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Title: Interrogating therapeutic manipulation of the endocannabinoid system in the human colon
Author: Vase, Hollie Francesca
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 2013
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The endocannabinoid system (ECS) is known to be involved in key aspects of cell maintenance within the human colon, as well as being dysregulated in pathophysiological conditions, including colon inflammation and cancer. However, the contribution of the ECS within each of these conditions has not been fully elucidated. This indicates that the current identification of key targets within the ECS that are involved in gut pathology could be used as potential novel therapeutics. Two experimental approaches were designed and optimised to give an insight into ECS signal regulation within the human colon and to screen ECS therapeutics, tetrahydrocannabinol (THC) and cannabidiol (CBD); a human colon ex vivo explant culture model and an innovative multiplexed quantitative gene expression technology, the GenomeLab GeXP system (Beckman Coulter). Gene targets were identified that are known markers of regulation and function in cells of healthy tissue. An assay, the hCellMarkerPlex was designed that incorporated twenty-three of these gene targets, epithelial (EZR, KRT18, SLC9A2), proliferation (PCNA, CCND1, MS4A12), differentiation (B4GANLT2, CDX1, CDX2), apoptotic (CASP3, NOX1, NTN1), fibroblast (FSP1, COL1A1), structural (ACTG2, CNN1, DES), gene transcription (HDAC1), stem cell (LGR5), endothelial (VWF) and mucin production (MUC2). The hCellMarkerPlex identified gene signatures which distinguished between normal, adenoma and carcinoma tissue, identifying cellular processes showing abnormal activity associated with pathological status. The resulting biomarker profiles were used to establish a human colon explant culture system. The human colon explant culture presents a novel model to study modulation of the ECS and screen ECS therapeutics. Combined with the GenomeLab GeXP System multiple components of the ECS were assessed at the gene regulatory level. A custom designed GeXP assay, the hECSplex, was developed. hECSplex gene expression signatures of EC receptors (CNR1, CNR2, GPR55 and TRPV1), ECS enzymes (NAPE-PLD, GDE1, DAGLA, DAGLB, FAAH, FAAH2 and PTGS2), inflammatory (IL1B, IL10, IL6, LEP, TNF and SOCS3), signalling pathway (ID1, BCL2, CFL1, BIRC5, TP53, MYC and KRAS), lipid production (SREBF1, ACACA), and plasma-membrane (OCLN) markers revealed altered expression of ECS components in carcinogenesis compared to normal tissue. Abstract vi . The hECSplex gene expression signature of colon explants showed that ECS was not altered during culture, emphasising the explant models capability as a pharmaceutical tool to test current and novel therapeutics. Applications of both THC and CBD to normal colon explants at different concentrations do not lead to any significant changes. Indicating the current pharmacological use of phytocannabinoids is causing no adverse effects in surrounding healthy colon tissue. The GenomeLab System presents new opportunities to interrogate multiple components of the endocannabinoid signalling system in small colon explant tissue samples, and in response to ECS therapeutics.
Supervisor: Not available Sponsor: Scottish Universities Life Sciences Alliance (SULSA)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Colon (Anatomy)