Immortalization of primary microglia: a new platform to study HIV regulation in the central nervous system
Immortalization of primary microglia: a new platform to study HIV regulation in the central nervous system
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DOI：
10.1007/s13365-016-0499-3
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发表时间：
2017-02-01
影响因子：
3.2
通讯作者：
Alvarez-Carbonell, David
中科院分区：
文献类型：
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作者：
Garcia-Mesa, Yoelvis;Jay, Taylor R.;Alvarez-Carbonell, David
中枢神经系统中HIV的主要储存库是小胶质细胞、血管周围巨噬细胞，在较小程度上是星形胶质细胞。为了研究控制HIV在小胶质细胞中表达的分子事件，我们开发了一种可靠且稳健的方法，以使来自新鲜CNS组织的原代胶质细胞和市售冷冻胶质细胞的小胶质细胞永生化。用表达SV 40 T抗原或SVR 40 T抗原和hTERT的组合的慢病毒载体转化原代人细胞，包括从成人脑组织获得的细胞。永生化细胞具有小胶质细胞样形态，并表达关键的小胶质细胞表面标志物，包括CD 11b、TGF β R和P2 RY 12。重要的是，通过测序证实这些细胞是人类来源的。通过RNA-seq鉴定的RNA表达谱也是小胶质细胞的特征。此外，这些细胞表现出预期的迁移和吞噬活性，以及产生原发性小胶质细胞特有的炎症反应的能力。永生化方法也已成功应用于广泛的小胶质细胞从其他物种（猕猴，大鼠和小鼠）。为了研究CNS中HIV分子调控的不同方面，用HIV报告病毒对细胞进行超感染，并选择了对炎症信号有反应的HIV的潜伏感染克隆。我们已经开发并严格表征的细胞系将为小胶质细胞中HIV感染的研究以及小胶质细胞功能的研究提供宝贵的资源。
The major reservoirs for HIV in the CNS are in the microglia, perivascular macrophages, and to a lesser extent, astrocytes. To study the molecular events controlling HIV expression in the microglia, we developed a reliable and robust method to immortalize microglial cells from primary glia from fresh CNS tissues and commercially available frozen glial cells. Primary human cells, including cells obtained from adult brain tissue, were transformed with lentiviral vectors expressing SV40 T antigen or a combination of SVR40 T antigen and hTERT. The immortalized cells have microglia-like morphology and express key microglial surface markers including CD11b, TGF beta R, and P2RY12. Importantly, these cells were confirmed to be of human origin by sequencing. The RNA expression profiles identified by RNA-seq are also characteristic of microglial cells. Furthermore, the cells demonstrate the expected migratory and phagocytic activity, and the capacity to mount an inflammatory response characteristic of primary microglia. The immortalization method has also been successfully applied to a wide range of microglia from other species (macaque, rat, and mouse). To investigate different aspects of HIV molecular regulation in CNS, the cells have been superinfected with HIV reporter viruses and latently infected clones have been selected that reactive HIV in response to inflammatory signals. The cell lines we have developed and rigorously characterized will provide an invaluable resource for the study of HIV infection in microglial cells as well as studies of microglial cell function.