Epstein Seth P, Wolosin J Mario, Asbell Penny A
Department of Ophthalmology and Black Family Stem Cell Institute, Mount Sinai School of Medicine, New York, New York, USA.
Trans Am Ophthalmol Soc. 2005;103:187-99; discussion 199.
Because stem cells exhibit high self-renewal capacity, slow cycling, and high proliferative potential, and one of many markers postulated for epithelial stem cells, p63, is challenged by widespread expression within stem cell-free regions, we examined p63 expression in these stem cell-associated cohorts compared with their controls.
Rabbit limbocorneal cryosections, cytospun cell-sorted (by fluorescence-activated cell sorter) side population (SP) and low side scatter (LSSC) cells, and limbal epithelial cells over feeders were stained for p63 by indirect immunofluorescence. Clones were fixed and stained daily for 7 days. Image analysis measured p63 intensity, plotting it against colony size.
All basal limbal cells were positive for p63, yet only 5% to 7% expressed high p63 intensities, 40% intermediate, and the majority low. Side population cells were less than 1% of total cells. The average intensity of SP staining was three times that of controls. Subpopulations displaying stemlike features exhibited highest p63 expression. Replication rates of isolated cells differed. Day 5 colonies contained 256 (16 hours/cycle) to two (96 hours/cycle) cells. Whereas all cells were positive for p63, intensity in slow-cycling cells was three to four times that in rapidly proliferating congeners. Increased cell doublings did not decrease fluorescence.
Results suggest that p63 concentration is maximal in stem cells and decreases with differentiation. High p63 levels seem to correlate with cells of the SP and LSSC phenotypes, indicating high cell stemness. With identification of stem cells, further studies can elucidate their use in supporting ocular surface health.
由于干细胞具有高自我更新能力、缓慢的细胞周期进程和高增殖潜力，并且上皮干细胞的众多假定标志物之一p63在无干细胞区域广泛表达，这对其作为上皮干细胞标志物提出了挑战，因此我们将这些与干细胞相关的队列中的p63表达与其对照进行了比较。
采用间接免疫荧光法对兔角膜缘冰冻切片、经细胞分选（通过荧光激活细胞分选仪）的侧群（SP）细胞和低侧向散射（LSSC）细胞以及饲养层上的角膜缘上皮细胞进行p63染色。克隆固定后每天染色，持续7天。图像分析测量p63强度，并将其与集落大小作图。
所有角膜缘基底细胞p63均呈阳性，但只有5%至7%的细胞表达高强度的p63，40%表达中等强度，大多数表达低强度。侧群细胞占总细胞的比例不到1%。SP染色的平均强度是对照的三倍。表现出干细胞样特征的亚群p63表达最高。分离细胞的复制速率不同。第5天集落包含256个（16小时/周期）至2个（96小时/周期）细胞。虽然所有细胞p63均呈阳性，但慢周期细胞中的强度是快速增殖同类细胞的三到四倍。细胞倍增增加并未降低荧光强度。
结果表明，p63浓度在干细胞中最高，并随着分化而降低。高p63水平似乎与SP和LSSC表型的细胞相关，表明细胞具有高干性。随着干细胞的鉴定，进一步的研究可以阐明它们在维持眼表健康中的作用。
