda DJ, Margolis DM. Administration of vorinostat disrupts HIV-1 latency in patients on antiretroviral therapy. Nature , 2012, 487(7408): 482-485.
[9] Rasmussen TA, Tolstrup M, Brinkmann CR, Olesen R, Erikstrup C, Solomon A, Winckelmann A, Palmer S, Dinarello C, Buzon M, Lichterfeld M, Lewin SR, Østergaard L, Søgaard OS. Panobinostat, a histone deacetylase inhibitor, for latent-virus reactivation in HIV-infected patients on suppressive antiretroviral therapy: a phase 1/2, single group, clinical trial. Lancet HIV , 2014, 1(1): e13-e21.
[10] Søgaard OS, Graversen ME, Leth S, Olesen R, Brinkmann CR, Nissen SK, Kjaer AS, Schleimann MH, Denton PW, Hey-Cunningham WJ, Koelsch KK, Pantaleo G, Krogsgaard K, Sommerfelt M, Fromentin R, Chomont N, Rasmussen TA, Østergaard L, Tolstrup M, Siliciano RF. The depsipeptide romidepsin reverses HIV-1 latency in vivo. PLoS Pathog , 2015, 11(9): e1005142.
[11] Keedy KS, Archin NM, Gates AT, Espeseth A, Hazuda DJ, Margolis DM. A limited group of class I histone deacetylases acts to repress human immunodeficiency virus type 1 expression. J Virol , 2009, 83(10): 4749-4756.
[12] Williams SA, Chen LF, Kwon H, Ruiz-Jarabo CM, Verdin E, Greene WC. NF-κB p50 promotes HIV latency through HDAC recruitment and repression of transcriptional initiation. EMBO J , 2006, 25(1): 139-149.
[13] Deeks SG. HIV: Shock and kill. Nature , 2012, 487(7408): 439-440.
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