Taheri Negar, Choi Egan L, Zhang Yuebo, Hayashi Yujiro
Department of Physiology and Biomedical Engineering, 200 First Street SW, Rochester, MN 55905, USA.
J Smooth Muscle Res. 2024;60:64-71. doi: 10.1540/jsmr.60.64.
Understanding the molecular interactions within the neuromuscular apparatus in the stomach is crucial for understanding their role in maintaining interstitial cells, such as the interstitial cells of Cajal (ICC), smooth muscle, and enteric neurons, as well as their contribution to gastric motility in both healthy and diseased states. Disruptions of these systems can lead to various gastric motor disorders and diseases, making it essential to explore their functions in detail. We herein present a protocol for gene knockdown using small interfering RNA (siRNAs) in organotypic culture. This ex vivo approach allows the precise manipulation of the gene expression in a tissue environment that closely mimics in vivo conditions, providing valuable insights into the gene function and its effects on gastric physiology. The protocol includes detailed steps for tissue preparation to ensure the preservation of the gastric muscles and the associated neuromuscular apparatus. We then describe the process of siRNA-mediated gene knockdown, offering tips for optimizing transfection efficiency and gene silencing. Additionally, we outline methods for analyzing the effectiveness of knockdown, including both quantitative and qualitative methods for the evaluation of the target gene expression. This protocol is adaptable to various research needs, allowing researchers to focus on specific genes of interest within the neuromuscular system of the stomach. By applying this approach, investigators can deepen their understanding of the molecular mechanisms underlying gastric motility and contribute to the development of new therapeutic strategies for treating gastric motor disorders and diseases.
了解胃神经肌肉装置内的分子相互作用对于理解它们在维持间质细胞（如 Cajal 间质细胞（ICC）、平滑肌和肠神经元）中的作用，以及它们在健康和疾病状态下对胃动力的贡献至关重要。这些系统的破坏可导致各种胃运动障碍和疾病，因此详细探索它们的功能至关重要。我们在此介绍一种在器官型培养中使用小干扰 RNA（siRNA）进行基因敲低的方案。这种体外方法允许在紧密模拟体内条件的组织环境中精确操纵基因表达，为基因功能及其对胃生理学的影响提供有价值的见解。该方案包括组织制备的详细步骤，以确保胃肌肉和相关神经肌肉装置的保存。然后我们描述 siRNA 介导的基因敲低过程，提供优化转染效率和基因沉默的提示。此外，我们概述了分析敲低有效性的方法，包括评估靶基因表达的定量和定性方法。该方案可适应各种研究需求，使研究人员能够专注于胃神经肌肉系统内感兴趣的特定基因。通过应用这种方法，研究人员可以加深对胃动力潜在分子机制的理解，并为开发治疗胃运动障碍和疾病的新治疗策略做出贡献。
