Abstract
Introduction: Nuclei of regenerating rat liver are able to incorporate precursors of DNA synthesis under suitable conditions in vitro. To evaluate the number of parenchymal nuclei which contribute to the incorporation of 3H-thymidine, autoradiograms of homogenized and incubated rat liver were analysed 23 hrs after a two third hepatectomy and the labelling index of 3H-TdR incorporating nuclei was determined. The influence of various conditions of incubation was investigated. Material and methods: The residual part of the livers of partially hepatectomized rats (Wistar AF/Han, male, 200–225 g) were homogenized in a Potter homogenizer with a teflon pestle and incubated in 4 volumes of TCM 199 containing 12 mM NaHCO 3 after addition of deoxythymidine-6- 3H and ATP. After 60 min of incubation at 37° C the homogenate was layered over sucrose and centrifuged at 600 × g for 10 min. The remainder was resuspended, recentrifugated and fixed with 10% formalin containing 0,4 mM of inactive deoxythymidine. Autoradiograms were made with stripping film (Kodak AR 10), and the percentage of labelled nuclei was determined. Results: The average labelling index of liver cell nuclei was 15% under the conditions of preparation and incubation as described. Elevating the concentration of ATP in the incubation medium did not significantly influence the amount of 3H-TdR labelled nuclei, but the number of silver grains overlying labelled parenchymal nuclei increased. After replacing TCM 199 by isotonic sucrose solution only a few silver grains were overlying a much reduced number of parenchymal cells. Discussion: The findings are compared with the results of regenerating liver in vivo. It can be assumed that DNA synthesis which has been started in vivo continues under adequate experimental conditions of incubation in vitro. Addition to the incubation medium of deoxyribonucleotides, however, is not required. ATP concentration is apparently not a limiting factor for the number of 3H-TdR-incorporating cells, but for the duration of 3H-TdR incorporation in vitro.
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