Akila, G (2013) Characterisation of Colonic Epithelial Stem Cells in Health and Inflammatory Bowel Disease. Doctoral thesis, The Tamilnadu Dr.M.G.R. Medical University, Chennai.
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Abstract
INTRODUCTION: The epithelium of the large intestine is a very rapidly replicating tissue with an enormous capacity for continuous proliferation, followed by differentiation, senescence and shedding. The proliferative capacity of the large intestinal epithelium is maintained through multipotent stem cells which are thought to be located towards the base of the colonic crypts. As yet poorly defined signals, that may include cytokines and growth factors which are secreted by the lamina propria cells or the intestinal subepithelial myofibroblasts, control the proliferation and differentiation of these epithelial stem cells. OBJECTIVES: 1. To isolate and characterize proliferative cells from the stem cell compartment of the colonic epithelium. 2. To determine whether the above proliferative epithelial cells can ameliorate experimental colitis in mice. 3. To determine whether there is an alteration in epithelial stem cell numbers and distribution in the colonic mucosa of patients with inflammatory bowel disease. METHODS: Human participants were recruited for these studies from those patients who were undergoing colonoscopy in the Department of Gastrointestinal Sciences of this hospital and were recruited into cases or controls depending on their satisfying inclusion criteria. Inclusion criteria: Ulcerative colitis: 1. Patients who were diagnosed to have ulcerative colitis, based on consensus Asian criteria that include clinical information, colonoscopy findings, biopsy findings consistent with idiopathic ulcerative colitis, and exclusion of infections (Ouyang et al, 2006). All patients seen in the Inflammatory Bowel Diseases Clinic and the Gastroenterology Clinic of this hospital underwent investigation according to a defined protocol that included colonoscopy and segmental ileo-colorectal biopsies. Colonoscopy was performed in the Endoscopy Unit and all biopsies were reported under the supervision of specialist gastrointestinal pathologists. 2. Age 18 years or older, of either sex. 3. Ability to give informed consent. Control participants: 1. Patients with irritable bowel syndrome or rectal bleeding undergoing screening colonoscopy to exclude inflammatory bowel disease, colonic polyps or cancer. 2. Normal colonoscopy and rectal biopsy. 3. Age 18 years or older, of either sex. 4. Ability to give informed consent. SUMMARY: In Summary, these studies provide new insight into colonic epithelial stem cell physiology including alterations in ulcerative colitis and in experimental colitis induced by dextran sodium sulphate in mice. Furthermore, alterations in stem cell signalling in the colonic mucosa of ulcerative colitis patients are pinpointed most of which appear to be specific to the disease and only a few secondary to inflammation. The specific Conclusions are as follows: 1. Expression of intracellular stem cell marker Musashi-1 and differentiation marker Hes-1 by immunocytochemistry and PCR revealed that proliferative cells are present in the pool of colonocytes isolated from mouse colon, and their distribution and numbers could be determined. In suspension cultures, we could identify the clones at the end of 14 days which were positive only for Musashi-1 and not Hes-1. So this confirms that zonal proliferative cells of the colon have the clonogenic potential by which it undergoes self-renewal by itself and forms progenitor cells. 2. Luminal infusion of proliferative colonic epithelial cells from donor mice into the colon of mice with dextran sodium sulphate colitis was not an effective method for epithelial stem cell transplantation from donor to recipient mice. 3. The comparison between proximal and distal colon of colonic epithelial stem cells represents that there was no difference observed in the numbers and distribution of these cells between proximal and distal colon of normal mouse as determined by immunostaining using the antibodies to Musashi-1 and Hes-1. 4. In mice with DSS colitis, there was no change in either proximal or distal colon in staining pattern against the antibodies Musashi-1 and Hes-1, even though the distal colon was more inflamed than the proximal colon. 5. There were significant changes observed between the colonic mucosal biopsies of control patients and colitis patients in both the numbers as well as in the location of stem cells in the colon. There was an increase in the numbers of stem cells in colitis patients when compared to control subjects and the stem cell location was also altered with the Musashi-positive cells being seen at the top of crypts in the colitis patients, while they were confined to the bottom of the crypts in normal controls. 6. There was no alteration in mRNA expression of genes related to stem cell multipotency, or in the Sonic and Indian Hedgehog pathways in ulcerative colitis. 7. mRNA expression of Fibroblast Growth Factor Receptor 1 was significantly down-regulated in active ulcerative colitis, while expression of Caudal type homeobox 2 gene was downregulated in UC in remission. The former is involved in tissue repair and the latter in cell differentiation in the crypt. 8. Notch2 and Notch3 expression was down-regulated in inactive ulcerative colitis, reflecting alterations in Notch signalling pathways in the disease. 9. Alterations in transforming growth factor (TGF)-β signalling pathways were reflected as down-regulation of SMAD3 and SMAD9 expression in both active and inactive UC possibly reflecting a reparative response to injury in colitis. Down-regulation of ACVRL1 in inactive UC may be a more specific change in a cell surface receptor for TGF- β. 10. Wnt signalling pathways were affected in inactive UC, including down-regulatino of Frizzled 7 & 8 and nuclear factor of activated T cells 4 (NFATC4), reflecting altered regulation of genes relating to crypt epithelial cell differentiation in the colon. NFACT1 expression was down-regulated in active UC.
| Item Type: | Thesis (Doctoral) |
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| Uncontrolled Keywords: | Characterisation, Colonic Epithelial Stem Cells, Health and Inflammatory Bowel Disease. |
| Subjects: | MEDICAL > Biochemistry |
| Depositing User: | Subramani R |
| Date Deposited: | 28 Oct 2020 13:49 |
| Last Modified: | 29 Oct 2020 05:33 |
| URI: | http://repository-tnmgrmu.ac.in/id/eprint/13164 |
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