Neural stem cells modified by human telomerase reverse transcriptase gene:superparamagnetic iron oxide labeling and in vitro MRI imaging

Dou Wen-guang; Yue Jun-yan; Hu Ying; Wu Qing-wu; Chen Jie

doi:10.3969/j.issn.2095-4344.2016.45.020

Abstract：BACKGROUND:Cel labeling in combination with in vitro MRI imaging can be used to noninvasively label transplanted neural stem cel s (NSCs), thereby exhibiting the existing site, existing way and some biological properties of the cel s. <br> OBJECTIVE:To investigate the effect of superparamagnetic iron oxide (SPIO) nanoparticles on the biological characteristics of NSCs modified by human telomerase reverse transcriptase (hTERT) gene and explore the changes of MRI after labeling in vitro. <br> METHODS:NSCs from rat bone marrow were cultured in vitro, and then were divided into normal NSCs group, control group (SPIO-labeled NSCs), negative control group (SPIO group) and hTERT transfection group (SPIO-labeled NSCs transfection group). Using electropration method, pcDNA3-hTERT recombinant plasmids were transfected into NSCs, fol owed by SPIO labeling. Afterwards, 4.7T MRI imaging was used to scan SPIO-labeled NSCs. Cel cycle and proliferation were detected using flow cytometry and MTT assay, respectively. Expression of hTERT at protein and gene levels was detected using RT-PCR and western blot. <br> RESULTS AND CONCLUSION:Prussian blue staining showed 97%of NSCs were labeled by SPIO. MRI results showed that compared with the normal NSCs group, T2 and T2*relaxation time was significantly declined in the control group, and the corresponding relaxation rate was significantly increased (P<0.01). Compared with the normal NSCs group, SPIO-labeled cel s in the other three groups showed stronger proliferation ability, and exhibited a cel cycle rest in G0-G1 and S stages. RT-PCR and western blot results showed that mRNA and protein expressions of hTERT were significantly higher in the hTERT transfection group than the control and negative control groups. These findings indicate that in vitro SPIO can efficiently label NSCs, and SPIO-labeled NSCs under hTERT modification can stably express hTERT gene and strengthen the proliferation ability. Additional y, 4.7T MR is effective for in vitro imaging of labeled cel s.