10 research outputs found
Adenoviral vector mediated ferritin over-expression in mesenchymal stem cells detected by 7T MRI in vitro
<div><p>Objective</p><p>The aim of the present work was to verify whether adenoviral vector mediated ferritin over-expression in mesenchymal stem cells could be detected by 7T MRI device, and to explore the relationship between ferritin content and MRI signal intensities.</p><p>Methods</p><p>A recombined adenoviral vector (rAdV) encoding ferritin heavy chain (FTH1) subunit was specially designed for the aim of infecting bone marrow mesenchymal stem cells (BMSCs). Ferritin over-expression in BMSCs was determined by cell immunocytochemistry and the ferritin content in cells was determined by ELISA assay. BMSCs were subjected to cell viability, proliferation and multi-differentiation analyses as well as 7T MRI test using fast spin-echo pulse sequence. The R2 value andδR2 were calculated according to T2 mapping images.</p><p>Results</p><p>As was confirmed by cell immunocytochemistry and ELISA assay, rAdV mediated ferritin was over-expressed in BMSCs. Ferritin over-expression did not interfere with stem cell viability or pluripotent differentiation but slowed cell proliferation. The R2 value of BMSCs<sub>-FTH1</sub> vs control BMSCs from 1–4 weeks was16.65±1.28 s<sup>-1</sup> vs 13.99±0.80 s<sup>-1</sup>, (t = 3.94, p = 0.004), 15.63±1.37 s<sup>-1</sup> vs 13.87±0.83 s<sup>-1</sup> (t = 2.47, p = 0.039), 15.53±0.88 s<sup>-1</sup> vs 14.25±0.53 s<sup>-1</sup> (t = 2.80, p = 0.023) and 14.61±1.28 s<sup>-1</sup> vs 13.69±1.03 s<sup>-1</sup> (t = 1.25, p = 0.24), respectively. δR2 gradually decreased from 1–4 weeks and the difference between the groups had statistical significance (F = 12.45, p<0.01).δR2 was positively correlated with OD value (r = 0.876, p<0.01) and ferritin concentration (r = 0.899, p<0.01) as determined by Pearson correlation.</p><p>Conclusions</p><p>Our study confirms that ferritin could be over-expressed in BMSCs as a result of rAdV mediated infection and could be quantitatively detected by 7T MRI device. The differences in T2 signal intensities and R2 values stem from internal contrast generated by endogenous ferritin over-expression. The correlation between δR2, OD and ferritin concentration suggests that MRI can detect ferritin signal change accurately.</p></div
Correlation of δR2 and OD value.
<p>The change in δR2 and OD value was positively correlated, with statistical significance (r = 0.876, p<0.01).</p
Correlation ofδR2 and ferritin concentration.
<p>The change in δR2 and ferritin concentration was positively correlated, with statistical significance (r = 0.899, p<0.01).</p
Correlation of δR2 and OD value.
<p>The change in δR2 and OD value was positively correlated, with statistical significance (r = 0.876, p<0.01).</p
Multilineage differentiation and ELLSA assay.
<p>(A) BMSCs<sub>-FTH1</sub> induced osteogenic differentiation formed alkaline phosphatase-positive aggregated nodules. (B) BMSCs<sub>-FTH1</sub> induced adipogenic differentiation showed positive Oil red O stained. (C) BMSCs<sub>-FTH1</sub> were positive to anti-rat ferritin heavy chain polyclonal antibody and stained yellow to dark brown, (D) Control BMSCs were negative stained to blue color.</p
MRI results of BMSCs<sub>-FTH1</sub> and BMSCs on 7T MRI device.
<p>(A) As revealed by the T2-mapping images of BMSCs<sub>-FTH1</sub> and control BMSCs acquired by 7T MRI, the signal intensity of BMSCs<sub>-FTH1</sub> (left) was darker than that of BMSCs (right).</p
The OD value, ferritin concentration (ng/ml), R2 value andδR2 at different time points.
<p>The OD value, ferritin concentration (ng/ml), R2 value andδR2 at different time points.</p
GFP gene expression in BMSCs confirmed by fluorescence microscope.
<p>(A-D) 24h, 48h, 72h and 96h after rAdV-FTH1 over-expressing virus (MOI: 150) infect BMSCs. GFP expression reached the peak in 72 hours as detected by fluorescence microscope.</p
The trend of OD, ferritin concentration and δR2.
<p>OD value, ferritin concentration andδR2 gradually decreased during 4 weeks’ observation.</p
Growth curve of BMSCs<sub>-FTH1</sub> and control BMSCs.
<p>Cell growth curve showed no difference between BMCs<sub>-FTH1</sub> and control BMSCs in the first 3 days but BMCs<sub>-FTH1</sub>slowed down in the last 4 days.</p