高內涵蛋白質晶片篩選技術: 結合大腸桿菌蛋白質晶片和高內涵篩選技術來找出能影響神經型態發生的細菌蛋白質

Abstract

越來越多的證據表明，腸道菌群可影響中樞神經系統的發展和發病機制。因此，在此研究中，我們試圖檢查每一大腸桿菌蛋白質對神經元形態的影響。為了從所有大腸桿菌蛋白質中，找出可以改變神經元形態的蛋白質，我們結合了蛋白質晶片技術（其最初被開發用於檢測蛋白質與蛋白質之間的相互作用）和高內涵篩選技術（最初被用於細胞形態變化之篩選）。為了驗證這種方法的可行性，我們首先建立了可以使小鼠皮層神經元生長在晶片表面上的培養方法。第二，我們證實了使用已知可抑制神經突生長的蛋白質點在晶片表面上可保留了其活性。第三，我們優化了免疫螢光染色的方法，提高了晶片上蛋白質點的偵測。最後，我們使用這種新穎的平台來篩選整個大腸桿菌蛋白質體，並找到一些可以降低神經突長度或神經貼附的蛋白質。

Increasing evidence suggest that intestinal microbiota can influence the development and pathogenesis of the central nervous system. Therefore, in this study we attempted to examine the effect of each and every protein in E. coli proteome on neuronal morphogenesis. To identify all E. coli proteins that can alter neuronal morphology, we combined the protein microarray technique (which was originally developed to detect protein-protein interactions) with high-content screening (which was originally used to analyze cell morphology changes upon perturbation). To verify the feasibility of this method, we first established the culturing method to maintain mouse cortical neurons on the microarray surface. Second, we verified that protein spotted on microarray surface retained its activity using a known neurite outgrowth inhibitor. Third, we optimized the immunofluorescence staining protocol to maximize protein spot detect on this microarray. Finally, we used this novel platform to screen through the entire E. coli proteome and identify some proteins which can reduce neurite length or neuronal attachment.