Sun Chi-Chin, Cheng Ching-Yi, Chien Chin-Sung, Pang Jong-Hwei Su, Ku Wan-Chen, Chen Phil Yeong-Fong, Yang Chuen-Mao
Department of Ophthalmology, Chang Gung Memorial Hospital, Keelung, Taiwan.
Invest Ophthalmol Vis Sci. 2005 Mar;46(3):808-15. doi: 10.1167/iovs.04-0370.
To investigate the expression and pivotal role of matrix metalloproteinase (MMP)-9 in the ex vivo expansion of human limbal explants with or without amniotic membrane (AM).
Corneoscleral buttons were cultured on intact, denuded AM or plastic dishes for 3 weeks. To determine the role of MMP-9 in cell migration, either the MMP inhibitor GM6001 or an MMP-9 antibody was used. Expression of MMP-9 was determined by gelatin zymography, reverse transcription-polymerase chain reaction, and immunohistochemical staining.
The expression of MMP-9 in all culture conditions increased in a time-dependent manner. However, the active form of MMP-9 emerged only in cultures on both intact and denuded AM from the second week. The averaged corrected ratio of MMP-9 expression in cultures on intact AM versus those on denuded AM or plastic dishes was 2.76 +/- 0.69- or 4.25 +/- 0.30-fold, respectively, when total RNA was used as an internal control. MMP-9 transcripts were upregulated in cultures on intact AM compared with the other two culture conditions. Immunohistochemical staining demonstrated that the MMP-9 protein was located on the limbal epithelial cells. Upregulation of MMP-9 associated with cell migration was significantly attenuated by both GM6001 and MMP-9 antibody, consistent with the inhibition of MMP-9 activity, as determined by gelatin zymography. In contrast, the sizes of limbal outgrowth were not different between the control and MMP-9 antibody-treated plastic dishes.
These results demonstrated that MMP-9 not only was upregulated, it was also involved in the outgrowth of limbal epithelial cells. These results suggest that cell-cell matrix interaction is involved in the expansion of limbal epithelial cells on intact AM, and MMP-9 may be a key element.
研究基质金属蛋白酶（MMP）-9在有无羊膜（AM）情况下人角膜缘外植体体外扩增中的表达及关键作用。
将角膜缘植片在完整、去上皮的羊膜或塑料培养皿上培养3周。为确定MMP-9在细胞迁移中的作用，使用了MMP抑制剂GM6001或MMP-9抗体。通过明胶酶谱法、逆转录-聚合酶链反应和免疫组织化学染色来测定MMP-9的表达。
在所有培养条件下，MMP-9的表达均呈时间依赖性增加。然而，仅从第二周起，MMP-9的活性形式才出现在完整和去上皮羊膜上的培养物中。当以总RNA作为内参时，完整羊膜上培养物中MMP-9表达的平均校正比值分别是去上皮羊膜或塑料培养皿上培养物的2.76±0.69倍或4.25±0.30倍。与其他两种培养条件相比，完整羊膜上培养物中的MMP-9转录本上调。免疫组织化学染色显示MMP-9蛋白位于角膜缘上皮细胞上。GM6001和MMP-9抗体均显著减弱了与细胞迁移相关的MMP-9上调，这与明胶酶谱法测定的MMP-9活性受到抑制一致。相反，对照和MMP-9抗体处理的塑料培养皿上角膜缘细胞生长的大小没有差异。
这些结果表明，MMP-9不仅被上调，还参与角膜缘上皮细胞的生长。这些结果提示，细胞-细胞基质相互作用参与了完整羊膜上角膜缘上皮细胞的扩增，而MMP-9可能是关键因素。
