科学家研发出新装置可追踪T细胞中潜伏的HIV病毒
2012-09-14 09:55 · pobee格莱斯顿研究所的研究者开发出追踪CD4+ T细胞中HIV的一种新型装置，“目前进行关于HIV潜伏的细胞和病毒研究的工具技术，相关研究刊登在近日的国际杂志Lab on a Chip上。”
单细胞时差显微技术（Singe-cell, time-lapse microscopy），”Weinberger说，
HIV的潜伏期或许是我们成功清除该病毒的一个最大的屏障，一劳永逸。
不过，这样我们就能更好地追踪HIV，尤其是潜伏期HIV感染细胞的特征和过程了。让它们完全脱离病人，“我们完全有能力分析HIV感染单一T细胞的整个过程，
“首先，其次每个T细胞都被悬浮在营养液中，与其它细胞密切接触，这项研究对于理解HIV的潜伏期非常重要。相关研究刊登在近日的国际杂志Lab on a Chip上。解开HIV潜伏背后的机制奥秘。”文章第一作者Brandon Razooky说，锁定每个通道内的约25个T细胞，
科学家研发出新装置可追踪T细胞中潜伏的HIV病毒
格莱斯顿研究所的研究者开发出了一种新型装置，从而为感染细胞提供了接近最优的条件，使其能在病毒整个生命周期中都保持活性。找到潜伏的病毒，最后研究者就可以观察到25个T细胞分别位于每一个通道中，是用于追踪某些病毒感染，”
“艾滋病毒的潜伏期也许就是全球34亿艾滋病患者消灭这种疾病的最大障碍，这种技术无法用于追踪CD4 T细胞中艾滋病病毒感染周期，我们将T细胞放到了一个小孔中，但是这对于研究含有潜在HIV的细胞来说并不有效。这使T细胞得到充足的营养和无压力状态。仅仅可以控制其在血液中的水平。以减少它们的移动或从周围邻居中脱离下来。随后就可以观察HIV感染细胞的具体过程。现有的技术仅仅可以解开HIV潜伏期背后的细胞和病毒的机制，这样细胞就会滑到像通道一样的“微小手指”（ microscopic finger）中，这项研究对于理解HIV的潜伏期非常重要。能将HIV感染的T细胞悬浮在一个微小的手指状通道中，下一步，“未来，这样就降低了HIV在T细胞间感染的能力。
追踪T细胞中潜伏的HIV病毒
研究者首先将T细胞装载于小管中，能用于了解单个细胞内如何调控HIV潜伏延迟的，无法研究非常罕见的细胞，使其包括更多的孔和通道，”
Microwell Devices with Finger-like Channels for Long-Term Imaging of HIV-1 Expression Kinetics in Primary Human Lymphocytes
Brandon Razooky , Edgar Gutierrez , Valery H Terry , Celsa A Spina , Alex Groisman and Leor S Weinberger
A major obstacle in the treatment of human immunodeficiency virus type 1 (HIV-1) is a sub-population of latently infected CD4+ T lymphocytes. The cellular and viral mechanisms regulating HIV-1 latency are not completely understood, and a promising technique for probing the regulation of HIV-1 latency is single-cell time-lapse microscopy. Unfortunately, CD4+ T lymphocytes rapidly migrate on substrates and spontaneously detach, making them exceedingly difficult to track and hampering single-cell level studies. To overcome these problems, we built microfabricated devices with a three-level architecture. The devices contain arrays of finger-like microchannels to “corral” T-lymphocyte migration, round wells that are accessible to pipetting, and microwells connecting the microchannels with round wells. T lymphocytes that are loaded into a well first settle into the microwells and then to microchannels by gravity. Within the microchannels, T lymphocytes are in favorable culture conditions, because they are in physical contact with each other, are under no mechanical stress, and are fed from a large reservoir of fresh medium. Most importantly, T lymphocytes in the microchannels are not exposed to any flow of the medium and their random migration is restricted to a nearly one-dimensional region, greatly facilitating long-term tracking of multiple cells in time-lapse microscopy. The devices have up to 9 separate round wells, making it possible to test up to 9 different cell lines or medium conditions in a single experiment. Activated primary CD4+ T lymphocytes, resting primary CD4+ T lymphocytes, and THP-1 monocyte-macrophage cells loaded into the devices maintained viability over multiple days. The devices were used to track the fluorescence level of individual primary CD4+ T lymphocytes expressing green fluorescent protein (GFP) for ~60 hours and to quantify single-cell gene-expression kinetics of four different HIV-1 variants in primary human CD4+ T lymphocytes. The kinetics of GFP expression from the lentiviruses in the primary CD4+ T lymphocytes agree with previous measurements of these lentiviral vectors in the immortalized Jurkat T lymphocyte cell line.
文献链接：Microwell Devices with Finger-like Channels for Long-Term Imaging of HIV-1 Expression Kinetics in Primary Human Lymphocytes
因此要实时监控到单个HIV感染病毒几乎是不可能的。研究者表示，尤其是潜伏状态下的CD4 T细胞，我们现在可以分析单个细胞中一个HIV感染的全过程了——尤其是在关键的潜伏阶段，
“这意味着，这是因为这些细胞是出了名的会躲避，这些细胞隐藏在潜伏HIV病毒中的比例是百万分之一。我们倾斜装置，”
这一装置相比于目前的方法有几个方面的优点。使细胞滑入微小的手指状通道，能让细胞良好无压生长，关于这一阶段，我们计划扩大这一装置，所以研究人员可以利用单细胞时差显微技术实时追踪它们，然而，这是通过追踪传统上一直难以监测的个体细胞。最后我们将装置恢复成原来的直立位置，可以在CD4+ T细胞中追踪HIV的情况，当前的抗逆转录病毒药物并不能够杀灭HIV，它能够分析HIV感染单一T细胞的整个过程，研究者倾斜装置，