ISOLATION AND MOLECULAR CHARACTERIZATION OF SEROUS AND MUCOUS GLAND-CELLS OF THE PORCINE AIRWAYS

Citation
R. Bals et al., ISOLATION AND MOLECULAR CHARACTERIZATION OF SEROUS AND MUCOUS GLAND-CELLS OF THE PORCINE AIRWAYS, American journal of respiratory cell and molecular biology, 18(4), 1998, pp. 548-553
Citations number
28
Categorie Soggetti
Cell Biology",Biology,"Respiratory System
ISSN journal
10441549
Volume
18
Issue
4
Year of publication
1998
Pages
548 - 553
Database
ISI
SICI code
1044-1549(1998)18:4<548:IAMCOS>2.0.ZU;2-0
Abstract
Secretory cells of the glands of the airways play important roles ih t he pathogenesis of several diseases. Little, however, is known about t he molecular biology of these cells. Here we describe a procedure for the separation of serous and mucous gland cells and the isolation of g enes specifically expressed in these cells. Mucosal tissue was prepare d from porcine large airways. Following enzymatic digestion, the cell types were separated by discontinuous Percoll density gradient centrif ugation. Cell purity was analyzed by electron microscopy. The cell fra ctions contained between 75 and 85% mucous and serous cells, respectiv ely. To isolate cell type-specific genes, poly(A)(+) RNA was isolated from serous and mucous cell fractions, reverse transcribed and used fo r differential display polymerase chain reaction (PCR). Out of about a total of 1,700 PCR products identified in horizontal polyacrylamide g els, most bands were found to be common to both cell fractions, indica ting that the transcript patterns in cells from both fractions are ver y similar. Eighteen PCR products, however, were consistently distinct in the two cell fractions, with eight products present only in RNA fro m the mucous cell fraction and 10 PCR products present only in RNA fro m the serous cell fraction. Dot-blot analysis of mRNA of serous and mu cous cells proved the cell type-specific expression of nine PCR produc ts. Northern blot analysis detected single transcripts for each PCR pr oduct. The development of a simple cell separation procedure for secre tory cells of the airways, combined with the ability to isolate numero us cell type-specific marker genes, should facilitate the molecular un derstanding of secretory cells of the airways.