Li Wei, He Hua, Kuo Ching-Liang, Gao Yingying, Kawakita Tetsuya, Tseng Scheffer C G
Ocular Surface Center and TissueTech, Inc., Miami, Florida 33173, USA.
Invest Ophthalmol Vis Sci. 2006 Jun;47(6):2381-9. doi: 10.1167/iovs.05-1491.
To investigate basement membrane (BM) formation during ex vivo expansion of limbal corneal epithelial cells on intact amniotic membrane (iAM) and epithelially denuded (d)AM.
Human limbal explants were cultured on iAM and dAM. Expression of BM components, including laminin-5, type IV collagen, type VII collagen, perlecan, integrin alpha6, and epithelial cell differentiation markers such as p63, cytokeratin 3 (K3), and cytokeratin 12 (K12), were investigated by immunostaining. Levels of matrix metalloproteinase (MMP)-2 and MMP-9 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 in the conditioned media were determined by ELISA and gelatin zymography.
All four BM components were preserved in both iAM and dAM before culturing, but dissolved 1 week afterward when MMP-2 was increased. Epithelial outgrowth correlated with increased expression of MMP-2 and -9 for both cultures. Resynthesis of BM began with laminin-5 followed by other components. This process took place at 1 week on iAM but at 2 weeks on dAM after culturing. At 4 weeks, BM was more maturely deposited as a linear band from the explant toward the leading edge on iAM and temporally correlated with a sharp decline of MMP-9 levels. In contrast, such BM deposition began at the leading edge on dAM only when TIMP-1 levels were increased. Epithelial cell outgrowth on iAM expressed more p63 but less K3 and K12 than did that on dAM.
After dissolution of original amniotic BM, new BM formed by ex vivo expanded human limbal corneal epithelial cells on iAM deposits much faster and is more mature, resulting in regeneration of a limbal epithelial phenotype. In contrast, BM deposition is delayed and remains immature on dAM, resembling wound healing by a corneal epithelial phenotype. Thus, BM resynthesis may be used as another objective readout for assessing the success of ex vivo expansion of limbal epithelial progenitor cells on AM.
研究角膜缘上皮细胞在完整羊膜（iAM）和上皮剥脱羊膜（dAM）上进行体外扩增时基底膜（BM）的形成情况。
将人角膜缘外植体培养于iAM和dAM上。通过免疫染色研究BM成分（包括层粘连蛋白-5、IV型胶原、VII型胶原、基底膜聚糖、整合素α6）以及上皮细胞分化标志物（如p63、细胞角蛋白3（K3）和细胞角蛋白12（K12））的表达。通过酶联免疫吸附测定法（ELISA）和明胶酶谱法测定条件培养基中基质金属蛋白酶（MMP）-2和MMP-9以及基质金属蛋白酶组织抑制剂（TIMP）-1的水平。
在培养前，iAM和dAM中的所有四种BM成分均得以保留，但在培养1周后溶解，此时MMP-2增加。两种培养物中上皮细胞的生长均与MMP-2和-9表达的增加相关。BM的重新合成始于层粘连蛋白-5，随后是其他成分。该过程在培养后iAM上于1周时发生，但在dAM上于2周时发生。在4周时，BM在iAM上作为一条从外植体向前缘的线性带更成熟地沉积，并且在时间上与MMP-9水平的急剧下降相关。相比之下，只有当TIMP-1水平增加时，dAM上的BM沉积才在前缘开始。iAM上的上皮细胞生长比dAM上的上皮细胞生长表达更多的p63，但表达更少的K3和K12。
在原始羊膜BM溶解后，由体外扩增的人角膜缘上皮细胞在iAM上形成的新BM沉积更快且更成熟，导致角膜缘上皮表型的再生。相比之下，BM沉积在dAM上延迟且仍不成熟，类似于角膜上皮表型的伤口愈合。因此，BM重新合成可作为评估角膜缘上皮祖细胞在羊膜上体外扩增成功与否的另一个客观指标。
