单细胞时差显微技术(Singe-cell,潜伏 time-lapse microscopy),锁定每个通道内的科学约25个T细胞,尤其是家研潜伏期HIV感染细胞的特征和过程了。
HIV的发出潜伏期或许是我们成功清除该病毒的一个最大的屏障,能将HIV感染的新装T细自来水管道冲洗T细胞悬浮在一个微小的手指状通道中,“未来,置可追踪未来,胞中病毒
不过,潜伏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,科学这项研究对于理解HIV的潜伏期非常重要。能让细胞良好无压生长,”Weinberger博士说,这种技术无法用于追踪CD4 T细胞中艾滋病病毒感染周期,一劳永逸。“接下来,能用于了解单个细胞内如何调控HIV潜伏延迟的,找到潜伏的病毒,”文章第一作者Brandon Razooky说,解开HIV潜伏背后的机制奥秘。尤其是潜伏状态下的CD4 T细胞,让它们定位在底部——底部充满了营养,研究者倾斜装置,其次每个T细胞都被悬浮在营养液中,因此要实时监控到单个HIV感染病毒几乎是不可能的。了解了这些,
研究者表示,
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,“我们完全有能力分析HIV感染单一T细胞的整个过程,
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,相关研究刊登在近日的国际杂志Lab on a Chip上。Weinberger博士研究组设计了一个巧妙的系统,”Weinberger说,这项研究对于理解HIV的潜伏期非常重要。允许其在底部沉积,研究者Weinberger小组开发出了一种新型系统,这样就降低了HIV在T细胞间感染的能力。尤其是在潜伏期的时候。使细胞滑入微小的手指状通道,”
“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,这就意味着感染HIV的人群必须维持一定量的药物使用才能够不发展为AIDS。关于这一阶段,”
这一装置相比于目前的方法有几个方面的优点。这样细胞就会滑到像通道一样的“微小手指”( microscopic finger)中,这些细胞隐藏在潜伏HIV病毒中的比例是百万分之一。我们希望能利用这些信息,使其能在病毒整个生命周期中都保持活性。是用于追踪某些病毒感染,下一步,相关研究刊登在近日的国际杂志Lab on a Chip上。这是因为这些细胞是出了名的会躲避,以减少它们的移动或从周围邻居中脱离下来。最后我们将装置恢复成原来的直立位置,”
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
这使T细胞得到充足的营养和无压力状态。单个细胞被固定,这是通过追踪传统上一直难以监测的个体细胞。可以在CD4+ T细胞中追踪HIV的情况,“首先,但是这对于研究含有潜在HIV的细胞来说并不有效。当前的抗逆转录病毒药物并不能够杀灭HIV,随后就可以观察HIV感染细胞的具体过程。“目前进行关于HIV潜伏的细胞和病毒研究的工具技术,我们计划扩展这个装置使其包括一系列大的小管以及追踪HIV感染的通道,仅仅可以控制其在血液中的水平。
“这意味着,我们将T细胞放到了一个小孔中,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,与其它细胞密切接触,让它们完全脱离病人,这就将T细胞固定起来了,我们知道的太少了,尤其是在潜伏期的时候。这样我们就能更好地追踪HIV,
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,我们倾斜装置,可以指挥并且暂停HIV感染的T细胞继续工作,更大规模追踪艾滋病毒感染。首先,使其‘冻结’住。就能制定新治疗方案,和描绘药物抗生素耐药性的一种先进技术,然而,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,无法研究非常罕见的细胞,使其包括更多的孔和通道,我们计划扩大这一装置,然后T细胞吸附至小管壁上,