THE ROLE OF STRETCH-INDUCED MECHANOTRANSDUCTION IN THE AIRWAY EPITHELIUM OF THE DEVELOPING LUNG
Date
2019-05
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
This study provides a quantitative methodology to measure and apply
controlled strain to a cellular system as a means of studying embryonic lung
development. The development of the type of model it provides will enable
researchers to obtain new insight on lung mechanotransduction pathways and to
determine the effects of various parameters of strain on developing lungs. An acrylic
strain device was used and calibrated to deliver repeatable strain to the MLE-12 cell
line.
The device was coated with fibronectin for one hour, which enabled cell
adhesion. Cells were then seeded and cultured overnight such that they reached 80-
90% confluency prior to the experiment. A novel LabView program was developed to
operate the strain device and enabled control of parameters including frequency of
stretch and amount of stretch to be applied. Assays were optimized for the MLE-12
cell line including: Fluo-8AM and K-Ras pulldown assay. Whole lung explants were
analyzed to determined baseline frequency of lung contractions and kymographs were
obtained. Cells were stretched at a frequency of 3.30 with a duty cycle of 3.33%,
which corresponds to a 10 second stretch, once every 5 minutes. Cells were analyzed
for proliferation with an Edu Assay Kit. Analysis was done using a novel MatLab
script created by our lab and modified. A GCamp cell line was created in order to
visualize calcium flux in real time during stretch. Effects were analyzed using ImageJ.
Proliferation data did not follow the trends suggested by the literature and
decreasing background fluorescence skewed fluorescence measurements in the
GCamp dataset. Deviations from suggested trends were likely due to flaws in the
device itself, as shown in calibration data. Overall, this study provides a methodology
for studying lung development with the creation of a novel strain device. The methods
described in this study could help to guide future experiments that will enable
researchers to elucidate pathways of mechanotransduction in the developing lung and
develop targeted therapeutics for developmental pulmonary diseases.
Description
Keywords
Neuroscience,Mechanotransduction, Lung