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K Musselmann, BP Kane, WR Kader, JR Hassell; A High Molecular Weight Factor Present in Cornea That Stimulates Keratocyte Proliferation . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1650.
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Purpose: Previous work has shown that a PBS extract of corneas stimulated growth of serum cultured corneal fibroblasts by 50%. In this study, we analyze the effects of a corneal extract on keratocyte proliferation and initiate characterization of the stimulating factor. Methods: Fresh corneas were minced and homogenized using a polytron in 5ml DMEM/g cornea. This tissue was extracted overnight by rocking at 4°C, centrifuged to pellet the insoluble material and the extract sterilized by filtration (.22µm). This extract was used either unfractionated or fractionated on an S-300 column and the fractions analyzed by SDS-page. Collagenase isolated keratocytes were plated at low density (∼110cells/mm2) and treated on day 1 with the DMEM extract. The cells were radiolabeled with 3H-thymidine (12.5µCi/ml DMEM) for either 24 or 44 hours and the incorporation measured by liquid scintillation or the DNA content measured by fluorometry. Results: Keratocytes cultured in corneal extract showed a dose dependent increase in 3H-thymidine incorporation (a maximum of 15-fold, p <.0001) while culture in 10%fetal bovine serum resulted in a 10-fold increase (p=.0006). Extract prepared from frozen corneas was equally active but heating the extract to 90°C for 10 minutes abolished the activity. The stimulating activity in the extract eluted at around 200 kD on S-300, while the activity in serum eluted at around 70 kD. The cells exposed to the extract showed an initial loss of processes similar to changes occurring in the presence of serum, but to a lesser degree. Conclusion: These results suggest that corneas contain unique, soluble, high molecular weight factors which stimulate keratocyte proliferation. The factors are readily present in the cornea, and are likely not the result of a cellular response to the homogenization, as indicated by the activity of extracts from frozen corneas. The proliferation without the complete loss of cell morpholgy as well as the molecular size could indicate that the stimulating factor in the corneal extracts is different than the factors present in serum.
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