CAJ | 학술논문

目的：观察小鼠角膜上皮祖细胞系TKE2在扩增以及分化状态下的角蛋白及干细胞标志物的表达情况。方法小鼠角膜上皮祖细胞系TKE2在无血清培养基Keratinocyte-SFM (KSFM)以及含10﹪胎牛血清（FBS）的DMEM培养基中培养，约70﹪融合时进行角蛋白10、12、14、15、16（K10、K12、K14、K15、K16）以及Connexin43、ABCG2的免疫荧光染色，以及Ki67、P63、PCNA的免疫细胞化学染色。结果无血清培养状态下的TKE2细胞呈克隆样生长，克隆内所有细胞呈ABCG2、K14、Ki67、PCNA以及P63阳性，K15阳性细胞散在分布，K16阳性细胞呈片状分布于克隆中央区，K10、K12以及Connexin43染色为阴性。在含有10﹪胎牛血清的DMEM中培养2 d后，细胞明显增大， ABCG2、K15、P63、Ki67以及PCNA转为阴性，克隆内只有少量细胞呈K16、K14阳性染色， K10、K12、Connexin43仍为阴性。结论 TKE2细胞具有角膜上皮干细胞特性，可以作为角膜缘上皮干细胞表型维持和分化诱导研究的良好工具。
목적：관찰소서각막상피조세포계TKE2재확증이급분화상태하적각단백급간세포표지물적표체정황。방법소서각막상피조세포계TKE2재무혈청배양기Keratinocyte-SFM (KSFM)이급함10﹪태우혈청（FBS）적DMEM배양기중배양，약70﹪융합시진행각단백10、12、14、15、16（K10、K12、K14、K15、K16）이급Connexin43、ABCG2적면역형광염색，이급Ki67、P63、PCNA적면역세포화학염색。결과무혈청배양상태하적TKE2세포정극륭양생장，극륭내소유세포정ABCG2、K14、Ki67、PCNA이급P63양성，K15양성세포산재분포，K16양성세포정편상분포우극륭중앙구，K10、K12이급Connexin43염색위음성。재함유10﹪태우혈청적DMEM중배양2 d후，세포명현증대， ABCG2、K15、P63、Ki67이급PCNA전위음성，극륭내지유소량세포정K16、K14양성염색， K10、K12、Connexin43잉위음성。결론 TKE2세포구유각막상피간세포특성，가이작위각막연상피간세포표형유지화분화유도연구적량호공구。
Objective To investigate the expression of stem cell markers and cytokeratins in murine corneal epithelial progenitor cell line TKE2 under proliferation and differentiation culture condition. Methods TKE2 cells were cultured in Keratinocyte-SFM (KSFM) and Dulbecco’s modified eagle medium (DMEM) with 10﹪fetal bovine serum (FBS). The cultures were terminated after TKE2 reached to 70﹪confluence. The expression patterns of stem cell markers (ABCG2, Ki67, P63, PCNA), proliferation related cytokeratins (K14, K15, K16), corneal terminal differentiation markers (K12, Connexin43) and epidermal epithelial specific marker (K10) on the TKE2 cells cultured under two conditions were evaluated by immunofluorescence and immunochemistry. Results In the KSFM medium, TKE2 cells could form uniform clones at the low seeding density. The expression of ABCG2, Ki67, PCNA, P63 and K14 were present in all cells of TKE2 clones. K15 were discretely expressed by TKE2 cells, while only sporadic cells in the central area of the clones expressed K16. After cultivation for 2 days in DMEM medium with 10﹪FBS, the cells became flat and enlarged. The expression of ABCG2, K14, K15, P63, Ki67 and PCNA were completely lost. K14 and K16 were weakly expressed in a small cluster of cells inside the TKE2 clones. K10, K12 and Connexin43 were negative in two culture conditions. Conclusions TKE2 processed the major characteristic of murine limbal stem cells. TKE2 cells may act as the potential cell model to study the mechanism of stemness maintenance as well as stem cell differentiation in limbal epithelium.