Introduction Crohn’sDisease is a chronic autoimmune condition characterised by abdominal pain and diarrhoea. 80% of patients will require surgery and half of these will be as a result of stricture formation. Strictures occur as a result of mesenchymalcell proliferation and deposition of extracellular matrix.
Epithelial-mesenchymaltransition (EMT) is a novel mechanism theorised to contribute to gut fibrosis and is a potential target to prevent stricture formation. EMT has been noted in vivo and in animal gut models. However, it has been previously shown that CaCo2 cells are non-responsive to TGFbeta1, the principle mediator of EMT. EMT has not been investigated in the CaCo2 BBE cells.
To determine the cell receptor and signalling phenotype of CaCo2 BBE cells
To establish EMT in CaCo2 BBE cells
Methods Cell culture -CaCo2 BBE cells were grown in DMEM and 10% FBS on plastic wells to 100% confluenencyand allowed to differentiate.
CaCo2 BBE cell phenotype -Western blotting and immunofluorescence was performed to according to manufacturer protocols to analyse the phenotype of CaCo2 BBE Cells.
TGF-beta mediated EMT -Cells were exposed to a 1 hour and 4 day treatment with TGF beta.
Results Cell phenotype Zo1 and E. Cadherinwere expressed on the cell membrane. Alpha-SMA was expressed in the cell nuclei.
Intracellularly, vimentinwas not expressed. E. Cadherinand zo1 were constituently expressed.
Cell Signalling In response to a 1 hour treatment with TGF beta 0.8ng/ml, 2.5ng/ml, 7.5ng/ml, an increase in p-SMAD2 was noted.
Response to TGF-beta Following a 4 day treatment with TGF beta (2.5ng/ml), no change in expression of alpha-SMA, E.cadherin, Vimentinwas observed by Western Blot or Immunofluorescence.
Conclusion EMT has been theorisedto play a central role in stricture formation in Crohn’sdisease. However, this mechanism in human gut cells has only demonstrated by one author.
This project has demonstrated that whilst CaCo2 BBE cells are able to respond to TGFbetaby increased phosphorylation of SMAD2, we were not able to definitively demonstrate EMT.
Disclosure of Interest None Declared
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