Winner 2001 - The IADR.CED Visiting Scholar Stipend
Elzbieta Dybizbanska
CED
/ IADR Visiting Scholar Stipend - Report
My
research project was undertaken at the University of Liverpool School of
Dentistry within the Department of Clinical Dental Sciences, between the dates
of 13th May to 16th August 2002 inclusive, and from the 7th October to 6th
November 2002, inclusive.
The
original theme of the project submitted concerned the influence of IL-1 genotype
on the activity of fibroblasts in periodontal disease, as evaluated by the
production of IL-1 in response to stimulation by bacterial antigens and / or by
the production of metalloproteinases in response to stimulation by IL-1b.
Due to the tight ethical control in Great Britain, it was not possible to
undertake research on cells taken from patients within the time given. For this
reason, the project was modified. The final project undertaken was a proteomic
evaluation of human gingival fibroblast stimulation by IL-1b,
and was supervised by Dr Anna Milan, Dr Rachel Hall and Prof. Graham Embery. The
aim of the project was to identify the proteins produced by unstimulated human
gingival fibroblasts from established cell lines in culture, and by cells
stimulated by IL-1b,
using 2-D electrophoresis. Secondary aims were to compare the protein profiles
of stimulated and unstimulated fibroblasts from 2 different cell lines, and to
establish optimal conditions for culturing the cells by evaluating the use of
different media, glucose concentrations and methods of passage. During my work
on the project I was able to learn the whole range of techniques involved in
cell culture and 2-D electrophoresis: resurrecting cells from liquid nitrogen,
different methods of subculturing cells, freezing cells from flasks,
determination of protein concentration in samples to be analysed, sample
preparation (protein precipitation), strip rehydration and first dimension
isoelectric focusing, preparation of different kinds of acrylamide gels and
second dimension SDS-PAGE, visualisation by the use of different staining
techniques and analysis of gels by use of image analysis software and in-gel
digestion of proteins, as a preparation for mass spectrometric analysis. This
last stage was undertaken by the University staff after my return to Poland.
Work
on this project was particularly valuable for me not only because it provided me
with a step-by-step opportunity to learn the techniques involved in cell culture
from people with great experience in this field, but also because, as far as I
know, proteomics is a field in which my Medical University has not had any
experience so far. Throughout my work on the project, my supervisors were very
supportive, patiently and willingly sharing their knowledge and experience.
Besides
working on my project, I had the opportunity to learn something about the
activities of the other research groups at the Department, and was fortunate to
be able to get to know scientists whom I had only known earlier from the dental
literature. Furthermore, I was able to take advantage of the University’s
well-stocked library and had access to dental journals which are unavailable in
Warsaw.
My
stay at the Department of Clinical Dental Sciences was a very valuable
experience. It has provided me with the knowledge and practical experience I
need in order to work as a partner with basic science departments at my own
University, as well as opening the gate to possible future co-operation between
our Universities and making valuable friendships. I would very much recommend
this kind of initiative.
Elzbieta Dybizbanska
1714
Proteomic Evaluation of Human Gingival Fibroblast Stimulation by Interleukin-1b
E.
DYBIZBANSKA1,
A.M. MILAN2, R.C. HALL2, J.B. STANBURY2, and G.
EMBERY2, 1 The Medical University of Warsaw, Poland, 2
The University of Liverpool, United Kingdom
Objectives:
Interleukin (IL)-1a,
IL-1b
and IL-1 receptor antagonist play a major role in the regulation of the
inflammatory response in periodontal tissues. The aim of this study was to
examine the response of isolated human gingival fibroblasts using proteomic
analysis to detail changes in the proteome in response to exposure to IL-1b.
Methods:
Two cells lines of human gingival fibroblasts (CRL-2014 ATCC; JWS an in-house
cell line) were cultured in DMEM/NUT mix F12 (HAM), 10% FCS, 1%
antibiotic-antimycotic at 5% CO2 at 37°C. Cells were passaged using
trypsin-EDTA. From passage 4, for 2 successive passages, half of the cultures
were supplemented with 5ng/ml recombinant human IL-1b, with the non-supplemented cultures acting as a control. Spent medium
was collected for evaluation. Samples containing equal concentrations (0.010-1
mg/ml) of proteins from the two groups were precipitated using PlusOne-2D clean
up Kit (Amersham Biosciences), and reconstituted in rehydration buffer. First
dimension isoelectric focussing was performed using 7cm immobilised gradient
strips (pH 3-10), with separation using the IPGphor System. Second dimension
SDS-PAGE was performed using 8% acrylamide gels. Gels were stained with
Coomassie blue and protein expression was comparatively analysed by use of image
analysis software. Proteins were identified by in-gel trypsin digestion followed
by MALDI-TOF mass spectrometric analysis.
Results:
A complex array of proteins was apparent, with differential expression being
noted in the cultures supplemented with IL-1b. The changes between the two groups were most marked in the 65-116 kDa
range. The presence of IL-1b clearly influences the metabolic activity of human
gingival fibroblasts in vitro.
Conclusions:
The proteomic approach employed in this preliminary study demonstrated the value
of utilising this global approach to examining protein expression, and may
increase our understanding of the influence of IL-1b on tissues of the periodontium. ED was supported by
CED/IADR Visiting Scholar stipend
81st General Session of the International Association
for Dental Research (June 25-28, 2003)