Saudi Cultural Missions Theses & Dissertations
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Item Restricted Investigation of PDI Involvement in Leukocyte Adhesion to Endothelial Cell in Inflammatory State Using Gene Editing (CRISPR/Cas9) Technology(Arabin Gulf university, 2025) almashhur, wedad awadh ali; Fathallah, Mohamed DahmaniLeucocyte adhesion to endothelial cells is a critical step in the inflammatory process, influenced by various molecular mediators, including Protein Disulfide Isomerase 1 (PDI1). This study aimed to investigate the broader roles of additional PDI family members in regulating endothelial cell activation and leucocyte adhesion under inflammatory conditions. We isolated endothelial cells with a cobblestone morphology from human peripheral blood and umbilical cords, achieving yields of 1.2 × 10⁵ and 3.5 × 10⁵ cells/mL, respectively. Neutrophils were isolated from blood samples using magnetic bead technology, and their phenotype was confirmed via flow cytometry. To induce inflammation, endothelial cells were stimulated with TNF-α (100 ng/mL). Quantitative real-time PCR was utilized to assess the expression of PDI genes and adhesion molecules, specifically ICAM-1, VCAM-1, and E-selectin, which serve as established markers of endothelial activation. CRISPR/Cas9 technology facilitated the knockout of PDIA2, PDIA3, PDIA4, and PDIA6 in endothelial cells, with validation achieved through sequencing and Western blot analysis. Efficient gene editing was confirmed by the significant reduction in expression levels of the targeted PDIs. The results demonstrated a 62.5% decrease in the expression of ICAM-1 and VCAM-1, while E-selectin expression was reduced by an average of 55% across all knockouts. Functional assays revealed significant reductions in neutrophil adhesion (p < 0.01) to knockout endothelial cells, with specific decreases of 25% in PDIA2, 20% in PDIA3, 35% in PDIA4, and 2% in PDIA6 knockout cells. These findings underscore the essential roles of various PDIs in the activation of endothelial cells and leucocyte adhesion during inflammation. The study illustrates the effectiveness of CRISPR/Cas9 technology in elucidating gene functions and highlights PDIs as potential therapeutic targets for inflammatory vascular diseases. This research provides valuable insights into the molecular mechanisms underlying inflammation and opens avenues for novel therapeutic strategies.2 0