Abstract 281 Poster Session IV, Tuesday, 5/4 (poster 322)

Surfactant protein A (SP-A) is the most abundant of the surfactant proteins and, along with the other surfactant proteins, is involved in the formation of tubular myelin, the modulation of the surface tension-reducing properties of surfactant phospholipids, and the metabolism of surfactant phospholipids. Because of the known interactions of SP-A with SP-B (eg., formation of tubular myelin), we hypothesized that elimination of SP-A mRNA would affect the regulation of SP-B gene expression. Midtrimester human fetal lung explants were maintained in serum-free Waymouth's medium for 3 to 5 days, during which time they undergo spontaneous type II cell differentiation. The explants were exposed to an antisense 18-mer phosphorothioate oligonucleotide (ODN) complimentary to a region 10 nucleotides downstream from the initiation codon region of SP-A mRNA. ODNs were used at a concentration of 90 µM. A similarly modified sense ODN was used as a control. Lipofectamine was used as a carrier and the media was changed daily with fresh ODN added. After 3 days in culture, no SP-A mRNA was detectable by Northern blot in the antisense treated group and compared to control there was no effect in either the sense or vehicle conditions. (ANOVA, n=3, p<0.02, Student-Newman-Keuls, p<0.05 for antisense ODN compared to other conditions). The densitometric data from the Northern blots were normalized to 18S rRNA to control for loading. After 5 days in culture, levels of immunoreactive SP-A were also significantly decreased after antisense treatment (n=2, p< 0.05). SP-B mRNA levels were measured by Northern blot after 3 days of culture when SP-A mRNA was absent. SP-B gene expression was not affected by the lack of SP-A mRNA. Tissue viability, as measured by the release of lactate dehydrogenase into the media, was also not affected by this ODN concentration. We conclude that selective elimination of SP-A mRNA with antisense ODNs results in a decrease in SP-A levels in human fetal lung without affecting steady-state SP-B mRNA levels. We speculate that during human lung development, the regulation of SP-B gene expression is independent of the level of SP-A mRNA or protein.