HIV-1储存库激活剂体外激活效应研究

Reactivation efficacies of HIV-1 latency reversing agents in vitro

目的探讨人类免疫缺陷病毒-1(HIV-1)储存库激活剂的体外激活效应,为临床应用提供参考。方法分离得到健康供血者外周血中的CD4^+T细胞,构建HIV-1潜伏感染的原代细胞模型。用不同浓度(0.5、1、2μmol/L)的帕比司他(Panobinostat)、伏立诺他(SAHA)(Vorinostat)、BIX-01294、JQ-1、Bryostatin-1、GS-9620、地西他滨(Decitabine)、戒酒硫(Disulfiram)、Sirolimus分别处理细胞,在不同作用时间(1、3、5 d)后利用流式细胞分析仪检测每种药物对HIV-1储存库细胞的绿色荧光蛋白(GFP)表达效率及细胞存活率,并确定每种激活剂的最佳激活浓度和作用时间。结果通过对由HIV-1启动子驱动的GFP表达效率及细胞存活率的统计分析,明确了在该细胞模型中这9种HIV-1储存库激活剂的最佳作用浓度和作用时间:Panobinostat的最佳激活浓度为1μmol/L,最佳作用时间为3 d;SAHA的最佳激活浓度为2μmol/L,最佳作用时间为1 d;JQ-1、Bryostatin-1和GS-9620在该模型中的最佳激活浓度均为1μmol/L且最佳作用时间均为1 d;BIX-01294、Decitabine、Disulfiram、Sirolimus在该模型中的最佳激活浓度均为0.5μmol/L且最佳作用时间均为1 d。横向对比发现,浓度为1μmol/L,作用时间为3 d的Panobinostat与浓度为0.5μmol/L,作用时间为1 d的Sirolimus在这9种药物中的激活效率最高,且对细胞存活无明显影响。结论系统比较确认了9种病毒储存库激活药物在体外细胞模型中的激活效率,及最佳作用浓度和时间,为后续这些药物的临床实验研究提供重要参考。

Objective In order to systematically compare the reactivation effects of human immunodeficiency virus-1(HIV-1)latency reversing agents in vitro,and provide reference for clinical application.Methods CD4^+T cells from peripheral blood of healthy donors were isolated to construct the primary cell model of HIV-1 latency.Different concentrations(0.5,1,2μmol/L)and different time of action(1,3,5 day)of panobinostat,SAHA,BIX-01294,JQ-1,bryostatin-1,GS-9620,decitabine,disulfiram and sirolimus were tested;flow cytometry was used to determine the GFP activation efficiency and cell survival of HIV-1 latency cells.Results Through the statistical analysis of GFP expression driven by HIV-1 promoter and cell survival,optimal concentration and time of action of 9 HIV-1 latency reversing agents in this cell model were determined:the optimal activation concentration of panobinostat was 1μmol/L,and the optimal activation time was 3 days.The optimum activation concentration of SAHA was 2μmol/L,and the optimum reaction time was 1 day.The optimal activation concentrations of JQ-1,Bryostatin-1 and GS-9620 in the model were 1μmol/L and the optimal time was 1 day.The optimal activation concentrations of BIX-01294,Decitabine,Disulfiram and Sirolimus in the model were 0.5μmol/L and the optimal reaction time was 1 day.Statistical analysis showed that panobinostat with a concentration of 1μmol/L for 3 days and sirolimus with a concentration of 0.5μmol/L for 1 day had the highest reactivation efficacy among the 9 drugs,with no significant effect on cell survival.Conclusion Reactivation efficacies,optimal concentrations and time of action were determined for 9 latency reversing agents,which provides important references for future clinical applications of these drugs.