Project description:The small intestinal epithelium mediates vital functions of nutrient absorption and host defense. The spatial organization of the epithelial cells along the crypt-villus axis segregates them into regions of specialized function. However, many of the mechanisms governing intestinal epithelial cell migration and the coordination of interactions with adjacent cells and the extracellular matrix are not fully understood. We have evaluated in vivo gene expression patterns of ileal epithelial cells in healthy human subjects, isolated by laser capture microdissection from either the villus epithelial or crypt cell regions of the small intestinal mucosa. Expression profiles in villus epithelium and Paneth cell lineages were determined by quantitative real-time PCR, DNA microarray, and immunohistochemistry based methods. Relative expression levels of selected epithelial biomarkers were compared between the ileum, jejunum, duodenum, colon, stomach, and esophagus. Previously established biomarkers as well as a novel and distinct set of genes believed to be linked to epithelial cell motility, adhesion, and differentiation were found to be enriched in each of the two corresponding cell populations. Additionally, high baseline expression levels of innate antimicrobials, alpha defensin 5 (HD5) and regenerating islet-derived 3 alpha (Reg3A), were detected exclusively within the small bowel, most notably in the ileum, in comparison to other sites along the gastrointestinal tract. Our findings provide new and important insights regarding the molecular machinery employed by small intestinal epithelial cells to mediate their function and spatial organization in vivo. Experiment Overall Design: Surgical specimens of human intestinal mucosa were obtained from 4 individuals undergoing surgery for colon cancer, bowel obstruction, or other non-inflammatory conditions. The samples were fixed overnight in 4% (w/v) paraformaldehyde, dehydrated in a graded alcohol series and paraffin-embedded. At least 500 epithelial cells from the crypt or upper villus regions were then captured by LCMD from unstained 6Âµm thick sections using a PALM MicroLaser System Total RNA was extracted for microarray-based gene expression analysis.

Project description:Gene expression along the crypt-villus (C-V) axis was analyzed using cryostat sectioning to isolate fractions representing the crypts (bottom) and villus tops (top). These fractions were used for analyzing gene expression in iron replete Wistar rats (++), iron deficient Wistar rats (low iron), and in iron deficient Wistar rats fed iron for 3 and 6 days (iron-fed). Differences were observed between the crypts and villus tops in the expression of genes associated with Wnt and BNP signaling, cell proliferation and apoptosis, lipid and iron transport and metabolism. Gene expression in villus crypts and tops was also compared between Wistar and Belgrade rats (bb) and Belgrade rats fed iron (iron-fed) particularly as related to iron absorption and metabolism to define the affects of the mutation in DMT1 in the Belgrade rat on the expression of genes related to iron absorption and metabolism and the response to iron feeding. Keywords: iron stress response A colony of Wistar strain rats (++) and Belgrade (bb) rats on a Wistar background maintained in our animal quarters was used for this study. Twelve one-month old ++ rats were separated into 3 groups of four rats each. Group 1 rats (samples 1-8) were fed a normal diet and served as control animals. Group 2 (samples 9-16) and Group 3 rats (samples 17-22) were fed a low iron diet and bled 2-3 ml every three days for 4 weeks and served as iron deficient rats (low-iron). Group 3 rats, of which one rat died during anesthesia while undergoing phlebotomy, were fed iron supplementation in drinking water (50 ÂµM Ferric ammonium citrate) for the three days before sacrifice (iron-fed). Rats were fasted overnight, killed by injection of pentobarbital sodium and a 1-cm duodenal segment 1 cm distal from the pylorus was cryostat sectioned at the right angle to the crypt-villus axis and the sections representing the top one-third of villus (top) and the bottom one-third of the villus were pooled for RNA isolation. For the study of Belgrade rats, eleven bb rats were divided into 3 groups with the 4 rats in Group 1 (samples 23-30) maintained on a normal diet, the 4 rats in Group 2 (samples 31-38) fed iron for 3 days prior to sacrifice, and the 3 rats in Group 3 (samples 39-44) fed iron for 6 days prior to sacrifice. All procedures for the bb rats were the same as described above for the ++ rats. An additional ++ rat (samples 45-46) was similarly analyzed in a separate experiment.

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