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Changes of Nuclear Matrix in Long-Term Culture of Limbal Epithelial Cells

Yam, Hin-Fai Ph.D.; Lam, Dennis Shun-Chiu F.R.C.Ophth.; Pang, Chi-Pui D.Phil.

Basic Investigations
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Purpose. This study was undertaken to investigate the changes of nuclear matrix in long-term culture of rabbit limbal epithelial cells.

Methods. Epithelial cells outgrown from limbal basal epithelium were serially cultivated. Nuclear matrices of early and late passages were extracted for morphologic study and protein analysis by two-dimensional gel electrophoresis and immunoblotting.

Results. Differential growth and changes in morphology were observed in limbal epithelial cells of early and late passages. Cytokeratin type 3 was expressed in cells of later passages, indicating corneal cell differentiation during the long-term culture. These cells also showed reduced density of nuclear matrix fibrils and thinning of nuclear lamina. They were shown by two-dimensional gel electrophoresis to have lost most nuclear matrix proteins, including lamin A/C and proliferating cell nuclear antigen. However, five new protein entities were also expressed.

Conclusion. The nuclear matrix appeared to change along with limbal epithelial cell differentiation in culture. Whether such changes may affect the growth and viability of limbal cells after transplantation requires in vivo tissue analysis.

From the Department of Ophthalmology and Visual Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong.

Submitted June 18, 2001;

Revision received November 13, 2001.

Accepted November 20, 2001.

Address correspondence and reprint requests to Dr. C.-P. Pang, Department of Ophthalmology & Visual Sciences, The Chinese University of Hong Kong, 3rd/F Hong Kong Eye Hospital, 147K Argyle Street, Hong Kong. E-mail: cppang@cuhk.edu.hk

© 2002 Lippincott Williams & Wilkins, Inc.