Læknablaðið : fylgirit - 03.01.2017, Blaðsíða 69

X V I I I V Í S I N D A R Á Ð S T E F N A H Í
F Y L G I R I T 9 1
LÆKNAblaðið/Fylgirit 91 2017/103 69
ing, NIPT and Rhesus genotyping. cfDNA holds promise in detecting
acquired somatic changes in cancer. We used a new technique, Northern
Lights Assay (NLA) to further investigate structure of cfDNA in body
fluids in healthy subjects.
Materials and Methods: NLA is based on Two-Dimensional Strand-
ness-Dependent Electrophoresis (2D-SDE), a technique of nucleic acid
separation based on size, strandness, and conformation changes induced
by damage. Each specimen is analyzed in sample pairs of non-digested
DNA to detect single- and double-stranded breaks and MboI-digested
DNA to detect various other lesions. NLA is run in microgel to improve
sensitivity and speed. We tested NLA on cfDNA isolated with gentle
methods from whole blood, plasma, saliva, urine sediment and cell-free
urine in healthy controls (7 males and 8 females) age 21 to 80.
Results: Yield from clinical volume samples was sufficient for sensitive
analysis with NLA. The cfDNA was variable within and between subjects,
but generally extensive damage was detected in various body fluids. cf-
DNA in plasma ranged from at least 3 kb fragments to a substantial fract-
ion of smaller DNA molecules representing apoptosis DNA fragments.
Conclusions: The findings have implications for cfDNA assays. Consistent
differences in rates of apoptosis in healthy subjects seem unlikely. A more
plausible explanation is time coordinated pulse release of cfDNA frag-
ments from apoptotic cells. This pulse would have previously gone undet-
ected using protein markers of cell death with half life of hours in plasma
instead of minutes for cfDNA.
V 23 Mesenchymal stem cells cultured on chitosan scaffold grafted
onto glass substrate
Ólafur E. Sigurjónsson1, Már Másson2, Adrianna Milewska3, Magda Ostrowska3, Jan
F. Biernat3
1Blood Bank, Landspitali-The National Universtiy Hospital of Iceland / Reykjavik University,
2Faculty of Pharamceutical Sciences, University of Iceland, 3Gdansk University of Technology
oes@landspitali.is
Introduction: One of the major problems that accompany implant sur-
gery, e.g. hip replacement and dental implant surgery is loosening of
the implant due to failure in osseointegration of the surrounding tissue
with the surface of the implant. To fix this, surfaces of implants have been
coated with various biomaterials to increase osseointegration and reduce
loosening of the implant. One such biomaterial is chitosan. In this project
we attached chitosan to glass surfaces using silylating reagents and ana-
lysed the bioactivity using mesenchymal stem cells and osteogenic differ-
entiation.
Methods: Glass surface was treated with silylating reagents, 3-amin-
opropyltrimethoxysilane (aminopropyl) and 3-glycidoxypropyltri-
ethoxysilane (glicydoxypropyl) dissolved in dry toluene.
The degree of modification was established spectrophotometrically us-
ing ninhydrin-hydrindantin buffered reagent. Mesenchymal stem cells
(MSCs) were cultured on the surface and differentiated for 14 days and
analyzed for morphology (Crystal violate blue and SEM) and Alkaline
Phosphatase activity.
Results: Osteogenic cells cultured on aminopropyl-chitosan glass plates
exhibited a slightly different morphology compared to cells grown on
glicydoxypropyl-chitosan glass plates showing ability to occur in a hig-
her number of cells per area. During osteogenic differentiation MSCs lost
their fibroblast-like morphology and became more cuboidal, indicating
successful osteogenesis as well as showing increase in ALP activity.
Conclusion: This pilot study indicates that glicydoxypropyl-chitosan and
aminopropyl-chitosan coated glass plates may serve as a coating material
for suregery implanst. More analysis is needed to confirm that.
V 24 The role of microRNA in TCEA1 expression
Linda H. Sighvatsdóttir1,2,3, Stefán Þ. Sigurðsson2,3
1Department of Biochemistry and Molecular Biology, UI, 2Faculty of Medicine, UI,
3Læknagarður
lhs4@hi.is
Introduction: Gene expression by RNA polymerase II (RNAPII) is not
exclusively regulated at the initiation step but also during the elongation
phase of transcription. Specific transcription factors such as TCEA1 en-
hance transcription elongation by reactivating paused or stalled RNAPII,
allowing transcription to proceed. Gene expression can also be regulated
by microRNAs by their binding to the 3’ untranslated (3´UTR) region of
target mRNA. This binding of the microRNA to the 3´UTR of the mRNA
results either in downregulation of protein translation or cleavage of the
mRNA target. Our studies are aimed at studying the role of microRNAs
in the regulation of TCEA1 expression.
Methods and materials: The 3´ UTR region of TCEA1 was cloned down-
stream of a luciferase reporter. This reporter plasmid was co-transfected
with different microRNA all found to have conserved binding sites in the
3´ UTR of TCEA1 based on microRNA target databases. Endogeneous
TCEA1 expression levels where measured by using highly specific TCEA1
antibody. Survival assay and wound healing assays have also been pre-
formed, for better understanding of the biological role these microRNA
have on TCEA1 function.
Result: Our data shows that TCEA1 expression is regulated by at least two
microRNAs. This regulation is seen throughout different experiments.
Conclusion: The microRNA family affecting TCEA1 expression is
frequently found to be downregulated in various types of cancer.
This raises a question regarding the role of TCEA1 in cancerous tissue,
specifically the importance of efficient transcription elongation and gene
expression.
V 25 Mitf gene mutations affect autophagy in mouse primary RPE
cells
Andrea García-Llorca1,2, Margrét H. Ögmundsdóttir3, Eiríkur Steingrímsson3, Þór
Eysteinsson3
1Physiology, 2Faculty of Medicine, 3Biochemistry and Molecular Biology, University of Iceland
agl7@hi.is
Introduction: Microphthalmia-associated transcription factor (MITF) reg-
ulates the differentiation and development of the retinal pigment epitheli-
um (RPE). The MITF protein is a member of the MYC supergene family of
basic-helix-loop-helix-leucine-zipper (bHLHZip) transcription factors and
is known to regulate the expression of cell-specific target genes by binding
DNA as a homodimer or as heterodimer with related proteins. The pur-
pose of this study was to examine if the Mitf gene plays a fundamental role
in regulating autophagy in primary RPE cells using various mutations in
the Mitf gene.
Methods: Primary RPE cells from wild type and MITF mutant mice (Mitf
mi-enu122(398), Mitf mi-wh/+ and Mitf mi-wh/Mitf mi-mi) were isolated
by enzymatic dissociation. Expression of LC3 and MITF was analyzed
with western blot analysis and confocal microscopy in primary RPE cells
from C57BL/6J mice, untreated or treated with the mTOR inhibitor Torin1
and starvation media. The levels of LC3 and MITF were measured and
compared by western blot in RPE cells from wild type and the Mitf mutant
mice.