Isolation and molecular characterization of serous and mucous gland cells of the porcine airways.

Abstract

Secretory cells of the glands of the airways play important roles in the pathogenesis of several diseases. Little, however, is known about the molecular biology of these cells. Here we describe a procedure for the separation of serous and mucous gland cells and the isolation of genes specifically expressed in these cells. Mucosal tissue was prepared from porcine large airways. Following enzymatic digestion, the cell types were separated by discontinuous Percoll density gradient centrifugation. Cell purity was analyzed by electron microscopy. The cell fractions contained between 75 and 85% mucous and serous cells, respectively. To isolate cell type-specific genes, poly(A)+ RNA was isolated from serous and mucous cell fractions, reverse transcribed and used for differential display polymerase chain reaction (PCR). Out of about a total of 1,700 PCR products identified in horizontal polyacrylamide gels, most bands were found to be common to both cell fractions, indicating that the transcript patterns in cells from both fractions are very similar. Eighteen PCR products, however, were consistently distinct in the two cell fractions, with eight products present only in RNA from the mucous cell fraction and 10 PCR products present only in RNA from the serous cell fraction. Dot-blot analysis of mRNA of serous and mucous cells proved the cell type-specific expression of nine PCR products. Northern blot analysis detected single transcripts for each PCR product. The development of a simple cell separation procedure for secretory cells of the airways, combined with the ability to isolate numerous cell type-specific marker genes, should facilitate the molecular understanding of secretory cells of the airways.