Effects of insulin and EGF on DNA synthesis in bovine endothelial cultures: flow cytometric analysis.
OBJECTIVE
To investigate the effects of insulin, epidermal growth factor (EGF), and the corneal storage media--DexSol--at 24 and 48 hours on DNA synthesis in confluent primary cultures of bovine corneal endothelial cells.


METHODS
Flow cytometry was used to measure changes in DNA synthesis. This technique allows a large number of cells to be counted and sorted into G1, S, and G2/M phases of the cell cycle.


RESULTS
Changing the normal culture media to DexSol had no effect on the cell cycle at 24 or 48 hours. The addition of insulin, EGF, or insulin + EGF to DexSol increased DNA synthesis within 24 hours. The mitotic indices for DexSol, DexSol + insulin, and DexSol + EGF were 0.134 (SE = +/- 0.022), 0.207 (+/- 0.027), and 0.205 (+/- 0.052), respectively. Adding insulin + EGF to the DexSol resulted in the most significant change in S and G2/M, increasing the mitotic index to 0.300 (+/- 0.072) (P = 0.0116). At 48 hours, the presence of the growth factors no longer had any effect.


CONCLUSIONS
Flow cytometry was a useful technique in separating cultured bovine corneal endothelial cells according to their DNA content. Analysis of the cultures after the addition of insulin and EGF showed an increase in DNA synthesis. The synergistic effects of the growth factors on corneal endothelial cells suggest that they stimulate mitotic activity by different mechanisms. The addition of mitogens to eye bank storage media may increase corneal endothelial cell densities in donor corneas.
Crow
J M
JM
Department of Ophthalmology, Ramsey Clinic, St. Paul, MN.
Lima
P H
PH
Agapitos
P J
PJ
Nelson
J D
JD
D000818:Animals; D002417:Cattle; D002453:Cell Cycle; D002478:Cells, Cultured; D002809:Chondroitin Sulfates; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis*; D004261:DNA Replication / Q000187:drug effects; D004357:Drug Synergism; D004728:Endothelium, Corneal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D005434:Flow Cytometry / Q000379:methods; D006531:HEPES; D007328:Insulin / Q000494:pharmacology*; D009930:Organic Chemicals
D016895:Culture Media, Serum-Free; C075735:Dexsol; D007328:Insulin; D009930:Organic Chemicals; D004815:Epidermal Growth Factor; D002809:Chondroitin Sulfates; D004247:DNA; D006531:HEPES