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dc.contributor.advisorButt, Grant
dc.contributor.authorvan Hout, Isabelle
dc.date.available2015-11-16T03:33:02Z
dc.date.copyright2015
dc.identifier.citationvan Hout, I. (2015). Modulation of colonic stem cell proliferation by bacterial components (Thesis, Bachelor of Biomedical Sciences with Honours). University of Otago. Retrieved from http://hdl.handle.net/10523/6080en
dc.identifier.urihttp://hdl.handle.net/10523/6080
dc.description.abstractThe intestine is home to a large number of commensal microbes, which are restricted to the intestinal lumen by the epithelial barrier. In the colon, which has the greatest number of commensal microbes, the epithelial barrier is maintained by turnover of the epithelium every 3-5 days by a small population of stem cells found at the base of the crypts. If bacteria are present in the lamina propria, it indicates a loss of barrier integrity so it is important for the epithelium to respond to this, to increase proliferation and repair the barrier to prevent inflammation. Previous research indicates that stimulating pattern recognition receptors, found on colonic stem cells, may be a mechanism by which epithelial proliferation can be altered. Here we have used human colonic organoids to determine if the colonic bacteria modulate the proliferation of the colonic stem cells and hence the properties of the colonic epithelium. Colonic organoids were grown from crypts isolated from healthy patients and suspended in Matrigel® overlaid with a specific stem cell growth media. Two distinct organoid types were seen in culture enteroids, which have a well-developed columnar epithelium and so appear to be representative of the native colonic epithelium, and spheroids, which have a poorly differentiated epithelium. The effect of the removal of the growth factor Wnt3A, removal of the inhibitors of the TGF-β pathway LY2157299 and SB202190, induction of differentiation by the addition of the γ-secretase inhibitor DAPT (10µm, [N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester), or inclusion of bacterial components including lipopolysaccharide (LPS), lipoteichoic acid (LTA) flagellin and muramyl dipeptide (MDP) (all 20ng ml-1), in the growth media, on cell proliferation in enteroids was assessed by measurement of the expression of proliferation-associated genes (Cylcin D, p15, LTBP1, Ki67 LGR5, FOXM1 and MYC) by qPCR. In addition, the incorporation of EdU (5-ethynul-2’-deoxyuridine) into the nuclei of dividing cells was used to quantify proliferation in whole mounts of both spheroids and enteroids. Statistical significance was determined by a Kruskal Wallis test and a Dunns post test or a Mann-Whitney test. Few changes in proliferation-associated genes at the transcript level in enteroids were seen, indicating environmental changes do not alter enteroid proliferation. Consistent with the qPCR findings, direct quantification of cell proliferation in whole mounts of enteroids, also found no significant change in proliferation between control and experimentally treated enteroids. In contrast, spheroids had a significantly higher rate of proliferation than enteroids (p<0.01). Furthermore, a significant decrease in proliferation was seen in spheroids either grown without Wnt3A (p<0.01), in the presence of DAPT (p<0.05), or with the addition of LPS (P<0.05) and flagellin (p<0.01) for 10 days, compared to control spheroids. These results show that environmental changes can affect spheroid proliferation; hence spheroids may be a better model of the crypt base, which is the site of proliferation in the native epithelium. They also show there is a basal level of proliferation maintained in the cultures, despite environmental manipulations. Finally, these results show that the bacterial components LPS and flagellin, which signal through TLR4 and TLR5 respectively, decrease colonic proliferation, by directly interacting with the epithelium. A decrease in proliferation under the conditions tested here was unexpected, and may highlight that other components such as immune or mesenchymal cells, may be important in controlling colonic proliferation.
dc.format.mimetypeapplication/pdf
dc.language.isoen
dc.publisherUniversity of Otago
dc.rightsAll items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.subjectColon
dc.subjectBacteria
dc.subjectProliferation
dc.subjectEnteroids
dc.subjectOrganoids
dc.subjectspheroids
dc.titleModulation of colonic stem cell proliferation by bacterial components
dc.typeThesis
dc.date.updated2015-11-16T03:21:29Z
dc.language.rfc3066en
thesis.degree.disciplinePhysiology
thesis.degree.nameBachelor of Biomedical Sciences with Honours
thesis.degree.grantorUniversity of Otago
thesis.degree.levelHonours
otago.openaccessOpen
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