HIV-1辅受体的配体细胞内双表达对病毒感染的阻断作用

Intracellular coexpression of CC- and CXC-chemokines,RANTES and SDF-1α for blocking HIV-1 infection

目的 观察Ⅰ型人免疫缺陷病毒 (HIV 1)辅受体的配体———RANTES和SDF 1α双表达于人淋巴细胞对各种嗜性HIV 1毒株感染的阻断作用。方法 用 pLNCX R K S K重组逆转录病毒液感染原代人外周血淋巴细胞 (PBLs) ,抗神经生长因子受体 (NGFR) 免疫磁珠法分离转化成功的PBLs,流式细胞仪检测筛选效率 ;HIV 1M嗜性、T嗜性和双嗜性毒株攻击转化PBLs ,检测HIV 1逆转录酶活性和 p2 4抗原分泌 ,以观察抗HIV 1感染的作用 ;同时进行转化PBLs表面CD3、CD4、CCR2、CCR5和CXCR4表达及破伤风毒素刺激后3 H 胸苷 (thymidine)掺入量检测 ,观察HIV 1辅受体配体的双表达对人PBLs正常生物学功能的影响。结果 抗 NGFR 免疫磁珠法获得了转化成功的PBLs,流式细胞仪检测发现pLNCX R K S K转染组 92 %以上的PBLs鼠抗NGFR标记物为阳性 ;HIV 1M嗜性、T嗜性和双嗜性毒株攻击后 ,pLNCX R K S K转化PBLs可以见到明显的逆转录酶活性和 p2 4抗原分泌抑制 ,并且在感染后第 12～ 2 0天时抑制作用最强 ;pLNCX R K S K转化PBLs表面CD3、CD4和CCR2表达水平无明显变化 ,而CCR5和CXCR4表达水平降低 ;破伤风毒素刺激后的转化PBLs仍具有主动增殖的能力。结论 HIV 1辅受体的配体通过在人PBLs内双表达 ,使HIV 1两类主要辅受体表型剔除 。

Objective To investigate the inhibitory effects on HIV-1 infection of the intracellular coexpression of the ligands of HIV-1 coreceptors—RANTES and SDF-1α. Methods Primary human PBLs were transduced with the recombinant vector pLNCX-R-K-S-K(△NGFR), followed by an anti-NGFR/anti-IgG-magnetic bead method selection and FCM detection. The transduced PBLs were infected with M-tropic, T-tropic or dual-tropic HIV-1 virus thereafter RT activities and p24 antigen detection were carried out to study anti-HIV-1 activity of the co-inactivation of CCR5 and CXCR4. Results pLNCX-R-K-S-K(△NGFR)-transduced PBLs were isolated with anti-NGFR/anti-IgG-magnetic bead method. After isolation, about 92% of the PBLs were positive for the NGFR marker. When the transduced PBLs were infected with M-tropic, T-tropic or dual-tropic HIV-1 virus, RT activities and p24 antigen secretions were very low in the cultures of pLNCX-R-K-S-K(△NGFR)-transduced PBLs, the best inhibitory effects of which were on day 12 to 20. What's more, there were comparable levels of the surface expression of CD3, CD4 and CCR2 and dramatically decreased expression of CCR5 and CXCR4 on the PBLs transduced with pLNCX-R-K-S-K(△NGFR). At the same time, the transduced PBLs actively proliferated after the tetanus antigens stimulation. Conclusions The intracellular coexpression of CC- and CXC-chemokines—RANTES and SDF-1α effectively protects primary human PBLs from M-tropic, T-tropic and dual-tropic viruses infection, but does not interfere with normal lymphocyte functions.