流式细胞术检测组蛋白乙酰化水平方法的建立与应用

摘要

Histone deacetylaseinhibitors, 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 andclinical research. In this study, we developed a flow cytometry-based method to de-tect histone acetylation levels by comparing different sample processing temperature(on ice vs. room temperature), perme-abilization method (intracellularvs. nuclear), antibody dose (antibody titration) and antibody incubation time (time gradient) using whole blood and peripheral blood mononuclear cells. In addition, we applied thisoptimizedmethod in in vitro ex-periment 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.%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。