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Y. Jeng Yuan; Expression of endostatin in human limbal epithelial cells cultivated on amniotic membrane . Invest. Ophthalmol. Vis. Sci. 2004;45(13):4812.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To understand the mechanisms by which endostatin (proteolytic product of collagen XVIII) is increased in human limbal epithelial cells (HLE) cultivated on preserved human amniotic membrane (AM). Methods: HLE were grown from explant culture on 35 mm dishes, or on dishes overlaid with intact or denuded AM. SHEM with 5% FBS was added until the epithelial sheet grew to 80 – 90% confluence. mRNA of HLE cultivated in serum and EGF–supplemented or deprived condition was collected and used for real time PCR study. In addition, serum–free conditioned medium (CM) was collected consecutively during a three–week period. The medium was condensed and used for Western blot analysis. Media conditioned by AM of various preparations was used for comparison. Results: Under serum and EGF supplemented condition, there was no elevation of collagen XVIII mRNA in HLE cultivated on AM. However, after 2 days of serum and EGF deprivation, HLE cultured on intact AM showed a substantial increase of collagen XVIII mRNA compared to HLE cultivated on dish. In addition, Western blot analysis of CM of HLE revealed weak bands around 21 kDa and 28 to 30 KDa. The signals became more prominent when HLE were cultivated on intact AM, and were most significant when HLE were cultivated on denuded AM. CM of viable AM also expressed strong signals for endostatin, but the signals weakened following cryopreservation, and became negative after 3 weeks incubation at 37 °C. Conclusion: The results suggested that both increased mRNA transcription and increased proteolytic cleavage of pre–existing collagen XVIII especially in the AM may contribute to increased endostatin level in HLE cultivated on AM.
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