인체각막윤부 상피세포 배양조건에서 retinoic acid가 각막상피분화와 점액발현에 미치는 조절효과

Dept. of Medicine/박사[한글] [영문]Damage to the ocular surface and corneal epithelial stem cells at the limbus from severe cicatricial ocular surface disorders such as chemical and thermal burns or Stevens-Johnson Syndrome has long posed a major challenge to ophthalmologists. The development of new surgical method using ex vivo cultivated epithelial sheets has brought in new era in ocular surface reconstruction. Maintenance of the healthy wet ocular surface is facilitated by the presence of mucins secreted on its surface as well as by the membrane-associated mucins in the apical cell glycocalyx. Given that the mucin layer contributes to epithelial barrier function, maintenance of the mucous phenotype may be a desirable property of transplantable epithelial cell sheets. However, regarding the various corneal epithelial cell culture methods, only a few studies have investigated mucin expression or other functional properties of epithelial cell sheets themselves. In this study, we sought to find an optimized serum-free condition for producing a fully differentiated corneal epithelial cell sheet with intact barrier function. Thus, we aimed to make our corneal epithelial cells maintain their mucous phenotypes, because membrane-associated mucins have critical roles in maintaining the healthy ocular surface by providing a barrier to penetration of pathogens. Retinoic acid (RA), a biological derivative of vitamin A, has been known to be essential for epithelial differentiation and for maintaining mucous phenotype. Thus, RA has been used as an essential element in serum-free culture media. It showed modulating effects on cellular proliferation and differentiation in various epithelial tissues. Thus, we investigated whether RA has a modulating effect on the differentiation of corneal epithelial cells. Furthermore, we investigated the effect of RA on the expression of membrane-associated mucins in the cultured corneal epithelial cells. Human corneal limbal epithelial (HCLE) cells were dissociated from donor eyes and grown in growth factor supplemented serum-free media. HCLE cells were cultured in RA-deficient or RA-supplemented media with various concentrations (10-9 to 10-6 M). Passage-3 cells were differentiated using the air-liquid interface technique after cells reached confluence in Transwell culture insert. As a morphologic characterization, overall thicknesses of cell layers and keratin layers were examined by hematoxylin/eosin staining and apical surface was examined by scanning electron microscopy (SEM). Stratified epithelial cells were then examined by immunohistochemistry using differentiation related markers such as p63, cytokeratin 3 (CK3), and MUC16. To examine the epithelial barrier function, HCLE cells were incubated with rose bengal dye. Furthermore, the effect of RA on the expression of three membrane-associated mucins (MUC1, -4, -16) was analyzed by quantitative real-time polymerase chain reaction and western blot analysis in a concentration and time dependent manner. Histologic and SEM findings suggested that cultured HCLE cells showed features of multi-layered squamous epithelium with numerous microvilli at the apical surface. RA-deficient culture resulted in overproduction of cornified keratin layer, whereas suitable concentrations of RA (10-8 to 10-7 M) induced normal appearing non-keratinized squamous epithelium. Under these conditions, p63, CK3, and MUC16 staining patterns were similar to those of limbal corneal epithelium. Higher concentration (10-6 M) of RA, however, resulted in fewer-layered epithelial cells with abnormal differentiation patterns. In barrier function test, rose bengal dye was excluded in the stratified epithelial cells grown in RA-supplemented media, whereas, it was easily penetrated in those grown in RA-deficient media. This result implied that cultured epithelial cells expressed functional MUC16. Furthermore, cultured HCLE cells expressed 3 membrane-associated mucins (MUC1, -4, -16) and their expressions were highly associated with RA concentrations and exposure times. Taken together, our results suggest that RA concentrations in the range of 10-8 M seem to be the most appropriate for obtaining functionally differentiated corneal epithelial cells with retaining similar phenotypes to those of limbal corneal epithelium. In conclusion, this study reported that human corneal epithelial cell was successfully cultured in serum-free, RA-supplemented media. Cultured HCLE cells retained physiologic features of fully differentiated corneal epithelium with barrier function. Our data suggested that such epithelial cells could be an ideal in vitro model for epithelial functional study. Furthermore, they may be suitable for epithelial cell sheet transplantation in ocular surface reconstruction, since they showed similar phenotypes to limbal epithelium. This study also demonstrated that RA was a key modulator in corneal epithelial differentiation and regulates mucin expression in a dose-dependent manner in cultured corneal epithelial cells.ope