美国能源部国家核安全管理局(National Nuclear Security Administration，NNSA) Sandia实验室的Anup Singh研究团队开发了一个新的方法，能观察到免疫细胞在最初数分钟到数小时内与入侵的致病菌搏斗的情况。此研究的合作伙伴还包括德州大学加尔威史东医学分部（University of Texas Medical Branch，简称UTMB at Galveston）以及加州大学圣地亚哥分校（University of California, San Francisco简称UCSF）。
Singh 教授说：「了解免疫细胞对付致病菌的初期阶段，有助于阻止疾病的形成也能在疾病形成初期进行有效的治疗。」由于每种细胞都有不同的生命周期，并非所有的细胞都在同一时间与致病菌相遇，因此，研究人员需要观察不同细胞在相同的生命周期及感染期的情况。Sandia实验室开发了许多工具才能完成这些实验，包括：”Microfludics”，能让研究人员进行单细胞的实验；”advanced imaging”，能观察到细胞更细致的信息；”powerful computational modeling”能更精确的仿真由影像或分析数据而得到的结果。
研究人员用两个互补的微流体芯片(microfluidic)组成一个平台，其中一个用来补捉及造像活细胞受到致病菌刺激时的情况，另一个则用来观察细胞备战的准备程序，包括选择什么细胞作战以及会用什么样的蛋白质等。当然还包括许多劳心劳力的技术，包括：共轭焦显微镜技术(Confocal Microscopy)、流式细胞仪（flow cytometer）以及免疫分析等。目标要同时观察到10~40个蛋白，并且能同时染色看到它们的3D分布情况。
Singh教授表示：「终极目标希望在二年内能制作出整合型的微型化微流体芯片系统，能将此系统置于生物安全等级3及4的实验室，以进行致病菌与免疫反应的研究，他强调这个整合的平台、生物性试剂和计算机仿真的应用絶不仅限于感染性疾病的研究。还可用来研究细胞内的讯息传递，例如：癌细胞等疾病的讯息传递，或者制药公司可用于生物指标的开发。」
(资料来源 : Bio.com)
英语原文：
Sandia Researchers Take New Approach to Studying How Cells Respond to Pathogens
04/03/07 -- A Sandia National Laboratories research team led by Anup Singh is taking a new approach to studying how immune cells respond to pathogens in the first few minutes and hours of exposure.
Their method looks at cells one at a time as they start trying to fight the invading pathogens.
Called the Microscale Immune Studies Laboratory (MISL) Grand Challenge, the work is in its second of three years of funding by the internal Laboratory Directed Research and Development (LDRD) program. Sandia is partnering on the project with the University of Texas Medical Branch (UTMB) at Galveston and the University of California, San Francisco (UCSF).
Sandia is a National Nuclear Security Administration (NNSA) laboratory.
Singh says the researchers are interested in studying the early events in immune response when a pathogen invades a body. Understanding the early steps could lead to better ways to diagnose and stop disease before there are symptoms and development of more effective therapeutics.
Most existing research into how immune cells respond has been done by looking at large cell populations. The Sandia researchers say information gathered from a large population of cells may mask underlying mechanisms at the individual cell level.
"Cells have different life cycles, just like any living being. And not all cells are exposed to the pathogen at the same time," Singh says. "We wanted to look at cells in the same life cycle and same infectious state. This can only be done cell by cell. We also want to study populations, but one cell at a time."
The research is possible because of advances in several Sandia-developed tools, including:
Microfluidics that allows researchers to do single-cell experiments
Advanced imaging that allows researchers to image individual cells with much higher information content than possible with current commercial imaging technologies
Powerful computational modeling that allows researchers to make sense of data obtained from microfluidic analysis and imaging
Real immune cells are short-lived outside of bodies. To do the type of experiments they wanted, the researchers needed cells that can stay alive more than a couple of hours, have the ability grow and represent a relevant model of human immune cells. They obtained "immortalized mouse immune cells" from a collaborator at UCSF that have the needed life span, and are accepted as a model system by the immunology research community.
"We're starting with robust and well-characterized cells, which really simplifies development of our new technologies and methods," Singh says. "We'll soon be working with other cell types, though, like white blood cells directly isolated from human patients. Our approach is designed to be flexible enough to handle many different cell types, and it also minimizes the number of cells needed for analysis, so it should enable us to do some unique studies on rare cell types."
Proteins in the cells of interest are tagged with fluorescent molecules, essentially colored dyes. The dyes range from green to red and give researchers the opportunity to track proteins and see, for example, the dynamic cellular production of proteins or protein-binding processes inside or on the surface of the cells.
The team is developing one platform with two complementary microfluidic modules -- one for trapping and imaging viable cells during stimulation with pathogens. The other combines cell preparation steps, cell selection and sorting followed by analysis of protein content in the selected cell subpopulations.
"In effect, we are taking many work-horse technologies such as confocal microscopy, flow cytometry and immunoassays and combining them into one compact, miniaturized platform using our unique microfluidic and imaging tools," Singh says.
Hyperspectral fluorescence imaging with multivariate curve resolution (MCR) is used to image the tagged proteins and provide quantitative measurements on multiple proteins simultaneously. The goal is to analyze as many as 10 to 40 proteins and cellular stains at a time in three dimensions.
The end results of the imaging and protein analysis are large amounts of data that must be categorized and understood. Computational modeling is then used to develop network models from experimental data and predictive modeling generates hypotheses to be tested next.
Singh says using an integrated microfluidic platform sets Sandia apart from the rest of the world. Sandia researchers have been working in the area of microfluidics -- the science of designing, manufacturing, and formulating devices and processes that deal with volumes of fluid on the order of nanoliters -- since the 1990s and have a good understanding about how to use microfluids to analyze cell activity. The microfluidic platform is fast and highly parallel and can perform hundreds of measurements 50 to 100 times faster than alternate methods.
Singh says the end goal is to make a benchtop miniaturized system expected in about two years. It would be placed in Biosafety Level 3 or 4 labs to study immune response to highly pathogenic organisms. He notes the integrated platform, biological reagents and computational models developed under this project have applicability beyond infectious disease research. These technologies can also be used for studying cellular signaling involved in diseases such as cancer or by pharmaceutical companies for biomarker discovery.
Source: DOE/Sandia National Laboratories
