干扰素β和γ基因修饰的人羊水间充质干细胞的制备
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摘要
目的利用慢病毒转染的方法将人干扰素β基因(hIFN-β)、人干扰素γ基因(hIFN-γ)分别导入人羊水来源的间充质干细胞(AFMSCs),以期获得稳定表达hIFN-β、hIFN-γ的AFMSCs。方法产前诊断获得羊水标本,培养出AFMSCs,对其增殖能力、细胞表面分子和体外分化能力进行鉴定。用携带hIFN-β、hIFN-γ基因的重组慢病毒颗粒感染AFMSCs,并用Elisa法对培养基上清中hIFN-β、hIFN-γ的分泌情况进行鉴定。结果人羊水间充质干细胞呈成纤维样形态,不表达CD34,高表达CD44、CD105和HLA-ABC,能稳定传代培养,具有成骨、成脂分化能力。ELISA结果显示hIFN-β、hIFN-γ修饰的AFMSCs能稳定表达hIFN-β、hIFN-γ,其表达量分别达1 830 pg/m L和65.33 pg/m L。结论利用慢病毒转染的方法成功制备了hIFN-β、hIFN-γ修饰的人羊水间充质干细胞。
Objective To obtain human amniotic fluid derived mesenchymal stem cells(AFMSCs)withstable expression of hIFN-β and hIFN-γ. Methods AFMSCs were infected with recombinant lentiviral particlescarrying hIFN-β or hIFN-γ gene. HIFN-β and hIFN-γ concentration in the culture medium was identified by ELISA.Results AFMSCs were fibroblast-like cells with no expression of CD34,but high expression of CD44,CD105 and HLA-ABC,and the cells had the ability of osteogenic and adipogenic differentiation. ELISA results showedthat the levels of hIFN-β or hIFN-γ in AFMSCs were 1830 pg/ml and 65.33 pg/ml,respectively. Conclusion ThehIFN-β and hIFN-γ modified AFMSCs were successfully obtained by lentiviral transfection.
Objective To obtain human amniotic fluid derived mesenchymal stem cells(AFMSCs)withstable expression of hIFN-β and hIFN-γ. Methods AFMSCs were infected with recombinant lentiviral particlescarrying hIFN-β or hIFN-γ gene. HIFN-β and hIFN-γ concentration in the culture medium was identified by ELISA.Results AFMSCs were fibroblast-like cells with no expression of CD34,but high expression of CD44,CD105 and HLA-ABC,and the cells had the ability of osteogenic and adipogenic differentiation. ELISA results showedthat the levels of hIFN-β or hIFN-γ in AFMSCs were 1830 pg/ml and 65.33 pg/ml,respectively. Conclusion ThehIFN-β and hIFN-γ modified AFMSCs were successfully obtained by lentiviral transfection.
引文
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[2]JOHNSON V,WEBB T,NORMAN A,et al.Activated mesen-chymal stem cells interact with antibiotics and host innate immune responses to control chronic bacterial infections[J].SciRep,2017,7(1):9575.
[3]KANTOR B,BAILEY R M,WIMBERLY K,et al.Methodsfor gene transfer to the central nervous system.Advances in ge-netics[J].2014,87:125-197.
[4]金潇,周松,胡建敏,等.间充质干细胞在肾脏移植中的应用研究进展[J].实用医学杂志,2018,34(3):499-502.
[5]DEVETZI M,GOULIELMAKI M,KHOURY N,et al.Geneti-cally-modified stem cells in treatment of human diseases:Tis-sue kallikrein(KLK1)-based targeted therapy[J].Int J MolMed,2018,41(3):1177-1186.
[6]张霞,都治香,王娜,等.白细胞介素12基因修饰骨髓间充质干细胞对卵巢癌细胞生长的影响[J].中国组织工程研究,2017,21(9):1346-1351.
[7]EHTESHAM M,KABOS P,GUTIERREZ M A,et al.Induc-tion of glioblastoma apoptosis using neural stem cell-mediateddelivery of tumor necrosis factor-related apoptosis-inducing li-gand[J].Cancer Res,2002,62(24):7170-7174.
[8]GAO Y,YAO A,ZHANG W,et al.Human mesenchymalstem cells overexpressing pigment epithelium-derived factor in-hibit hepatocellular carcinoma in nude mice[J].Oncogene,2010,29(19):2784-2794.
[9]STEIN B L,TIU R V.Biological rationale and clinical use ofinterferon in the classical BCR-ABL-negative myeloproliferativeneoplasms[J].J Interferon Cytokine Res,2013,33(4):145-53.
[10]LAPENTA C,DONATI S,SPADARO F,et al.NK cell activa-tion in the antitumor response induced by IFN-αdendritic cellsloaded with apoptotic cells from follicular lymphoma patients[J].J Immunol,2016,197(3):795-806.
[11]ELKORD E,BURT D J,SUNDSTEDT A,et al.Immunologi-cal responseand overall survival in a subset of advanced renalcell carcinomapatients from a randomized phase 2/3 study ofnaptumomab estafenatox plus IFN-αversus IFN-α[J].Oncotar-get,2015,6(6):4428-4439.
[12]BORDEN E C,JACOBS B,HOLLOVARY E,et al.Gene reg-ulatory and clinical effects of interferonβin patients with meta-static melanoma:A PhaseⅡTrial[J].Interferon Cytokine Res,2011,31(5):433-440.
[13]HAN X,YANG Q,LIN L,et al.Interleukin-17 enhances im-munosuppression by mesenchymal stem cells[J].Cell DeathDiffer,2014,21(11):1758-1768.
[14]HUANG Y,YU P,LI W,et al.p53 regulates mesenchymalstem cell-mediated tumor suppression in a tumor microenviron-ment through immune modulation[J].Oncogene,2014,33:3830-3838.
[15]SHOU P,CHEN Q,JIANG J,et al.Type I interferons exertanti-tumor effect via reversing immunosuppression mediated bymesenchymal stromal cells[J].Oncogene,2016,35(46):5953-5962.
[16]BURKE S J,UPDEGRAFF B L,BELLICH R M,et al.Regula-tion of i NOS gene transcription by IL-1βand IFN-γrequires acoactivator exchange mechanism[J],Mol Endocrinol,2013,27(10):1724-1742.