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纸质出版:2014
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贺小娅,蔡金华,秦勇,高雅丽.磁共振报告基因FTH1慢病毒载体构建及其在人神经母细胞瘤细胞中的表达[J].陆军军医大学学报(原第三军医大学学报),2014,36(23):2338-2342.
He Xiaoya,Cai Jinhua,Qin Yong,Gao Yali.Construction of recombinant lentivirus vector carrying FTH1 as an endogenous MRI reporter gene and its expression in SK-N-SH cells[J].J Amry Med Univ (J Third Mil Med Univ),2014,36(23):2338-2342.
贺小娅,蔡金华,秦勇,高雅丽.磁共振报告基因FTH1慢病毒载体构建及其在人神经母细胞瘤细胞中的表达[J].陆军军医大学学报(原第三军医大学学报),2014,36(23):2338-2342. DOI:
He Xiaoya,Cai Jinhua,Qin Yong,Gao Yali.Construction of recombinant lentivirus vector carrying FTH1 as an endogenous MRI reporter gene and its expression in SK-N-SH cells[J].J Amry Med Univ (J Third Mil Med Univ),2014,36(23):2338-2342. DOI:
目的 构建携带人铁蛋白重链多肽1(fevritin heavy chaik 1
FTH1)基因的慢病毒载体,并检测其在人神经母细胞瘤细胞(SK-N-SH)中的表达。 方法 以慢病毒为载体,将FTH1基因转入SK-N-SH细胞中。实验组(SK-N-SH-FTH1)、空载组(SK-N-SH-GFP)和空白组(SK-N-SH),均用含500 μmol/L枸橼酸铁铵(FAC)的培养基培养并连续4 次传代。CCK-8试剂检测细胞增殖活性;普鲁士蓝染色及透射电镜检测细胞聚铁效能;MR成像(MRI)观察T
2
WI信号变化;Western blot检测FTH1表达情况。 结果 CCK-8试剂检测发现3组细胞未添加FAC培养时,其增殖能力无显著差异[实验组(0.99±0.03),空载组(0.99±0.02),空白组(0.98±0.05);P0.05
]
;然而,添加500 μmol/L FAC后,其增殖能力[实验组(0.73±0.08),空载组(0.76±0.02),空白组(0.73±0.39)
]
均较前明显减低(P0.05)。普鲁士蓝染色及透射电镜显示实验组细胞内聚集较多的铁颗粒;MR扫描显示在500 μmol/L FAC培养条件下,实验组T
2
WI信号显著降低;Western blot结果显示实验组FTH1表达,且经4次传代后仍持续稳定表达。 结论 成功构建携带FTH1基因的慢病毒载体,并证明FTH1在SK-N-SH细胞中长期稳定表达及聚铁能力。
Objective To construct a lentiviral vector carrying human fevritin heavy chaik 1 (FTH1) gene and observe its expression in neuroblastoma SK-N-SH cells. Methods The FTH1 gene was transferred into SK-N-SH cells by using a lentiviral vector. The cells in experimental group (SK-N-SH-FTH1)
mock group (SK-N-SH-GFP) and blank group (SK-N-SH) were cultured in
the medium containing 500 μmol/L ferric ammonium citrate (FAC) for 4 passages
respectively. The effect of FTH1 over-expression or FAC on cell viability was tested by CCK-8 assay. Prussian blue staining and transmission electron microscopy were used to determine iron accumulation in the cells. MR imaging was performed to determine the changes of T
2
WI signal intensity of cells in vitro. The expression of FTH1 gene was detected by Western blot analysis. Results CCK-8 assay showed there was no significant difference in cell viability among the 3 groups of cells cultured in the medium without FAC (SK-N-SH-FTH1: 0.99±0.03
SK-N-SH-GFP: 0.99±0.02
SK-N-SH: 0.98±0.05
P0.05). However
when the culture medium contained 500 μmol/L FAC for 48 h
the cell viability was significantly reduced (SK-N-SH-FTH1:0.73±0.08; SK-N-SH-GFP: 0.76±0.02; SK-N-SH: 0.73±0.39
P0.05). Prussian blue staining and transmission electron microscopy showed a great large amount of iron particles were accumulated in the experimental cells. Accordingly
T
2
WI signal intensity was significantly decreased in the experimental cells than the other 2 types of control cells. Western blot analysis confirmed that FTH1 was expressed continuously and stably in the experimental cells even at the 4th passage. Conclusion The recombinant lentivirus vector carrying FTH1 gene is constructed successfully
and its stable over-expression results in iron accumulation in SK-N-SH cells after infection.
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