摘要
背景:细胞标记与体外MRI成像联合,可无创性活体标记移植的神经干细胞存在部位、存在方式及其一些生物学特性。目的:探讨超顺磁氧化铁纳米颗粒(SPIO)标记对人端粒酶反转录酶(hT ERT)基因修饰神经干细胞生物学特性的影响及标记后MR成像效果。方法:体外培养大鼠骨髓来源的神经干细胞,将其分为正常神经干细胞组、SPIO标记神经干细胞组、SPIO标记空载病毒组及SPIO标记hT ERT转染组。采用电穿孔法将pcD NA3-hT ERT重组质粒转染至神经干细胞,并进行SPIO标记,当神经干细胞标记成功后即行4.7T MR扫描,流式细胞仪、MTT法检测细胞周期、细胞增殖能力,RT-PCR和Western blot检测hT ERT基因和蛋白的表达。结果与结论:(1)普鲁士蓝染色显示SPIO对神经干细胞的标记率为97%;(2)MRI成像显示,与正常神经干细胞组相比,SPIO标记神经干细胞组的T2及T2*弛豫时间均显著下降,相应的弛豫率则显著升高(P<0.01);(3)与正常神经干细胞比较,SPIO标记的3组神经干细胞增殖能力相比明显增强,处于G0-G1及S期的比例明显增多;(4)与SPIO标记神经干细胞组、SPIO标记空载病毒组比较,SPIO标记hT ERT转染组hT ERT mR NA及蛋白的表达均明显增强;(5)结果表明,在体外超顺磁性氧化铁能够高效标记神经干细胞,经hT ERT基因修饰后能够稳定表达h TERT基因,细胞增殖能力显著增强,4.7T MR仪能够对标记后的细胞有效进行体外成像。
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. 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, 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. 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.
出处
《中国组织工程研究》
CAS
北大核心
2016年第45期6821-6826,共6页
Chinese Journal of Tissue Engineering Research
