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O'Loughlin, Jonathan (2018) Production of a sustainable primary airway cell model for respiratory research. MRes thesis, University of Nottingham.

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Abstract

The airway epithelium is one of the body’s front-line defences against environmental factors. It is known that in diseases such as asthma and COPD the epithelial layer is susceptible to damage which propagates inflammation and airway remodelling. Whilst primary airway epithelial cells have been key to furthering our knowledge of respiratory diseases, their practicality in research is limited by their short lifespan when cultured in vitro. Work completed by our research group previously highlighted the potential of BMI-1 in extending the lifespan, whilst maintaining plasticity, of bronchial epithelial cells for research purposes. The aim of this project was to build further on this work and determine whether HBECs engineered to express BMI-1 are comparable to normal human bronchial epithelial cells (NHBECs).



Using lentivirus transduction, eight primary human bronchial epithelial cell (HBEC) donors were infected to stably overexpress BMI-1. Cells overexpressing BMI-1 had an evident proliferative advantage in comparison to cells engineered to express an empty vector, as these cells underwent senescence within 3 passages, following lentivirus infection. By performing an MTT assay, cells modified to overexpress BMI-1 were shown to have greater metabolic activity than NHBECs. The impact of BMI-1 on cell properties were assessed using electric cell substrate impedance sensing (ECIS) and a two-step cell cycle assay. BMI-1 upregulation does not significantly alter cell adherence and cell spreading of HBECs. In addition, overexpression of BMI-1 did not impact the ability of HBECs to form a resistive barrier. It was also shown that the frequency of cells in the G1/G0 phase of the cell cycle was significantly reduced in cells engineered to overexpress BMI-1 in comparison to NHBECs. Preliminary RNA-sequencing analyses suggest alterations in gene expression exist as anticipated but these are modest and identifiable, representing just 2.3% of the genome (5% FDR).

Together these results show that the use of lentivirus to deliver and promote BMI-1 expression in HBECs is an effective genetic manipulation system. BMI-1 expression provides cells with a proliferative advantage and enhanced metabolic activity within cells. Furthermore, it has been shown that HBECs engineered to overexpress BMI-1 behave almost identically to NHBECs when cultured in vitro.

Item Type:	Thesis (University of Nottingham only) (MRes)
Supervisors:	Sayers, Ian Bosquillon, Cynthia
Keywords:	BMI-1; Bronchial epithelial cells; Lentivirus; Cell culture
Subjects:	W Medicine and related subjects (NLM Classification) > WF Respiratory system
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Medicine
Item ID:	49344
Depositing User:	O'Loughlin, Jonathan
Date Deposited:	03 Apr 2019 13:49
Last Modified:	07 May 2020 14:02
URI:	https://eprints.nottingham.ac.uk/id/eprint/49344

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