Marker development for mosquito hemocytes

描述（由申请人提供）：昆虫免疫系统由细胞和体液成分组成，它们天生就能识别各种外来入侵者。虽然在过去几年中，在识别诸如抗菌肽和调节其合成的信号通路等体液因子方面取得了很大进展，但对细胞防御反应的控制知之甚少。这在很大程度上是由于许多昆虫的体积小，这使得收集血细胞和鉴定血细胞产生的效应分子变得困难。在体内对血细胞介导的防御反应进行操控性实验，或者分离出特定的血细胞群体用于体外实验，通常也很困难。对于像蚊子这样的病媒节肢动物来说尤其如此，因为关于不同类型血细胞的起源、分化或功能的信息很少。蚊子血细胞的分类主要基于形态学，只有有限的数据将这些形态学特征与功能特征联系起来。对于极其重要的病媒，如冈比亚按蚊，在任何生命阶段对血细胞的个体发生几乎一无所知。在这里，我们的目标是使用血细胞样细胞系（4a-3B细胞），完全测序的An。利用我们对其他昆虫的血细胞谱系标记的了解，开发了一套强大的特征来识别冈比亚线虫。冈比亚按蚊通过姬氏。考虑到应用程序的探索性和An的有限背景。因此，我们认为将该申请作为R21提案提交是合适的。具体目标是：1.；克隆和表征4a-3B细胞亚群，2。鉴定An的候选基因标记和基因特异性标记。2 .冈比亚红细胞和4a-3B细胞；对亚克隆4a-3B细胞进行蛋白质组学分析，鉴定新的候选标记物。本研究结果将为了解不同血细胞在豚鼠细胞免疫中的作用提供重要工具。冈比亚和其他种类的蚊子。

DESCRIPTION (provided by applicant): The insect immune system consists of both cellular and humoral elements that innately recognize broad classes of foreign intruders. While great progress has been made over the last several years in identifying humoral factors like antimicrobial peptides and the signaling pathways that regulate their synthesis, much less is known about control of cellular defense responses. This is due in large part to the small size of many insects which makes collection of hemocytes and identification of hemocyte-produced effector molecules difficult. It is also often difficult to conduct manipulative experiments on hemocyte-mediated defense responses in vivo or to isolate defined populations of hemocytes for use in experiments in vitro. This is particularly true for vector arthropods like mosquitoes where little information exists on the origins, differentiation or functions of different hemocyte types. Mosquito hemocytes are classified primarily on the basis of morphology with only limited data relating these morphological characters to functional features. In the case of critically important vectors, like Anopheles gambiae, virtually nothing is known about hemocyte ontogeny in any life stage. Here, our goal is to use hemocyte-like cell lines (4a-3B cells), the fully sequenced An. gambiae genome, and our knowledge of hemocyte lineage markers from other insects to develop a robust suite of characters for identifying An. gambiae hemocytes. Given the exploratory nature of the application and limited background available on An. gambiae hemocytes, we think it appropriate to submit the application as an R21 proposal. Specific aims are: 1. Clone and characterize subpopulations of 4a-3B cells, 2. Characterize candidate generic and gene specific markers in An. gambiae hemocytes and 4a-3B cells, and 3. Conduct a proteomic analysis of the subcloned 4a-3B cells to identify new candidate markers. The results of this study will provide essential tools for understanding the role of different hemocytes in cellular immunity of An. gambiae and likely other species of mosquitoes.