Department of Medicine, Division of Gastroenterology and Hepatology, Keck School of Medicine of USC, Los Angeles, California, United States of America.
USC Libraries Bioinformatics Services, University of Southern California, Los Angeles, California, United States of America.
PLoS One. 2020 Sep 14;15(9):e0238852. doi: 10.1371/journal.pone.0238852. eCollection 2020.
The pathogenesis of esophageal injury in gastroesophageal reflux disease (GERD) is incompletely understood. We modeled exposure of human esophageal myofibroblasts (HEMFs) to gastroesophageal reflux by repeated treatment with pH 4.5 and pH 4.5 bile salts and determined the effects on the epithelium in a 3D organotypic-like air-liquid interface model. Total, basal and supra-basal thickness of the epithelium were measured and immunostaining for p63, for basal (CK 14) and supra-basal (CK 4) squamous differentiation markers, and for cell proliferation (PCNA) were performed. Epithelial cell proliferation in response to HEMF conditioned media was also assessed in 2D culture. In the 3D organotypic model, total epithelial thickness increased similarly with pH 4.5 and pH 4.5 bile salt treated versus untreated and bile salt treated HEMF conditioned media. Epithelial p63 immunostaining was increased and multilayered. There was expansion of the CK14+ basal and CK4+ supra-basal layers in the epithelium established with conditioned media from pH 4.5 and pH 4.5 bile salt treated HEMFs versus untreated HEMF conditioned media. PCNA + cells per μm of tissue were unchanged in the basal layer across all treatment conditions while PCNA + cells per total DAPI + cells were decreased. In 2D culture, basal epithelial proliferation decreased with conditioned media from pH 4.5 and pH 4.5 bile salt treated HEMFs compared to conditioned media from untreated HEMF conditioned media. Secreted factors from HEMFs treated with acidic stimuli encountered in GERD increase epithelial thickness compared to secreted factors from untreated HEMFs and expand both basal and supra-basal layers. Our findings demonstrate for the first time paracrine regulation of the squamous epithelium from acid stimulated HEMFs. The effects of secreted factors from acid treated HEMFs on basal cell proliferation in this model and the mechanism mediating the increase in epithelial thickness merit further investigation.
胃食管反流病（GERD）中食管损伤的发病机制尚不完全清楚。我们通过重复用 pH4.5 和 pH4.5 胆汁盐处理来模拟人食管成纤维细胞（HEMFs）暴露于胃食管反流，并在 3D 器官样气液界面模型中确定其对上皮的影响。测量上皮的总厚度、基底层和超基底层厚度，并进行 p63、基底（CK14）和超基底层（CK4）鳞状分化标志物以及细胞增殖（PCNA）的免疫染色。还在 2D 培养中评估了对 HEMF 条件培养基中上皮细胞增殖的反应。在 3D 器官样模型中，与未处理和胆汁盐处理的 HEMF 条件培养基相比，pH4.5 和 pH4.5 胆汁盐处理的上皮总厚度增加相似。上皮 p63 免疫染色增加且呈多层状。与未处理的 HEMF 条件培养基相比，用 pH4.5 和 pH4.5 胆汁盐处理的 HEMF 产生的条件培养基建立的上皮中 CK14+基底和 CK4+超基底层扩张。在所有处理条件下，基底层中每个组织μm 的 PCNA+细胞保持不变，而总 DAPI+细胞中 PCNA+细胞减少。在 2D 培养中，与未处理的 HEMF 条件培养基相比，用 pH4.5 和 pH4.5 胆汁盐处理的 HEMF 产生的条件培养基中的基底上皮增殖减少。与未处理的 HEMF 相比，来自 GERD 中遇到的酸性刺激物处理的 HEMF 分泌的因子会增加上皮厚度，并扩大基底和超基底层。我们的研究结果首次证明了酸刺激的 HEMF 的旁分泌调节鳞状上皮。该模型中酸处理的 HEMF 分泌因子对基底细胞增殖的影响及其介导上皮厚度增加的机制值得进一步研究。
