Article
Searching for the molecular benchmark of physiological intestinal anastomotic healing in rats: an experimental study
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Published: | April 26, 2013 |
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Objective: Intestinal anastomotic healing processes differ greatly from analogous more widely-researched cutaneous phenomena. To date, we still lack comprehension of the particularities of the physiology of intestinal healing. This investigation focuses on the mRNA response time course to formation of an anastomosis.
Materials & Methods: In 8 rats, end-end ilio-ilial anastomoses were performed (n=2 per group). At days (d) 0 (control), 2, 4 and 8, 10mm perianastomotic segments were resected and examined by Affymetrix microarray (RG-230; > 31000 probe sets) to assess changes in gene regulation. Microarray findings were validated using real-time PCR for selected genes. In addition to screening global gene expression during anastomotic healing, we identified genes particularly intensely regulated during healing as potential new target genes for further investigation. We also subjected our data sets to an overrepresentation analysis using Gene Ontology and Kyoto Encyclopedia for Genes and Genomes (KEGG).
Results: Compared to the control group, we observed a biological time curve of differentially regulated genes peaking on day 2 with a total of 2238 genes decreasing by day 4 to 1687 genes and levelling at 1407 genes by day 8. PCR validation for MMP3 and MMP13 showed not only an identical expression polarity, but analogous regulation intensity. When setting the cut-off of up-regulation at e.g. 10-fold to identify more likely relevant genes, the total gene count was significantly lower with 58, 47 and 39 genes on days 2, 4 and 8 respectively. Over the time course of healing, near 947 Gene Ontology subcategories were significantly overrepresented. As well, 23 overrepresented KEGG pathways were identified.
Conclusion: This study is the first of its kind that focuses explicitly on gene expression during intestinal anastomotic healing under standardized conditions. Our work sets a foundation for further studies toward a more profound understanding of the physiology of anastomotic healing.