This study aims to develop a novel serum-free culture strategy containing only two small molecules, Y27632 and SB431542 (2C), for expansion of mouse lacrimal gland epithelial cells (LGECs) and investigate an innovative therapeutic approach for lacrimal gland (LG) injury. LGECs proliferative capacity was assessed by cell counting, crystal violet staining, qRT-PCR and immunofluorescence. Cell differentiation was achieved by manipulating culture conditions and assessed by qRT-PCR and AQP5 immunofluorescence. LGECs were seeded in Matrigel for three-dimensional culture and assessed by qRT-PCR and immunofluorescence. Secretory function of the cultures was assayed by ELISA. , 2C injection verified its reparative capacity in a mouse LG injury model. Corneal fluorescein staining, phenol red cotton thread, H&E, immunofluorescence and Western blot were used to assess LG injury repair. LGECs cultured with 2C exhibited high expression of stemness/proliferation markers and maintained morphology and proliferative capacity even after the tenth passage. Removal of 2C was efficacious in achieving LGECs differentiation, characterized by the increased AQP5 expression and LTF secretion. 3D spheroids cultured with 2C demonstrated differentiation potential, forming microglandular structures containing multiple LG cell types with secretory functions after 2C removal. , 2C improved the structural integrity and function of the injured LG. We present a small molecule combination, 2C, that promotes LGECs expansion and differentiation and accelerates LG injury repair . This approach has potential applications for providing a stable source of seed cells for tissue engineering applications, providing new sights for LG-related diseases treatment.

中文翻译：

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独特的小分子混合物：体外促进泪腺上皮细胞增殖，体内加速泪腺损伤修复


本研究旨在开发一种仅包含两种小分子 Y27632 和 SB431542 (2C) 的新型无血清培养策略，用于扩增小鼠泪腺上皮细胞 (LGEC)，并研究泪腺 (LG) 损伤的创新治疗方法。通过细胞计数、结晶紫染色、qRT-PCR 和免疫荧光评估 LGEC 的增殖能力。通过控制培养条件实现细胞分化，并通过 qRT-PCR 和 AQP5 免疫荧光进行评估。 LGECs 接种在 Matrigel 中进行三维培养，并通过 qRT-PCR 和免疫荧光进行评估。通过ELISA测定培养物的分泌功能。 ，2C注射在小鼠LG损伤模型中验证了其修复能力。采用角膜荧光素染色、酚红棉线、H&E、免疫荧光和Western blot来评估LG损伤修复情况。用 2C 培养的 LGEC 表现出干性/增殖标记的高表达，甚至在第十代后仍保持形态和增殖能力。去除 2C 可有效实现 LGEC 分化，其特征是 AQP5 表达和 LTF 分泌增加。用 2C 培养的 3D 球体表现出分化潜力，在去除 2C 后形成含有多种具有分泌功能的 LG 细胞类型的微腺体结构。 ，2C 改善了受伤 LG 的结构完整性和功能。我们提出了一种小分子组合 2C，它可以促进 LGEC 的扩增和分化，并加速 LG 损伤的修复。该方法具有潜在的应用前景，可为组织工程应用提供稳定的种子细胞来源，为 LG 相关疾病的治疗提供新的前景。