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

doi:10.3969/j.issn.2095-4344.2016.45.020

Abstract

Abstract:

BACKGROUND: Cell labeling in combination with in vitro MRI imaging can be used to noninvasively label transplanted neural stem cells (NSCs), thereby exhibiting the existing site, existing way and some biological properties of the cells.
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.
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, followed by SPIO labeling. Afterwards, 4.7T MRI imaging was used to scan SPIO-labeled NSCs. Cell 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.
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 cells in the other three groups showed stronger proliferation ability, and exhibited a cell 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. Additionally, 4.7T MR is effective for in vitro imaging of labeled cells.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Neural Stem Cells, Telomerase, Organisms, Genetically Modified, Tissue Engineering

CLC Number:

R394.2

Dou Wen-guang, Yue Jun-yan, Hu Ying, Wu Qing-wu, Chen Jie. Neural stem cells modified by human telomerase reverse transcriptase gene: superparamagnetic iron oxide labeling and in vitro MRI imaging[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(45): 6821-6826.

Figures/Tables 3

2.1 骨髓源神经干细胞形态接种24 h内，倒置显微镜下可见细胞形态呈圆形，悬浮生长(图1A)；4-6 d后可见贴壁细胞明显增加，细胞形态由圆形逐渐变成梭形；两三次传代后，细胞形态趋向一致，大部分呈梭形，伴长轴突样纤维生长，选取第3代神经干细胞进行实验(图1B)。 2.2 普鲁士蓝染色观察神经干细胞的标记率标记后的细胞胞浆内有蓝染铁颗粒(图2)，未经标记的细胞胞浆内无蓝染颗粒存在，SPIO对神经干细胞的标记率为97%。 2.3 MR成像检测结果采用4.7 T磁共振成像，扫描序列包括T1WI、T2WI及T2* WI 3种，与未标记的神经干细胞相比，标记的神经干细胞在T2WI序列的信号强度平均下降50.66%，而在T2* WI序列上二者的信号强度相比平均下降53.70%，T2WI序列与T2* WI序列相比后者的信号强度变化明显。未标记神经干细胞和标记神经干细胞的T2弛豫时间分别为510 ms及76 ms，其弛豫率R2(1/T2)分别为1.94 s-1及12.98 s-1，T2*弛豫时间分别为109.0、22.9 ms，其弛豫率R2*(1/T2*)分别为9.17 s-1、43.67 s-1，由以上结果可见标记细胞的T2及T2*弛豫时间均显著下降，相应的弛豫率则显著升高。 2.4 各组神经干细胞的细胞周期与普通神经干细胞组相比较，SPIO标记3组神经干细胞处于G0-G1及S期的比例明显增多，处于G2-M期的比例明显减少，SPIO标记hTERT转染组更为显著(P < 0.05)，见表1。 2.5 各组神经干细胞的增殖能力细胞于传代后24 h内缓慢生长，48 h后生长迅速，120 h时细胞密度最高，结果表明SPIO标记的3组神经干细胞与正常神经干细胞相比较，增殖能力明显增强，差异有显著性意义(P < 0.05)，见表2。 2.6 各组神经干细胞hTERT mRNA表达 SPIO标记神经干细胞组、SPIO标记空载病毒组及SPIO标记hTERT转染组细胞的hTERT mRNA相对吸光度值分别为0.31±0.02，0.37±0.03，0.39±0.08。SPIO标记hTERT转染组hTERT mRNA的表达显著增强，见图3。 2.7 各组神经干细胞hTERT蛋白的表达 SPIO标记神经干细胞组、SPIO标记空载病毒组及SPIO标记hTERT转染组hTERT蛋白条带灰度分别为23%，25%，78%，各组间差异有显著性意义(P < 0.05)，见图4。"

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