PEG3启动子启动铁蛋白报告基因在干细胞瘤变时特异表达及体外磁共振成像
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摘要
目的探讨利用肿瘤特异性启动子PEG3驱动MRI报告基因铁蛋白重链(ferritin heavy chain 1, FTH1)在干细胞瘤变时特异性表达并进行MRI检测的可行性。方法构建PEG3启动子控制FTH1表达的病毒载体PEG3-FTH1-LV,感染大鼠骨髓间充质干细胞(mesenchymal stem cells, MSCs),获得MSCs-PEG3-FTH1细胞,MSCs和MSCs-CMV-FTH1作为对照,通过与大鼠胶质瘤细胞C6间接共培养2周诱导其瘤变为TMSCs、TMSCs-PEG3-FTH1、TMSCs-CMV-FTH1。观察细胞形态,检测细胞迁移能力、细胞增殖速度及细胞表面抗体CD29、CD34、CD45、CD90的表达,裸鼠皮下成瘤实验验证细胞瘤变,Western blot、普鲁士蓝染色、细胞透射电镜和磁共振成像(MRI)检测细胞诱导前后FTH1基因的表达。结果 TMSCs细胞较MSCs细胞变小,多呈纺锤状,增殖速度加快,迁移能力增强,CD34、CD45表达增加,CD90表达减少(P<0.05),裸鼠皮下成瘤阳性;Western blot结果显示:MSCs、MSCs-PEG3-FTH1、TMSCs细胞不表达或少量表达FTH1蛋白,而MSCs-CMV-FTH1、TMSCs-PEG3-FTH1、TMSCs-CMV-FTH1细胞均大量表达FTH1蛋白(P<0.05);普鲁士蓝染色和细胞透射电镜观察到MSCs-CMV-FTH1、TMSCs-PEG3-FTH1、TMSCs-CMV-FTH1细胞内铁颗粒,而在MSCs、MSCs-PEG3-FTH1、TMSCs细胞内无明显铁颗粒,MRI显示TMSCs-PEG3-FTH1细胞信号较MSCs-PEG3-FTH1细胞信号明显降低。结论 PEG3启动子可驱动报告基因FTH1在干细胞瘤变时特异性表达并能被MRI检测。
Objective To explore the feasibility of using progression elevated gene-3(PEG3) promoter, a tumor-specific promoter, to drive the specific expression of the reporter gene ferritin heavy chain 1(FTH1) and the detection of FTH1 expression using in vitro magnetic resonance imaging(MRI) in bone marrow mesenchymal stem cells(MSCs) after malignant transformation. Methods A viral vector carrying the reporter gene FTH1 driven by PEG3 promoter was transfected into rat MSCs to obtain MSCs-PEG3-FTH1 cells, and the MSCs transfected with a viral vector carrying FTH1 driven by the CMV promoter(MSCs-CMV-FTH1 cells) and the MSCs without transfection served as the controls. The non-transfected MSCs, MSCs-PEG3-FTH1 cells, and MSCs-CMV-FTH1 cells were indirectly co-cultured with rat glioma C6 cells for 2 weeks to induce their malignant transformation(TMSCs, TMSCs-PEG3-FTH1 and TMSC-PEG3-FTH1 cells, respectively). The morphology, migration and proliferation, and the cell surface antigens CD29, CD34, CD45 and CD90 of the transformed cells were observed and analyzed, and their capacity of subcutaneous tumor formation was evaluated in nude mice. Western blotting, Prussian blue staining, transmission electron microscopy, and in vitro MRI were used to detect the changes in the expression of FTH1 gene in the cells after the transformation. Results Compared with MSCs, most of TMSCs were small in size with a spindle-like morphology and showed strong proliferation and migration abilities with increased expression of CD34 and CD45, decreased expression of CD90(P<0.05), and an ability of subcutaneous tumor formation in nude mice. Western blotting showed that FTH1 protein was not expressed or expressed at low levels in MSCs, MSCs-PEG3-FTH1 cells and TMSCs, while high FTH1 expression was detected in MSCs-CMV-FTH1, TMSCs-PEG3-FTH1 and TMSCs-CMV-FTH1 cells(P<0.05). Prussian blue staining and transmission electron microscopy identified the presence of iron particles in MSCs-CMV-FTH1, TMSCs-PEG3-FTH1 and TMSCs-CMV-FTH1 cells, but not in MSCs, MSCs-PEG3-FTH1 cells or TMSCs. In vitro MRI showed obviously decreased T_2 signal in TMSCs-PEG3-FTH1 cells as compared with MSCs-PEG3-FTH1 cells. Conclusion PEG3 promoter can drive the specific expression of FTH1 that allows MRI detection in rat MSCs after malignant transformation.
Objective To explore the feasibility of using progression elevated gene-3(PEG3) promoter, a tumor-specific promoter, to drive the specific expression of the reporter gene ferritin heavy chain 1(FTH1) and the detection of FTH1 expression using in vitro magnetic resonance imaging(MRI) in bone marrow mesenchymal stem cells(MSCs) after malignant transformation. Methods A viral vector carrying the reporter gene FTH1 driven by PEG3 promoter was transfected into rat MSCs to obtain MSCs-PEG3-FTH1 cells, and the MSCs transfected with a viral vector carrying FTH1 driven by the CMV promoter(MSCs-CMV-FTH1 cells) and the MSCs without transfection served as the controls. The non-transfected MSCs, MSCs-PEG3-FTH1 cells, and MSCs-CMV-FTH1 cells were indirectly co-cultured with rat glioma C6 cells for 2 weeks to induce their malignant transformation(TMSCs, TMSCs-PEG3-FTH1 and TMSC-PEG3-FTH1 cells, respectively). The morphology, migration and proliferation, and the cell surface antigens CD29, CD34, CD45 and CD90 of the transformed cells were observed and analyzed, and their capacity of subcutaneous tumor formation was evaluated in nude mice. Western blotting, Prussian blue staining, transmission electron microscopy, and in vitro MRI were used to detect the changes in the expression of FTH1 gene in the cells after the transformation. Results Compared with MSCs, most of TMSCs were small in size with a spindle-like morphology and showed strong proliferation and migration abilities with increased expression of CD34 and CD45, decreased expression of CD90(P<0.05), and an ability of subcutaneous tumor formation in nude mice. Western blotting showed that FTH1 protein was not expressed or expressed at low levels in MSCs, MSCs-PEG3-FTH1 cells and TMSCs, while high FTH1 expression was detected in MSCs-CMV-FTH1, TMSCs-PEG3-FTH1 and TMSCs-CMV-FTH1 cells(P<0.05). Prussian blue staining and transmission electron microscopy identified the presence of iron particles in MSCs-CMV-FTH1, TMSCs-PEG3-FTH1 and TMSCs-CMV-FTH1 cells, but not in MSCs, MSCs-PEG3-FTH1 cells or TMSCs. In vitro MRI showed obviously decreased T_2 signal in TMSCs-PEG3-FTH1 cells as compared with MSCs-PEG3-FTH1 cells. Conclusion PEG3 promoter can drive the specific expression of FTH1 that allows MRI detection in rat MSCs after malignant transformation.
引文
[1] LITWINOWICZ R,KAPELAK B,SADOWSKI J,et al.The use of stem cells in ischemic heart disease treatment[J].Kardiochir Torakochirurgia Pol,2018,15(3):196-199.DOI:10.5114/kitp.2018.78446.
[2] B?OGOWSKI W,BODNARCZUK T,STARZY?SKA T.Concise review:pancreatic cancer and bone marrow-derived stem cells[J].Stem Cells Transl Med,2016,5(7):938-945.DOI:10.5966/sctm.2015-0291.
[3] KATSUKAWA M,NAKAJIMA Y,FUKUMOTO A,et al.Fail-safe therapy by gamma-ray irradiation against tumor formation by human-induced pluripotent stem cell-derived neural progenitors[J].Stem Cells Dev,2016,25(11):815-825.DOI:10.1089/scd.2015.0394.
[4] CLéMENT F,GROCKOWIAK E,ZYLBERSZTEJN F,et al.Stem cell manipulation,gene therapy and the risk of cancer stem cell emergence[J].Stem Cell Invest,2017,4:67.DOI:10.21037/sci.2017.07.03.
[5] SU Z Z,SARKAR D,EMDAD L,et al.Targeting gene expression selectively in cancer cells by using the progression-elevated gene-3 promoter[J].Proc Natl Acad Sci U S A,2005,102(4):1059-1064.DOI:10.1073/pnas.0409141102.
[6] 刘慧娟,胡若愚,戴王娟,等.绿色荧光蛋白转基因大鼠骨髓间充质干细胞培养及鉴定[J].中国医学科学院学报,2016,38(1) :9-15.DOI:10.3881/j.issn.1000-503X.2016.01.002.LIU H J,HU R Y,DAI W J,et al.Culture and identification of bone marrow mesenchymal stem cells from enhanced green fluorescent protein-transgenic rats[J].Acta Acad Med Sin,2016,38(1):9-15.DOI:10.3881/j.issn.1000-503X.2016.01.002.
[7] LI X Y,ZHANG Y,QI G X.Evaluation of isolation methods and culture conditions for rat bone marrow mesenchymal stem cells[J].Cytotechnology,2013,65(3):323-334.DOI:10.1007/s10616-012-9497-3.
[8] 廖一凡,蔡金华,钟毅,等.NSE启动子在骨髓间充质干细胞成神经分化中启动FTH1基因特异性表达及体外磁共振成像[J].第三军医大学学报,2018,40(13):1171-1178.DOI:10.16016/j.1000-5404.201712135.LIAO Y F,CAI J H,ZHONG Y,et al.Specific expression and in vitro magnetic resonance imaging of NSE-promoter in initiating FTH1 gene during neural differentiation of bone marrow mesenchymal stem cells[J].J Third Mil Med Univ,2018,40(13):1171-1178.DOI:10.16016/j.1000-5404.201712135.
[9] BHANG H E,GABRIELSON K L,LATERRA J,et al.Tumor-specific imaging through progression elevated gene-3 promoter-driven gene expression[J].Nat Med,2011,17(1):123-129.DOI:10.1038/nm.2269.
[10] CHAN I,LEBEDEVA I V,SU Z Z,et al.Progression elevated gene-3 promoter (PEG-Prom) confers cancer cell selectivity to human polynucleotide phosphorylase (hPNPase(old-35))-mediated growth suppression[J].J Cell Physiol,2008,215(2):401-409.DOI:10.1002/jcp.21320.
[11] SONG K,HUANG M Q,SHI Q,et al.Cultivation and identification of rat bone marrow-derived mesenchymal stem cells[J].Mol Med Rep,2014,10(2):755-760.DOI:10.3892/mmr.2014.2264.
[12] TAN B,SHEN L J,YANG K,et al.C6 glioma-conditioned medium induces malignant transformation of mesenchymal stem cells:Possible role of S100B/RAGE pathway[J].Biochem Biophys Res Commun,2018,495(1):78-85.DOI:10.1016/j.bbrc.2017.10.071.
[13] CARETTI V,SEWING A C,LAGERWEIJ T,et al.Human pontine glioma cells can induce murine tumors[J].Acta Neuropathol,2014,127(6):897-909.DOI:10.1007/s00401-014-1272-4.
[14] KONG X Y,GUAN J,MA W B,et al.CD34 over-expression is associated with gliomas’ higher WHO grade[J].Medicine (Baltimore),2016,95(7):e2830.DOI:10.1097/MD.0000000000002830.
[15] Transcriptional gene silencing limits CXCR4-associated depletion of bone marrow CD34+ cells in HIV-1 infection:Erratum[J].AIDS,2018,32(18):2857-2858.DOI:10.1097/01.aids.0000547984.16868.93.
[16] MILDMAY-WHITE A,KHAN W.Cell surface markers on adipose-derived stem cells:a systematic review[J].Curr Stem Cell Res Ther,2017,12(6):484-492.DOI:10.2174/1574888X11666160429122133.
[17] KOBOLAK J,DINNYES A,MEMIC A,et al.Mesenchymal stem cells:Identification,phenotypic characterization,biological properties and potential for regenerative medicine through biomaterial micro-engineering of their niche[J].Methods,2016,99:62-68.DOI:10.1016/j.ymeth.2015.09.016.
[18] AVRIL T,ETCHEVERRY A,PINEAU R,et al.CD90 expression controls migration and predicts dasatinib response in glioblastoma[J].Clin Cancer Res,2017,23(23):7360-7374.DOI:10.1158/1078-0432.CCR-17-1549.
[19] SUKOWATI C H,ANFUSO B,TORRE G,et al.The expression of CD90/Thy-1 in hepatocellular carcinoma:an in vivo and in vitro study[J].PLoS ONE,2013,8(10):e76830.DOI:10.1371/journal.pone.0076830.
[20] MORAES D A,SIBOV T T,PAVON L F,et al.A reduction in CD90 (THY-1) expression results in increased differentiation of mesenchymal stromal cells[J].Stem Cell Res Ther,2016,7(1):97.DOI:10.1186/s13287-016-0359-3.
[2] B?OGOWSKI W,BODNARCZUK T,STARZY?SKA T.Concise review:pancreatic cancer and bone marrow-derived stem cells[J].Stem Cells Transl Med,2016,5(7):938-945.DOI:10.5966/sctm.2015-0291.
[3] KATSUKAWA M,NAKAJIMA Y,FUKUMOTO A,et al.Fail-safe therapy by gamma-ray irradiation against tumor formation by human-induced pluripotent stem cell-derived neural progenitors[J].Stem Cells Dev,2016,25(11):815-825.DOI:10.1089/scd.2015.0394.
[4] CLéMENT F,GROCKOWIAK E,ZYLBERSZTEJN F,et al.Stem cell manipulation,gene therapy and the risk of cancer stem cell emergence[J].Stem Cell Invest,2017,4:67.DOI:10.21037/sci.2017.07.03.
[5] SU Z Z,SARKAR D,EMDAD L,et al.Targeting gene expression selectively in cancer cells by using the progression-elevated gene-3 promoter[J].Proc Natl Acad Sci U S A,2005,102(4):1059-1064.DOI:10.1073/pnas.0409141102.
[6] 刘慧娟,胡若愚,戴王娟,等.绿色荧光蛋白转基因大鼠骨髓间充质干细胞培养及鉴定[J].中国医学科学院学报,2016,38(1) :9-15.DOI:10.3881/j.issn.1000-503X.2016.01.002.LIU H J,HU R Y,DAI W J,et al.Culture and identification of bone marrow mesenchymal stem cells from enhanced green fluorescent protein-transgenic rats[J].Acta Acad Med Sin,2016,38(1):9-15.DOI:10.3881/j.issn.1000-503X.2016.01.002.
[7] LI X Y,ZHANG Y,QI G X.Evaluation of isolation methods and culture conditions for rat bone marrow mesenchymal stem cells[J].Cytotechnology,2013,65(3):323-334.DOI:10.1007/s10616-012-9497-3.
[8] 廖一凡,蔡金华,钟毅,等.NSE启动子在骨髓间充质干细胞成神经分化中启动FTH1基因特异性表达及体外磁共振成像[J].第三军医大学学报,2018,40(13):1171-1178.DOI:10.16016/j.1000-5404.201712135.LIAO Y F,CAI J H,ZHONG Y,et al.Specific expression and in vitro magnetic resonance imaging of NSE-promoter in initiating FTH1 gene during neural differentiation of bone marrow mesenchymal stem cells[J].J Third Mil Med Univ,2018,40(13):1171-1178.DOI:10.16016/j.1000-5404.201712135.
[9] BHANG H E,GABRIELSON K L,LATERRA J,et al.Tumor-specific imaging through progression elevated gene-3 promoter-driven gene expression[J].Nat Med,2011,17(1):123-129.DOI:10.1038/nm.2269.
[10] CHAN I,LEBEDEVA I V,SU Z Z,et al.Progression elevated gene-3 promoter (PEG-Prom) confers cancer cell selectivity to human polynucleotide phosphorylase (hPNPase(old-35))-mediated growth suppression[J].J Cell Physiol,2008,215(2):401-409.DOI:10.1002/jcp.21320.
[11] SONG K,HUANG M Q,SHI Q,et al.Cultivation and identification of rat bone marrow-derived mesenchymal stem cells[J].Mol Med Rep,2014,10(2):755-760.DOI:10.3892/mmr.2014.2264.
[12] TAN B,SHEN L J,YANG K,et al.C6 glioma-conditioned medium induces malignant transformation of mesenchymal stem cells:Possible role of S100B/RAGE pathway[J].Biochem Biophys Res Commun,2018,495(1):78-85.DOI:10.1016/j.bbrc.2017.10.071.
[13] CARETTI V,SEWING A C,LAGERWEIJ T,et al.Human pontine glioma cells can induce murine tumors[J].Acta Neuropathol,2014,127(6):897-909.DOI:10.1007/s00401-014-1272-4.
[14] KONG X Y,GUAN J,MA W B,et al.CD34 over-expression is associated with gliomas’ higher WHO grade[J].Medicine (Baltimore),2016,95(7):e2830.DOI:10.1097/MD.0000000000002830.
[15] Transcriptional gene silencing limits CXCR4-associated depletion of bone marrow CD34+ cells in HIV-1 infection:Erratum[J].AIDS,2018,32(18):2857-2858.DOI:10.1097/01.aids.0000547984.16868.93.
[16] MILDMAY-WHITE A,KHAN W.Cell surface markers on adipose-derived stem cells:a systematic review[J].Curr Stem Cell Res Ther,2017,12(6):484-492.DOI:10.2174/1574888X11666160429122133.
[17] KOBOLAK J,DINNYES A,MEMIC A,et al.Mesenchymal stem cells:Identification,phenotypic characterization,biological properties and potential for regenerative medicine through biomaterial micro-engineering of their niche[J].Methods,2016,99:62-68.DOI:10.1016/j.ymeth.2015.09.016.
[18] AVRIL T,ETCHEVERRY A,PINEAU R,et al.CD90 expression controls migration and predicts dasatinib response in glioblastoma[J].Clin Cancer Res,2017,23(23):7360-7374.DOI:10.1158/1078-0432.CCR-17-1549.
[19] SUKOWATI C H,ANFUSO B,TORRE G,et al.The expression of CD90/Thy-1 in hepatocellular carcinoma:an in vivo and in vitro study[J].PLoS ONE,2013,8(10):e76830.DOI:10.1371/journal.pone.0076830.
[20] MORAES D A,SIBOV T T,PAVON L F,et al.A reduction in CD90 (THY-1) expression results in increased differentiation of mesenchymal stromal cells[J].Stem Cell Res Ther,2016,7(1):97.DOI:10.1186/s13287-016-0359-3.