Serum-free organ culture of suckling rat jejunum: effect of regulatory hormones.
To facilitate the study of regulators of differentiation and proliferation of small intestinal epithelium in the suckling rat we have developed a serum-free organ culture system and used it to examine epithelial responsiveness to various regulatory hormones. These hormones included the insulin-like growth factors (IGFs) whose action can be blocked by binding proteins in serum. Jejunal explants from 5-day-old suckling rats maintained better brush border enzyme activity and better histology when cultured under hyperbaric conditions for 24 h in serum-free Dulbecco's modified Eagle's medium/F12 medium than in RPMI 1640 plus 10% fetal bovine serum. Tissue responsiveness to various regulatory hormones was then tested in the serum-free medium. Insulin had no significant effect on morphology, proliferation rate, or enzyme activity in 5-day explants after 24 h in culture. However, insulin did increase lactase activity and induce the early appearance of sucrase in 10- and 12-day explants after 48 h in culture. Dexamethasone increased specific activities of alkaline phosphatase (30%, P < 0.001) and lactase (15%, P < 0.001), and reduced shedding of alkaline phosphatase into the medium (P < 0.001), in explants of 5-day-old rats cultured over 24 h. Dexamethasone combined with insulin had no obvious effect on the rate of protein or DNA synthesis but did increase villus height (P = 0.04) and crypt depth (P = 0.001) and acted synergistically to further increase lactase activity above levels obtained by either alone. IGF-I and IGF-II, des-(1-3)IGF-I, fibroblast growth factor (FGF), and growth hormone (GH) had no effect on morphology or biochemical activity of explants after 24 or 48 h culture.(ABSTRACT TRUNCATED AT 250 WORDS)
University of Melbourne Department of Medicine, Royal Melbourne Hospital, Victoria, Australia.
V
V
Albert
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Barkla
G P
GP
Young
D000818:Animals; D000833:Animals, Suckling; D002455:Cell Division; D016895:Culture Media, Serum-Free; D004247:DNA / Q000096:biosynthesis; D003907:Dexamethasone / Q000494:pharmacology*; D004848:Epithelium / Q000187:drug effects; D005260:Female; D013006:Growth Hormone / Q000494:pharmacology; D006133:Growth Substances / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology*; D007413:Intestinal Mucosa / Q000166:cytology / Q000187:drug effects*; D007583:Jejunum / Q000166:cytology / Q000187:drug effects*; D008297:Male; D008871:Microvilli / Q000187:drug effects / Q000201:enzymology; D009924:Organ Culture Techniques / Q000379:methods*; D014176:Protein Biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley
D016895:Culture Media, Serum-Free; D006133:Growth Substances; D007328:Insulin; D003907:Dexamethasone; D013006:Growth Hormone; D004247:DNA