目的:本研究旨在开发一种仅包含两个小分子的新型无血清培养策略,Y27632和SB431542(2C),用于小鼠泪腺上皮细胞(LGECs)的体外扩增,并研究了泪腺(LG)损伤的创新治疗方法。
方法:通过细胞计数评估LGECs的增殖能力,结晶紫染色,qRT-PCR和免疫荧光。通过操纵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。将LGEC接种在基质胶中进行三维培养,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。在体内,2C注射验证了其在小鼠LG损伤模型中的修复能力。角膜荧光素染色,酚红棉线,H&E,免疫荧光和Westernblot用于评估LG损伤修复。
结果:用2C培养的LGECs即使在第十次传代后仍表现出高表达的干性/增殖标志物,并保持形态和增殖能力。去除2C对实现LGECs分化是有效的,以AQP5表达和LTF分泌增加为特征。用2C培养的3D球状体显示出分化潜力,2C去除后,形成含有多种LG细胞类型的微腺体结构,具有分泌功能。在体内,2C改善了受损LG的结构完整性和功能。
结论:我们提出了一种小分子组合,2C,在体外促进LGEC的扩增和分化,并在体内加速LG损伤修复。这种方法在为组织工程应用提供稳定的种子细胞来源方面具有潜在的应用。为LG相关疾病的治疗提供了新的视野。
OBJECTIVE: This study aims to develop a novel serum-free culture strategy containing only two small molecules, Y27632 and SB431542 (2C), for in vitro expansion of mouse lacrimal gland epithelial cells (LGECs) and investigate an innovative therapeutic approach for lacrimal gland (LG) injury.
METHODS: 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. In vivo, 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.
RESULTS: 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. In vivo, 2C improved the structural integrity and function of the injured LG.
CONCLUSIONS: We present a small molecule combination, 2C, that promotes LGECs expansion and differentiation in vitro and accelerates LG injury repair in vivo. 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.