CAA-Midgut Growth Factors Inducing Stem Cell-Mediated Epithelial Regeneration

由于消化过程中涉及的剪切力，肠上皮细胞具有最高的细胞更新速率之一，并且上皮完整性通过由干细胞增殖和分化控制的组织愈合机制来保持。在昆虫中肠上皮中，这种愈合过程仅在蜕皮期间或损伤后活跃，并且已经假设它是对生物杀虫剂的抗性机制。该项目的主要目标是识别激活中肠上皮愈合反应的特定生长因子，并表征该过程中导致毒素抗性的变化。该研究将集中在用来自细菌苏云金芽孢杆菌的Cry毒素攻击后，Heliothis virescens毛虫（烟草夜蛾）中肠中引起的再生反应。原代中肠细胞培养物将用作实验模型，并且将使用液相色谱、干细胞分化测定和基于凝胶的差异蛋白质组学的组合来鉴定在Cry毒素处理后激活其分化为成熟细胞的因子。根据这种方法，还将表征抗Cry烟草芽虫中肠再生反应的改变。激活中肠再生的因子的鉴定将提供关于干细胞控制上皮愈合的相关信息，并且通过提供控制细胞培养更新以开发稳定的中肠细胞系的工具而使科学界受益匪浅。该项目的更广泛影响还包括确定用于开发新的生物防治方法和杀虫剂的关键愈合途径。少数民族研究生将接受最先进的蛋白质组学和细胞培养技术的培训，这种培训将提供一个坚实的核心工具，以追求未来在生物和医学研究领域的研究机会。主要研究者和学生将在为代表性不足的群体服务的当地教育机构的社区外联活动中介绍该项目的成果，以培养参与科学研究的兴趣。

Due to the shear forces involved in the digestive process, the intestinal epithelium has one of the highest rates of cell renewal, and epithelial integrity is preserved by tissue healing mechanisms controlled by stem cell proliferation and differentiation. In the insect midgut epithelium, this healing process is only active during molting or following injury and has been hypothesized that it is a mechanism of resistance to biological insecticides. The main goal of this project is the identification of the specific growth factors activating the midgut epithelium healing response and the characterization of alterations in this process that result in resistance to toxins. The study will be focused on the regenerative response elicited in midguts from Heliothis virescens caterpillar (tobacco budworm) after being challenged with Cry toxins from the bacterium, Bacillus thuringiensis. Primary midgut cell cultures will be used as the experimental model and the factors activating their differentiation to mature cells upon Cry toxin treatment will be identified using a combination of liquid chromatography, stem cell differentiation assays, and gel-based differential proteomics. Following this approach, alterations in the midgut regenerative response in Cry-resistant tobacco budworms will also be characterized. Identification of the factors activating midgut regeneration will provide relevant information on control of epithelial healing by stem cells and greatly benefit the scientific community by providing tools to control cell culture renewal for developing stable midgut cell lines. The broader impacts of this project also include the identification of crucial healing pathways for the development of novel biocontrol methods and insecticides. A minority graduate student will be trained in state-of-the-art proteomic and cell culturing technologies and this training will provide a solid core of tools to pursue future research opportunities in biological and medical research fields. Both the principal investigator and the student will present the results of this project during community outreach events at local educational institutions serving underrepresented groups in order to foster an interest in participating in scientific research.