经长期HAART治疗的中国HIV—AIDS患者外周血细胞内HIV-1前病毒DNA的检测及其意义

Analysis of HIV-1 proviral DNA in peripheral blood cells from long-term HAART-treated AIDS pa- tients in China

目的探索NK细胞是否是HIV-1病毒的储存库细胞之一，并观察在长期HAART治疗后，T淋巴细胞及单核细胞内是否仍有HIV-1前病毒DNA存在。方法对15例规范接受HAART治疗4～7年的HIV—AIDS患者采集血标本进行横断面研究。使用磁珠分选法从外周血单个核细胞（PBMC）中分离出T淋巴细胞、单核细胞和NK细胞，通过HIV实时荧光定量检测外周血HIV-1RNA病毒载量和NK细胞、单核细胞、T淋巴细胞内的HIV-1 DNA水平，流式细胞术检测外周CD4＋T细胞数量，对数据进行统计学处理。结果15例接受4～7年[平均（62．45±13．53）个月]HAART治疗的HIV／AIDS患者外周血HIV-1RNA均低于检测下限（〈50拷贝／ml）；T淋巴细胞、NK细胞中和单核细胞中HIV-1前病毒DNA的平均含量分别为2．03±0．48、1．82±0．32和1．754-0．19（10g10拷贝／10。个细胞）；3种细胞中HIV-1DNA阳性（〉50拷贝／m1）者比例分别为6／15、4／15、2／15。结论中国HIV-1感染者外周血中除了T淋巴细胞和单核细胞外，NK细胞也可能是HIV-1的病毒储存库之一。在长期HAART治疗（4～7年）后，尽管表现出明显的抗病毒效果和免疫重建效果，仍然未能彻底清除NK细胞、T淋巴细胞和单核细胞中的HIV-1前病毒DNA。

Objective The aim of this study was to verify that NK cells were one type of the virus reservoir cells, and to observe whether NK cells, T lymphocytes and monocyte are long-term viral reservoir cells of HIV-1. Methods A group of 15 chronic HIV-l-infected adults, who received 4 - 7 years-HAART treatment [ mean （ 62. 45 ± 13.53 ） months ], participated in this study for erosssectional study. After the eth- ical approval was obtained, a volume （40 ml） of peripheral whole blood was obtained from each patient. The number of T cell subgroup was detected with flow eytometry. The NK ceils, T lymphocytes, and monocytes were separated from peripheral blood mononuclear cells （PBMC） using microbead sorting method （ CD3 ＋ , CD14 ＋ and CD56 ＋ antibody microbeads）. DNA was extracted with human DNA kit. Real-time fluorescent quantitative PCR was used to detect the serum HIV RNA and DNA in the three types of cells. Graphpad prism5 software was used to analyze the collected data. Results Among 15 patients with 4 - 7- year-HAART treatment, plasma HIV-1 RNA of all the patients are undetectable; however, HIV-1 DNA was detected in 6 patients＇T lymphocyte [ mean （ 2. 03 ± 0. 48 ） log10copies/106 cells ], in 4 patients ＇ Nk cells [ mean（ 1.82 ± 0. 32 ） logl0copies/106 cells ], and in 2 patients＇ monoeytes [ mean（ 1.75 ± 0. 19 ） log10copies/10^6 cells]. Conclusions These findings revealed that NK cells were another important HIV cellular reser- voir besides T-lymphocytes and monocytes, and T-lymphocytes might be the main long-lasting HIV reserdvoir.