人类免疫缺陷病毒-1感染者外周血单个核细胞体外培养的免疫学及病毒学特点

目的观察人类免疫缺陷病毒(HIV)感染者外周血单个核细胞(PBMC)体外培养过程中免疫学及病毒学的动态变化特点,并评价培养的细胞体系抗病毒活性。方法采集14例HIV感染者和6名健康人的PBMC,体外经多种细胞因子诱导培养,每3天观察细胞增殖情况,同时检测细胞表型、上清中细胞因子浓度和病毒载量。结果培养的健康人PBMC在(35±5)d最大增殖(61±8)倍;7例培养成功的HIV感染者PBMC在(21±6)d最大增殖(17±13)倍,另7例HIV感染者PBMC培养失败;同时发现HIV感染者PBMC体外培养最大增殖时间与其培养前外周血基础CD4/CD8比值呈明显正相关(P<0.05)。表型分析发现,培养的PBMC为优先CD8细胞增殖的异质T细胞群,主要由CD4、CD8及CD3CD56细胞组成;部分HIV感染者PBMC培养期间分泌白细胞介素(IL)1α、IL12、肿瘤坏死因子(TNF)α和IL10等细胞因子能力较高;而11/12例HIV感染者PBMC体外培养初期可扩增出大量病毒,但随着培养时间的延长,病毒载量逐渐降低甚至低于检测限。结论体外大量扩增基础CD4/CD8比值较高的HIV感染者外周血PBMC是可行的,扩增的细胞具备较强的Th1细胞免疫反应能力,为进一步开展艾滋病的免疫细胞治疗提供必要的实验数据。

Serial analyses of viral levels and immunocytes during in vitro incubation of peripheral blood mononuclear cells from HIV-infected individuals

OBJECTIVE: To investigate the dynamic changes of viral loads and immunocytes during the in vitro culture of peripheral blood mononuclear cells (PBMC) from HIV carriers. METHODS: The PBMCs from 14 HIV-infected individuals and 6 healthy persons were incubated in serum-free AIM-V medium containing cocktail cytokines. The phenotype of CD3, CD4, CD8, CD3CD56 and CD25 was identified by flow cytometric analysis every two days. The production of cytokines in the supernatants, including IL-1alpha, IL-12, TNF-alpha and IL-10 was measured by ELISA. The supernatant HIV-1 RNA load was also determined by Real-time fluorescent PCR. RESULTS: During a 21-day incubation period, The PBMCs multiplied approximately 60.7-fold and 16.8-fold respectively in the healthy controls and 7 out of the 14 HIV-infected subjects, however failed to multiply in the remaining 7 HIV-infected subjects. The expanded cells were phenotypically shown a heterogeneous cellular population with 23.3%-35% for CD3(+)CD4(+) T cells and 58.7%-72% for CD3(+)CD8(+) T cells, and approximate 17% CD3(+)CD56(+) cells at 16-day incubation for HIV-infected cases. HIV-1-positive PBMCs were found to produce an elevated ratios (value range 6.01 - 48.04) of IL-12:IL-10 compared to healthy individuals (6.65 - 10.2) at 16-day incubation. Furthermore, serial analyses of HIV-1 RNA levels showed an inverted V type dynamic change during 16 day in vitro incubation period. CONCLUSION: In vitro expansion of functional immunocytes of HIV-1 carrier origin is feasible and may facilitate the autologous antiviral immune therapy for HIV-infected patients.