The use of exfoliated cells to study the effect of diet upon the colon

• Main Author: Barrow, T. M.
• Published: University of Cambridge 2010
• Subjects: 616.994
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.596429

This thesis investigates whether changes in colonic gene expression can be detected by the use of exfoliated cells, in the context of a study into the effect of a processed meat diet upon the colon. The yields of colonocytes and squamous cells from the stools of 16 healthy volunteers were established using cytokeratins 8 and 10 as markers of tissue lineage. Yields were insufficient for the purpose of studying gene expression at the mRNA level. RNA extraction directly from stool was observed to be highly degraded and displayed poor marker stability. Using a novel method of isolating exfoliated cells directly from the rectum (‘direct cell sampling’), colonocyte, yields were in the range of 10³ – 10⁴ cells, with little contamination by squamous cells or faecal debris. Few cells were observed to be proliferative, as determined by the absence of Ki67 and PCNA. RNA extracted from the cells showed satisfactory marker stability, with samples analysed in quadruplicate by qPCR displaying mean ranges of 0.72 and 1.03 in the Ct values for the <i>β</i>-actin and cytokeratin 8 genes. The approach was taken forward for validation against results from biopsies. To study the effect of a processed meat diet upon gene expression, biopsies were collected from 17 volunteers fed processed meat and vegetarian diets in a random cross-over design. For the validation of the direct cell sampling approach, exfoliated cells were collected from 7 volunteers for comparison. Microarray analysis performed on the biopsy samples revealed no differential gene expression between the diets. Stratification of the data set, to reduce variance, revealed increased expression of six inflammatory genes (CCL2, CCL18, CCL20, HBEGF, IL1B, VNN1) in response to the vegetarian diet. This unexpected finding requires further investigation.