自噬在巨噬细胞抵抗HIV Vpr诱导的凋亡中的作用及机制研究

Persistence of latent viral reservoirs is the huge obstacles that prevent eradication and cure of HIV infection. Macrophages represent major viral reservoirs. Macrophages are insensitive to HIV cytopathic effect and support viral replication, which contributes to viral persistence and reservoir formation. One of the factors that contribute to HIV persistence in macrophages is their resistance to various apoptotic stimuli. Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) is a small 96-amino acid multifunctional protein. Vpr can induce apoptosis in several cell types, including lymphocytes, monocytes, astrocytes, and neurons. However, HIV-1 infected macrophages are resistant to Vpr-induced apoptosis, and the mechanisms that render macrophages resistant to Vpr-induced apoptosis are poorly understood..Autophagy is a self-digestion process essential for balancing sources of energy at critical times in development and in response to nutrient stress. A lot of data indicates autophagy is involved in numerous pathologic and physiologic progresses of many diseases. Autophagy has dual characteristics of promoting cell survival and death, in the context of disease, it has been seen as an adaptive response to survival, whereas in other cases it appears to promote cell death and morbidity. To some extent, autophagy plays a pivotal role in deciding whether a cell will live or die. However, it has not been demonstrated whether autophagy is involved in macrophages resistant to Vpr-induced apoptosis. Here, we used a THP1-derived macrophages (THP1-MACs) line to study the effects of HIV-Vpr on autophagy and interconnection between autophagy and macrophage resistance to Vpr-induced apoptosis. The study may provide a new theoretical basis and experimental data for HIV persistence in macrophages.

HIV病毒储存库的形成是艾滋病治疗的主要障碍。巨噬细胞是HIV重要的病毒储存库，其形成机制复杂，近年来HIV Vpr蛋白参与巨噬细胞HIV病毒库的形成受到关注。Vpr蛋白是HIV的一个辅助蛋白，可以诱导多种细胞的凋亡，却不能促使巨噬细胞凋亡，这有助于HIV巨噬细胞储存库的形成，但其机制尚未明了。细胞自噬的主要功能之一是细胞受到应激性死亡威胁时保持细胞的存活。那么自噬是否介导了巨噬细胞抵抗Vpr诱导的凋亡？我们在前期工作中观察到Vpr能诱导巨噬细胞发生自噬现象，且抑制自噬后能明显增加巨噬细胞的凋亡。我们推论：巨噬细胞通过上调自噬水平来抵抗Vpr诱导的凋亡，从而利于病毒库的形成。该结果在国际上尚无报道，本研究将在前期工作的基础上深入研究自噬与巨噬细胞抵抗Vpr凋亡的关系，探讨其调控机制，将使我们对HIV巨噬细胞病毒库的形成有更深一步的了解，也为清除HIV储存库提供新的干预靶点和策略。

自噬参与了巨噬细胞抵抗HIV Vpr诱导的凋亡，本课题运用细胞生物学和分子生物学等方法与技术，对自噬参与调控巨噬细胞抵抗HIV Vpr诱导的凋亡分子机制进行了初步研究。首先通过核转染技术将Vpr质粒转染至THP1源巨噬细胞，观察Vpr对THP1源巨噬细胞自噬和凋亡的影响；再上调和下调自噬的水平，了解自噬是否介导THP1源巨噬细胞抵抗Vpr诱导的凋亡。进而初步探讨了自噬介导THP1源巨噬细胞抵抗Vpr诱导凋亡的机制。结果显示自噬能诱导THP1源巨噬细胞自噬；抑制自噬水平能够增加THP1源巨噬细胞的凋亡水平。本研究使我们对HIV巨噬细胞病毒库的形成有更深一步的了解，也为清除HIV储存库提供新的干预靶点和策略。项目资助发表SCI检索论文1篇，待发表两篇。共同培养硕士研究生2名。项目投入经费23万元，支出：8.8961万元，余额14.1039万元。由于该结论在临床细胞标本（健康人来源巨噬细胞）方面验证难度较大，暂未开展，准备改变实验室方法再验证此结论，因此各项支出与预算不完全相符，剩余经费计划用于本项目后续支出。