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Abstract：
By employing an innovative biohybrid membrane, the present study aimed at elucidating the mechanistic role of the focal adhesion kinase (FAK) in epithelial morphogenesis in vitro over 4, 7, and 10 days. The consequences of siRNA-mediated FAK knockdown on epithelial morphogenesis were monitored by quantifying cell layers and detecting the expression of biomarkers of epithelial differentiation and homeostasis. Histologic examination of FAK-depleted samples showed a significant increase in cell layers resembling epithelial hyperplasia. Semiquantitative fluorescence imaging (SQFI) revealed tissue homeostatic disturbances by significantly increased involucrin expression over time, persistence of yes-associated protein (YAP) and an increase of keratin (K) 1 at day 4. The dysbalanced involucrin pattern was underscored by ROCK-IISer1366 activity at day 7 and 10. SQFI data were confirmed by quantitative PCR and Western blot analysis, thereby corroborating the FAK shutdown-related expression changes. The artificial FAK shutdown was also associated with a significantly higher expression of filaggrin at day 10, sustained keratinocyte proliferation, and the dysregulated expression of K19 and vimentin. These siRNA-induced consequences indicate the mechanistic role of FAK in epithelial morphogenesis by simultaneously considering prospective biomaterial-based epithelial regenerative approaches.
摘要：
通过采用创新的生物杂交膜，本研究旨在阐明粘着斑激酶（FAK）在体外4、7和10天的上皮形态发生中的机制作用。通过定量细胞层并检测上皮分化和稳态的生物标志物的表达来监测siRNA介导的FAK敲低对上皮形态发生的影响。FAK耗尽样品的组织学检查显示类似上皮增生的细胞层显着增加。半定量荧光成像（SQFI）显示组织体内平衡紊乱显著增加的表达随着时间的推移，Yes相关蛋白（YAP）的持续存在和第4天角蛋白（K）1的增加。在第7天和第10天，ROCK-IISer1366的活性强调了失衡的总合蛋白模式。SQFI数据通过定量PCR和Westernblot分析证实，从而证实FAK关闭相关表达式的变化。在第10天，人工FAK关闭还与聚丝团蛋白的表达显着升高有关，持续的角质形成细胞增殖，以及K19和波形蛋白的表达失调。通过同时考虑基于生物材料的预期上皮再生方法，这些siRNA诱导的结果表明FAK在上皮形态发生中的机制作用。
