Kim Mee Kum, Lee Jae Lim, Oh Joo Youn, Shin Mi Sun, Shin Kyeong Seon, Wee Won Ryang, Lee Jin Hak, Park Ki Sook, Son Young Sook
Department of Ophthalmology, Seoul National University College of Medicine, and Seoul ARtificial Eye Center, Seoul National University Hospital Clinical Research Institute, Seoul, Korea.
J Korean Med Sci. 2008 Oct;23(5):864-9. doi: 10.3346/jkms.2008.23.5.864.
To compare the stem niche in different culture conditions of limbal epithelial cells, the suspended human limbal epithelial cells (HLECs) were seeded on the 3T3-pretreated plates and the other suspended cells were plated on amniotic membranes (AMs) which were either cryo-preserved or freeze-dried. All were cultured for 10 to 12 days. Reverse transcription-polymerase chain reaction (RT-PCR) for ATP-binding cassette, subfamily G, member 2 (ABCG2), p63, cytokeratin 12, and connexin 43 were performed in cultivated HLECs and their expression levels were compared. The mRNA expression of all markers examined showed no statistically significant differences between the cells on cryo-preserved and on freeze-dried AM. The expression of p63 and cytokeratin 12 in cultivated cells on AMs were significantly lower than those in 3T3-cocultured cells on RT-PCR and immunofluorescent staining. Cultivated HLECs on AMs showed reduced proliferation and differentiation while maintaining stem-property regardless of the preservative method of AM.
为了比较不同培养条件下角膜缘上皮细胞的干细胞龛，将悬浮的人角膜缘上皮细胞（HLECs）接种在经3T3预处理的培养板上，将其他悬浮细胞接种在冷冻保存或冻干的羊膜（AMs）上。所有细胞均培养10至12天。对培养的HLECs进行ATP结合盒转运体G亚家族成员2（ABCG2）、p63、细胞角蛋白12和连接蛋白43的逆转录-聚合酶链反应（RT-PCR），并比较它们的表达水平。所有检测标志物的mRNA表达在冷冻保存的羊膜和冻干羊膜上的细胞之间无统计学显著差异。在RT-PCR和免疫荧光染色中，羊膜上培养细胞中p63和细胞角蛋白12的表达明显低于3T3共培养细胞中的表达。无论羊膜的保存方法如何，羊膜上培养的HLECs在维持干细胞特性的同时，增殖和分化能力降低。
