A METHOD FOR EXAMINING DIFFERENTIAL MESSENGER-RNA EXPRESSION ALONG THE CRYPT-VILLUS AXIS OF THE HUMAN SMALL-INTESTINE

1. The aim of th is study was to devise a method of segregating crypt and villus cell subpopulations from endoscopic human small intestinal biopsies which might be used to examine changes associated with functi onal differentiation at the molecular level. 2. Routine endoscopic bio psies from the human small intestine were subjected to a modified prot ocol of mechanical disruption and chelation to yield subpopulations of different cell types. The purity and character of the cell population s isolated was assessed by measuring enzyme activity and thymidine inc orporation and by histology. A guanidinium isothiocyanate method was a dapted for small samples to extract RNA from the isolated subpopulatio ns, and probes for RNA with a known predilection for crypt and villus cells were used to further investigate the application and usefulness of the technique. 3. Sequential histological examination during the se gregation protocol demonstrated that different cell types were removed serially from the biopsy samples. Cell-type enrichment of the segrega ted subpopulations was demonstrated by differential alkaline phosphata se activity and by differences in thymidine incorporation in the sampl es isolated. Sufficient quantities of RNA could be extracted from the segregated subpopulations for Northern blot analysis and the different ial expression of mRNA for sucrase-isomaltase and transferrin receptor was demonstrated in the villus and crypt subpopulations respectively. 4. Messenger RNA can be successfully extracted from different cell ty pes segregated from routine hu man endoscopic small intestinal biopsie s. This technique should prove useful for investigating the mechanisms regulating the functional differentiation of epithelial cells in the small intestine and the regulatory mechanisms governing absorption of macromolecules.