An open microfluidic device to study chemotaxis of eukaryotic cells

描述（由申请人提供）：拟议的研究项目旨在研究细胞迁移的机制，并帮助我们进一步了解极化在定向细胞迁移中的作用。这些研究将使用社会阿米巴Dictyosteelium discoideum，并将使用新开发的开放式微流体装置由聚二甲基硅氧烷制成。该装置具有多个微米尺寸的通道，其可用于使形态上不同的细胞彼此竞争，并且通道还能够限制细胞用于极性研究。了解更多关于D。这些发现可能与控制其他真核细胞如中性粒细胞的趋化过程相关，也阐明了调节癌症转移的途径。为了实现这些目标，确定了三个具体目标。第一个具体目标集中在进一步开发可用于趋化性测定的开放式微流体装置，并允许可视化稳定的被动化学梯度。第二个具体目标是通过比较极化和非极化D.盘状突细胞向其各自的化学引诱物。表达荧光细胞骨架和信号传导蛋白的细胞将在微流体通道的一端暴露于化学引诱物，之后梯度将被逆转，并且随着细胞去增殖和再增殖，这些荧光分子的再分布将被分析。第三个具体目标集中在真核细胞中的梯度感应机制。将梯度浓度（输入）与迁移速度和信号蛋白定位（输出）进行比较。根据国家普通医学科学研究所（NIGMS）的使命，这项研究计划旨在了解真核细胞的趋化过程。这些实验的发现将有助于阐明细胞感知化学梯度和迁移的基本机制，并进一步了解癌细胞如何转移。拟议的研究计划将要求PI。应用在课堂上和科学文献中学到的理论。将在实验室采用适当的研究技术，并在研究申办者的指导下进行实验。Janetopoulos实验室的合格人员和范德比尔特大学医学中心的工作人员将促进适当设备和技术使用的培训和实施。 公共卫生相关性：这项研究与公共卫生有关，因为它试图了解真核细胞在化学梯度上向上或向下迁移的基本原理。这是一种在许多疾病状态下变得异常的行为，例如关节炎期间的炎症和原发性肿瘤的癌症转移。通过了解调节这种运动的机制，应该有可能设计出阻止流氓细胞向健康器官运动的治疗方法，从而防止致命和衰弱疾病的传播。

DESCRIPTION (provided by applicant): The proposed research project seeks to investigate the mechanisms by which cells polarize and help further our understanding of the role of polarization in directed cell migration. These studies will use the social amoeba Dictyostelium discoideum and will be conducted using a newly developed open microfluidic device made out of Polydimethylsiloxane. This device has multiple, micron-sized channels which can be used to race morphologically distinct cells against each other and the channels are also capable of confining cells for polarity studies. By learning more about D. discoideum cells, these findings can be correlated with the chemotaxis processes controlling other eukaryotic cells such as neutrophils and also elucidate the pathways that regulate cancer metastasis. In order to achieve these goals, three specific aims have been set. The first specific aim focuses on the further development of an open microfluidic devices that can be used for chemotaxis assays, and allows the visualization of a stable, passive chemical gradient. The second specific aim targets the role and mechanisms of polarity in chemotaxing cells by comparing the migration of polarized and unpolarized D. Discoideum cells towards their respective chemoattractants. Cells expressing fluorescent cytoskeletal and signaling proteins will be exposed to chemoattractant at one end of a microfluidic channel, after which the gradient will be reversed and the redistribution of these fluorescent molecules will be analyzed as the cells de-polarize and re-polarize. The third specific aim focuses on the gradient sensing mechanism in eukaryotic cells. The concentration of the gradient (input) will be compared to the migration speeds and signaling protein localizations (output). In line with the mission of the National Institute of General Medical Sciences (NIGMS), this research proposal seeks to understand the chemotaxis processes of eukaryotic cells. The findings from these experiments will help elucidate the basic mechanisms by which cells sense chemical gradients and migrate and further our understanding of how cancer cells metastasize. The proposed research program will require the P.I. to apply theory learned in both the classroom and from scientific literature. Appropriate research techniques will be picked up in the laboratory and the experiments will be performed under the guidance of the research sponsor. Training and implementation on the use of appropriate equipment and techniques will be facilitated by qualified personnel in the Janetopoulos laboratory and the staff at Vanderbilt University medical center. PUBLIC HEALTH RELEVANCE: This research is relevant to public health as it seeks to understand the basic principles by which eukaryotic cells migrate up or down a chemical gradient. This is a behavior that becomes aberrant in many diseases states such as inflammation during arthritis, and metastasis of cancer from the primary tumor. By gaining knowledge about the mechanisms that regulate this motility, it should be possible to design treatments that block the movement of rogue cells toward healthy organs and hence prevent the spread of deadly and debilitating diseases.