Biomedical Engineering Reference
In-Depth Information
21.2.6
Establishment of GDF-15-silenced cell lines
To investigate the effect of GDF-15 in radiation response we tried to silence the
GDF-15 gene in immortalized human fibroblast cells using the MISSION Lentiviral
Transduction system bought from SIGMA (Sigma, St Louis, MO, USA). The
system contains five different shRNA for GDF-15 gene. Immortalized F11-hTERT
human fibroblasts were transduced with the lentiviral vectors and stable clones were
selected on the base of their puromycin resistance. Expression of GDF-15 mRNA
was measured by quantitative PCR. We named the stable shGDF-15 expressing F11-
hTERT cells as “shGDF-15”-: #1, #2, #3 and #4. The stable transfected F11-hTERT
cells were propagated in the presence of puromycin
.0:75
g
=
ml
/
.
21.2.7
Data analysis
Except where otherwise specified, results are shown as mean and standard deviation
of three separate experiments and P values are calculated using paired Student's
Ttest.
One-way analysis of variance and two-tailed t tests were used to compare
differences among groups. P
<0:05
was considered statistically significant. Data
were presented as means
˙
SE.
21.3
Results
21.3.1
Differentially expressed genes following radiation
exposure
Three hundred sixty six differentially expressed genes were identified after 0.1 Gy
gamma radiation in F11 primary fibroblast cells [
15
]. Of these 366 genes, 347 genes
were induced, and 19 were repressed. Five hundred two differentially expressed
genes were found after irradiating the cells by 0.5 Gy gamma radiation [
16
]. Of
these, 450 genes were up-regulated, and 52 genes were down-regulated (Table
21.2
).
Remarkably, 126 of differentially expressed genes responded both after 0.1 and
0.5 Gy exposures (Fig.
21.1
).
To elucidate the involvement of specific biological processes in cellular re-
sponses to low dose exposures at the transcriptional level, the gene ontology
(GO) annotations of the genes were performed. The results of GO analysis of
the differentially expressed genes are summarized in Table
21.2
. Several genes
encoding the subunits of ribosomal complex were up-regulated in F11 cells
both after 0.1 and 0.5 Gy radiations. Other significantly up-regulated biological
