Wirtschafter J D, Ketcham J M, Weinstock R J, Tabesh T, McLoon L K
Department of Ophthalmology, University of Minnesota, Minneapolis 55455, USA.
Invest Ophthalmol Vis Sci. 1999 Dec;40(13):3138-46.
The conjunctival epithelium performs an important role in the homeostasis and integrity of the eye. To protect the integrity of the ocular surface, these cells must be replaced from locally concentrated or randomly distributed foci of stem cells. These slow-cycling stem cells produce transient amplifying cells that undergo further divisions before becoming mature conjunctival epithelial cells. In the current study, the source of palpebral conjunctival cells was determined.
Adult rabbits were injected intraperitoneally with bromodeoxyuridine (BrdU) at a dose of 50 mg/kg body weight and killed after 1, 3, 5, and 7 days and 2 months. The orbital contents and eyelids were exenterated en bloc, frozen to maintain the orientation between the eyelids and globe, and sectioned in a parasagittal plane. Random midglobe sections were stained for the presence of proliferating cell nuclear antigen (PCNA). Additional sections were immunostained to detect BrdU-labeled conjunctival epithelial cells. BrdU-positive cells were counted in a series of 0.4-mm zones from the mucocutaneous junction of the eyelid, through the fornix and bulbar conjunctiva. A second set of rabbits received daily injections of BrdU for 2 or 4 weeks followed by a 2-month BrdU-free period before death and processing.
In all eyelid sections examined, there was a focus of PCNA-positive cells in the mucocutaneous junction and a few scattered PCNA-positive cells along the length of the palpebral conjunctiva toward the fornix. In both the upper and lower eyelids, the peak concentration of BrdU-labeled cells/0.4-mm zone was located at progressively greater distances from the mucocutaneous junction in the animals killed at 1, 3, and 5 days respectively and was unidentifiable by 7 days. A focus of BrdU-labeled conjunctival cells remained within 1 to 2 mm of the mucocutaneous junction at all postinjection intervals. These were always found within one cell height of the basement membrane in the basal layer of the epithelium. In the long-term studies, BrdU-labeled nuclei were retained at the mucocutaneous junction.
The mucocutaneous junction of the conjunctival epithelium is a source of actively dividing transient amplifying cells that migrate toward the fornix at a rate of approximately 1.7 mm/d with a transit time of approximately 6 days. Long-term retention of label at the mucocutaneous junction indicates that slow-cycling stem cells are present at this location. It appears that most palpebral conjunctival epithelial stem cells are located near the mucocutaneous junction. These results are not necessarily at variance with previous studies, but they diminish the relative importance of the forniceal region in palpebral conjunctival homeostasis. The mucocutaneous junction may provide a therapeutically significant source of replacement conjunctival cells.
结膜上皮在眼的稳态和完整性中发挥重要作用。为保护眼表的完整性，这些细胞必须从局部集中或随机分布的干细胞灶得到补充。这些慢周期干细胞产生短暂增殖细胞，后者在成为成熟结膜上皮细胞之前会进一步分裂。在本研究中，确定了睑结膜细胞的来源。
成年兔腹腔注射剂量为50mg/kg体重的溴脱氧尿苷（BrdU），分别在1、3、5、7天和2个月后处死。将眼眶内容物和眼睑整块摘除，冷冻以保持眼睑与眼球之间的方位，然后在矢状旁平面切片。随机取眼球中部切片，染色检测增殖细胞核抗原（PCNA）的存在。另取切片进行免疫染色，以检测BrdU标记的结膜上皮细胞。从眼睑的黏膜皮肤交界处开始，穿过穹窿和球结膜，在一系列0.4mm区域内计数BrdU阳性细胞。第二组兔每天注射BrdU，持续2或4周，然后在处死和处理前有2个月的无BrdU期。
在所有检查的眼睑切片中，黏膜皮肤交界处有一个PCNA阳性细胞灶，沿睑结膜向穹窿方向有一些散在的PCNA阳性细胞。在上、下眼睑中，分别在注射后1、3和5天处死的动物中，BrdU标记细胞/0.4mm区域的峰值浓度位于距黏膜皮肤交界处逐渐增大的距离处，到7天时无法识别。在所有注射后的时间间隔内，BrdU标记的结膜细胞灶都保留在黏膜皮肤交界处1至2mm范围内。这些细胞总是在上皮基底层基底膜的一个细胞高度内被发现。在长期研究中，BrdU标记的细胞核保留在黏膜皮肤交界处。
结膜上皮的黏膜皮肤交界处是活跃分裂的短暂增殖细胞的来源，这些细胞以约1.7mm/d的速度向穹窿迁移，转运时间约为6天。标记物在黏膜皮肤交界处的长期保留表明该位置存在慢周期干细胞。看来大多数睑结膜上皮干细胞位于黏膜皮肤交界处附近。这些结果不一定与先前的研究相矛盾，但它们降低了穹窿区域在睑结膜稳态中的相对重要性。黏膜皮肤交界处可能提供具有治疗意义的结膜替代细胞来源。
