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
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摘要
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-SHFTH1)、空载组(SK-N-SH-GFP)和空白组(SK-N-SH),均用含500μmol/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T2WI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);P>0.05];然而,添加500μmol/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(P<0.05)。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500μmol/L FAC培养条件下,实验组T2WI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 T2 WI signal intensity of cells in vitro. The expression of FTH1 gene was detected by Western blot analysis. Results CCK-8assay 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,P > 0. 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,P < 0. 05). Prussian blue staining and transmission electron microscopy showed a great large amount of iron particles were accumulated in the experimental cells. Accordingly,T2 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.
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 T2 WI signal intensity of cells in vitro. The expression of FTH1 gene was detected by Western blot analysis. Results CCK-8assay 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,P > 0. 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,P < 0. 05). Prussian blue staining and transmission electron microscopy showed a great large amount of iron particles were accumulated in the experimental cells. Accordingly,T2 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.
引文
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[12]Cozzi A,Corsi B,Levi S,et al.Overexpression of wild type and mutated human ferritin H-chain in Hela cells:in vivo role of ferritin ferroxidase activity[J].J Biol Chem,2000,275(33):25122-25129.
[13]Liu J,Cheng E C,Long R C,et al.Noninvasive monitoring of embryonic stem cells in vivo with MRI transgene reporter[J].Tissue Eng Part C Methods,2009,15(4):739-747.
[2]Vandsburger M H,Radoul M,Cohen B,et al.MRI reporter genes:applications for imaging of cell survival,proliferation,migration and differentiation[J].NMR Biomed,2013,26(7):872-884.
[3]Naumova A V,Reinecke H,Yarnykh V,et al.Ferritin overexpression for noninvasive magnetic resonance imaging-based tracking of stem cells transplanted into the heart[J].Mol Imaging,2010,9(4):201-210.
[4]Kim H S,Joo H J,Woo J S,et al.In vivo magnetic resonance imaging of transgenic mice expressing human ferritin[J].Mol Imaging Biol,2013,15(1):48-57.
[5]Hasegawa S,Furukawa T,Saga T.Molecular MR imaging of cancer gene therapy:ferritin transgene reporter takes the stage[J].Magn Reson Med Sci,2010,9(2):37-47.
[6]Kim H S,Cho H R,Choi S H,et al.In vivo imaging of tumor transduced with bimodal lentiviral vector encoding human ferritin and green fluorescent protein on a 1.5T clinical magnetic resonance scanner[J].Cancer Res,2010,70(18):7315-7324.
[7]Kim D,Chung N K,Allen S,et al.Ferritin-based new magnetic force microscopic probe detecting 10 nm sized magnetic nanoparticles[J].ACS Nano,2012,6(1):241-248.
[8]Mc Laughlin R,Hylton N.MRI in breast cancer therapy monitoring[J].NMR Biomed,2011,24(6):712-720.
[9]Lee S W,Lee S H,Biswal S.Magnetic resonance reporter gene imaging[J].Theranostics,2012,2(4):403-412.
[10]Ziv K,Meir G,Harmelin A,et al.Ferritin as a reporter gene for MRI:chronic liver over expression of H-ferritin during dietary iron supplementation and aging[J].NMR Biomed,2010,23(5):523-531.
[11]Oshtrakh M I,Alenkina I V,Vinogradov A V,et al.Mossbauer spectroscopy of the iron cores in human liver ferritin,ferritin in normal human spleen and ferritin in spleen from patient with primary myelofibrosis:preliminary results of comparative analysis[J].Biometals,2013,26(2):229-239.
[12]Cozzi A,Corsi B,Levi S,et al.Overexpression of wild type and mutated human ferritin H-chain in Hela cells:in vivo role of ferritin ferroxidase activity[J].J Biol Chem,2000,275(33):25122-25129.
[13]Liu J,Cheng E C,Long R C,et al.Noninvasive monitoring of embryonic stem cells in vivo with MRI transgene reporter[J].Tissue Eng Part C Methods,2009,15(4):739-747.