Tei M, Moccia R, Gipson I K
Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, MA 02114, USA.
Invest Ophthalmol Vis Sci. 1999 Aug;40(9):1944-51.
To determine site and time of initiation of expression of the membrane-spanning mucin ASGP (rMuc4) and the goblet cell-specific, gel-forming mucin rMuc5AC by the developing rat ocular surface epithelium.
Newborn Sprague-Dawley rat pups were killed at 1, 7, and 14 days after birth. Adult rats (weight, 200 g) were used as controls. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect ASGP mRNA using beta-actin as an internal control. Competitive RT-PCR was performed to quantitate rMuc5AC mRNA using an rMuc5AC-competitive reference standard (CRS) as an internal control. In situ hybridization was performed to localize ASGP and rMuc5AC mRNA. Goblet cells were detected by staining with periodic acid-Schiff (PAS) reagent.
ASGP mRNA was detected by RT-PCR at 1 day after birth. Compared with beta-actin, the amount of ASGP mRNA showed a progressive increase from 1 to 14 days of postnatal development. By in situ hybridization, the expression of ASGP was first clearly detected at 14 days after birth at the lid margin, where the most stratification of epithelium was seen, and along the adjacent palpebral conjunctiva. This pattern was seen in rat eyelids that were not yet open but appeared about to open. In rat eyelids already open at 14 days after birth, ASGP mRNA was diffusely spread in the apical cell layer of both conjunctival and corneal epithelia. The expression of rMuc5AC was detected by RT-PCR in ocular surface epithelium in rat pups 1 day after birth. Quantitative RT-PCR showed a low level of rMuc5AC RNA expression in conjunctiva of 1-, 7-, and 14-day-old rats followed by a large increase in expression between 14 days and adulthood. The expression of rMuc5AC was first detected by in situ hybridization in a few goblet cells at 7 days after birth. One or two labeled cells were present in the fornical area; some were on the palpebral side of the fornix; others were present on the bulbar side. The distribution and time of appearance of rMuc5AC correlated with that of PAS staining of goblet cells.
The developmental expression of the membrane-spanning mucin ASGP (rMuc4) and the gel-forming mucin rMuc5AC are regionally and temporally separated. Expression of the gel-forming mucin begins at the fornix at 7 days after birth and is correlated with the appearance of goblet cells, whereas, expression of the membrane-spanning mucin begins later at the lid margin at day 14. Expression of the membrane-spanning mucin correlates to eyelid opening.
确定发育中的大鼠眼表上皮细胞中跨膜黏蛋白ASGP（rMuc4）和杯状细胞特异性凝胶形成黏蛋白rMuc5AC的表达起始部位和时间。
新生的Sprague-Dawley大鼠幼崽在出生后1天、7天和14天处死。成年大鼠（体重200 g）用作对照。以β-肌动蛋白为内对照，通过逆转录聚合酶链反应（RT-PCR）检测ASGP mRNA。以rMuc5AC竞争性参考标准品（CRS）为内对照，通过竞争性RT-PCR定量rMuc5AC mRNA。进行原位杂交以定位ASGP和rMuc5AC mRNA。通过过碘酸-希夫（PAS）试剂染色检测杯状细胞。
出生后1天通过RT-PCR检测到ASGP mRNA。与β-肌动蛋白相比，ASGP mRNA的量在出生后1至14天呈逐渐增加。通过原位杂交，在出生后14天首次在睑缘清楚地检测到ASGP的表达，睑缘上皮分层最明显，且在相邻的睑结膜也有表达。这种模式在尚未睁开但似乎即将睁开的大鼠眼睑中可见。在出生后14天已经睁开的大鼠眼睑中，ASGP mRNA在结膜和角膜上皮的顶端细胞层中呈弥漫性分布。出生后1天在大鼠幼崽的眼表上皮中通过RT-PCR检测到rMuc5AC的表达。定量RT-PCR显示，1日龄、7日龄和14日龄大鼠结膜中rMuc5AC RNA表达水平较低，随后在14天至成年期表达大幅增加。出生后7天通过原位杂交首次在少数杯状细胞中检测到rMuc5AC的表达。穹窿部有一两个标记细胞；一些在穹窿部的睑侧；另一些在球侧。rMuc5AC的分布和出现时间与杯状细胞的PAS染色情况相关。
跨膜黏蛋白ASGP（rMuc4）和凝胶形成黏蛋白rMuc5AC的发育性表达在区域和时间上是分开的。凝胶形成黏蛋白的表达在出生后7天始于穹窿部，与杯状细胞的出现相关，而跨膜黏蛋白的表达在出生后第14天较晚始于睑缘。跨膜黏蛋白的表达与眼睑睁开相关。
