【作者】 黄秀艳；

【导师】 曾耀英； Howard E. Gendelman；

【作者基本信息】 暨南大学 ， 生物医学工程， 2010， 博士

【摘要】 HIV相关的神经认知障碍(HIV-1 Associated Neurocognitive Disorders, HAND)是HIV病的中枢神经系统(Central Nervous System, CNS)常见并发症,然而其发病机理还不清楚。为此,本文进行了以下的研究：1)HIV-1中枢神经系统致病性机理研究；2)调节性T细胞的神经保护作用的机理研究。第一部分旨在为HAND的治疗药物研发奠定基础；第二部分旨在为HAND的以免疫细胞为基础的干预提供科学依据。众所周知,HIV-1需要靶细胞表面的受体(CD4)和共受体(CCR5和CXCR4)的介导才能入胞,因此在CNS只有小胶质细胞(Microglia)和血管旁巨噬细胞(Perivascular Macrophage)才能被HIV-1感染。已有的研究表明,在HAND的发生发展中,小胶质细胞的过度活化和由此而引发的神经炎症起着重要的作用。要在体外进行HIV-1致病性机理的研究,小胶质细胞的分离、纯化和培养是本研究的前提条件(第一部分—第一章)。HIV-1糖蛋白gp120(gp120)是HIV-1病毒膜蛋白,HIV-1通过gp120与宿主细胞表面的CD4分子结合而进入细胞内,推测gp120参与了HAND的发生发展；为此,论文第一部分的第二章呈现了gp120刺激人的小胶质细胞钙离子内流的结果。从gp120能够诱发细胞外钙离子进入小胶质细胞细胞的事实推断小胶质细胞的活化与此有关,这一推断进一步为gp120可使小胶质细胞ERK发生磷酸化和NF-kB发生核转位的结果所证实。另外,HIV-1颗粒被看作是一个超分子,它的表面蛋白包括自身基因组编码的蛋白(gp120)和出胞时所携带的宿主蛋白,因此,第一部分的第三章呈现了灭活HIV-1颗粒刺激人的小胶质细胞钙离子内流的结果。结果表明,灭活HIV-1颗粒所引发的钙内流效应明显强于gp120,推论HIV-1颗粒表面的宿主蛋白在此起了一定的生物学作用。由此可见,能够阻断gp120和HIV-1颗粒相关的钙离子内流的药物有望遏制HAND的发生发展。已有的体内研究发现：向HIVE小鼠模型过继输入CD3活化的调节性T细胞(Regulatory T Cell, Treg)能够产生神经保护,但是其保护机制的细节尚不清楚。本论文的第二部分是在此基础上,通过分析体外共培养HIV-1/VSV感染的巨噬细胞和Treg,探讨Treg对感染的巨噬细胞的功能调节,旨在阐明Treg的神经保护作用的机理。第二部分的三个章节分别呈现了相关研究的结果。我们发现：1)Treg可以上调HIV-1/VSV感染的巨噬细胞的抗病毒免疫应答；2)Treg可以杀伤HIV-1/VSV感染的巨噬细胞；3)Treg可以诱导HIV-1/VSV感染的巨噬细胞发生Apoptosis,而不是Pyroptosis; 4)活化的Treg能够表达颗粒酶A,颗粒酶B和穿孔素；5)Treg可以通过granzyme/perforin杀伤HIV-1/VSV感染的巨噬细胞；6)Treg可以通过cAMP途径诱导HIV-1/VSV感染的巨噬细胞功能转化(M1-M2)。由此,Treg可以通过以上机制影响HIV-1/VSV感染的巨噬细胞的功能,从而化解感染的巨噬细胞引发的神经炎症。这些发现推翻了认为Treg功能仅局限于其免疫抑制的教条,并为探讨适应性免疫监控如何改善HAND提供了新的思路。更多还原

【Abstract】 The pathogenesis of HIV-1-associated neurocognitive disorders (HAND), the most common complication of HIV disease in CNS, is still unclear. Therefore, this study was designed to explore the underlying mechanisms. The Part One of this thesis is devoted to the contributions of inactivated HIV-1 particle to microglial activation, which aims to providing scientific basis for therapeutic drug discovery. The Part Two of this thesis is focus on the mechanisms of Treg induced neuroprotection, which sets stage for immune intervention of HAND.As we known, the entry of HIV-1 to target cells is mediated by its receptor (CD4) and co-receptors (CCR5 and CXCR4). Therefore, in the CNS, only microglia and perivascular macrophage express those molecules and are infected by HIV-1. Previous studies demonstrated that the neuroinflammation triggered by activated microglia/macrophage plays a key role in the initiation and development of HAND. In order to decipher the effects of HIV-1 particle on microglia, it is very important to obtain highly purified human microglia, which is described in the First Chapter of Part One. HIV-1 glycoprotein, gp120, amongst the most studied viral proteins in the pathogenesis of HIV disease, is shown to mediate the virus entry and involve in neuroinflammation. In the Second Chapter of Part One, we found that gp120 could activate microglia via triggering calcium influx, inducing ERK phosphorylation and stimulating NF-кB translocation in human microglia. Moreover, HIV-1 particle is considered as a supermolecule, because its surface incorporates both virus and host derived proteins. The comparison study showed that inactivated HIV-1 particles trigger much stronger calcium influx than gp120, so the host derived proteins must play an important role in microglial activation. Taken together, the drugs, which could block the microglial calcium influx triggered by either gp120 or HIV-1 particles, may incite neuroprotection.Recent research has demonstrated a neuroprotective role for Treg in a mouse model of HIVE. However, the mechanism is only partially understood, which are believed to resolve neuroinflammation elicited by HIV-1 infected microglia/macrophage. In Part Two of this thesis, we cocultured HIV-1/VSV infected macrophage with Treg to find how Treg modulate macrophage function. We demonstrated that Treg promote neuroprotection by inhibiting virion release, killing infected macrophages and inducing phenotypic switch. Surprisingly, Treg inhibits progeny virion release through upregulating ISG15, an ubiquitin-like protein involving in interferon-mediated antiviral immunity. Importantly, Treg kills infected macrophages through both caspase-3 and granzyme/perforin pathways. Independently, Treg transforms infected macrophages from M1 to M2 phenotype as demonstrated by downregulated inducible nitric oxide synthase and unregulated arginase 1 expression. Taken together, Treg affacts infected macrophage function and achieves neuroinflammation resolution during the course of HIV disease. These findings challenge the dogma of a solitary Treg immune suppressor function and provide novel insights into how adaptive immunosurviellance limits the severity of HAND.更多还原