去分化联合IDO基因修饰增强人脐带间充质干细胞的抗氧化应激存活及Treg细胞诱导能力
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摘要
【目的】研究去分化处理联合吲哚胺-2,3-双加氧酶(IDO)基因修饰,对人脐带间充质干细胞(hMSC)抗氧化应激生存能力及诱导调节性T淋巴细胞(Treg)能力的影响。【方法】通过对hMSC的短暂成脂分化诱导和恢复培养,获得去分化hMSC(De-hMSC)。实验组De-hMSC感染携带IDO基因的重组逆转录病毒,对照组为感染绿色荧光蛋白(ZsGreen1)基因的De-hMSC以及正常培养的未感染De-hMSC和hMSC。通过Western blot检测被感染细胞的IDO蛋白表达,应用流式细胞术测定IDO基因修饰型De-hMSC(IDO/De-hMSC)的免疫表型,同时鉴定其成骨和成脂分化能力。利用Annexin V-FITC/PI双染流式细胞术比较各组细胞在300μmol/L t-BHP作用下的抗氧化应激生存能力,采用三色荧光标记流式细胞术测定含各组细胞培养上清的条件培养基对正常人外周血单个核细胞(PBMC)中Treg细胞含量的影响。【结果】IDO/De-hMSC仍具有间充质干细胞的免疫表型和成骨、成脂分化能力。在300μmol/L t-BHP作用下,De-hMSC的细胞存活率较hMSC明显增加(P<0.05),经IDO基因修饰后De-hMSC的细胞存活优势无明显改变(P>0.05)。与未修饰的各对照组相比,含IDO/De-hMSC上清的条件培养基能明显上调PBMC中CD4~+CD25~+CD127~(low)Treg细胞的含量(P<0.05)。【结论】成脂去分化联合IDO基因修饰不影响hMSC的干细胞特性,并能同时增强h MSC的抗氧化应激生存能力及其对Treg细胞的诱导能力,是一种有望在免疫抑制治疗中发挥作用的间充质干细胞修饰策略。
【Objective】To investigate whether the dedifferented human umbilical cord mesenchymal stem cells(hMSC)modified by IDO gene can get improved ability to survive oxidative stress as well as to induce regulatory T(Treg)lymphocytes.【Methods】The dedifferentiated h MSC(De-hMSC)were obtained by a transient adipogenic induction and subsequent recovery culture in normal medium.The IDO gene modified De-hMSC(IDO/De-hMSC)were prepared by retroviral infection using recombinant retrovirus harboring IDO gene.The De-hMSC infected by retrovirus containing ZsGreen1 gene,the non-infected De-hMSC and h MSC were set as controls.Exogenous expression of IDO protein wasconfirmed by Western blot.Flow cytometry analysis was performed to detect the immunophenotype of IDO/De-hMSC,and their osteogenic/adipogenic differentiation abilities were also assessed.Cell survival rates under the oxidative stress of300μmol/L t-BHP were determined by Annexin V-FITC/PI double staining flow cytometry.Human peripheral blood mononuclear cells(PBMC)were isolated and treated with conditioned medium containing the culture supernatant of hMSC,De-hMSC,Mock/De-hMSC and IDO/De-hMSC,respectively.Changes in the proportion of CD4~+CD25~+CD127~(low) Treg cells in PBMC were determined by triple fluorescent labeling flow cytometry.【Results】The De-hMSC modified by IDO gene still have the immunophenotype as well as the osteogenic/adipogenic differentiation abilities that are typical of mesenchymal stem cells.When challenged by 300μmol/L t-BHP,the number of viable cells in De-hMSC significantly elevated compared with hMSC(P<0.05),and the survival advantage of De-hMSC was not obviously affected by IDO gene modification(P>0.05).Conditioned medium containing the supernatant from IDO/De-hMSC dramatically up-regulated the percentage of CD4~+CD25~+CD127~(low)Treg cells in PBMC in contrast to the control groups(P<0.05).【Conclusions】IDO/De-hMSC have the same immunophenotype and differentiation capacity as the native hMSC,and can simultaneously enhance the ability of hMSC to survive against oxidative stress and to induce Treg cells,which may be a potential modification strategy of mesenchymal stem cells for immunosuppressive therapy.
【Objective】To investigate whether the dedifferented human umbilical cord mesenchymal stem cells(hMSC)modified by IDO gene can get improved ability to survive oxidative stress as well as to induce regulatory T(Treg)lymphocytes.【Methods】The dedifferentiated h MSC(De-hMSC)were obtained by a transient adipogenic induction and subsequent recovery culture in normal medium.The IDO gene modified De-hMSC(IDO/De-hMSC)were prepared by retroviral infection using recombinant retrovirus harboring IDO gene.The De-hMSC infected by retrovirus containing ZsGreen1 gene,the non-infected De-hMSC and h MSC were set as controls.Exogenous expression of IDO protein wasconfirmed by Western blot.Flow cytometry analysis was performed to detect the immunophenotype of IDO/De-hMSC,and their osteogenic/adipogenic differentiation abilities were also assessed.Cell survival rates under the oxidative stress of300μmol/L t-BHP were determined by Annexin V-FITC/PI double staining flow cytometry.Human peripheral blood mononuclear cells(PBMC)were isolated and treated with conditioned medium containing the culture supernatant of hMSC,De-hMSC,Mock/De-hMSC and IDO/De-hMSC,respectively.Changes in the proportion of CD4~+CD25~+CD127~(low) Treg cells in PBMC were determined by triple fluorescent labeling flow cytometry.【Results】The De-hMSC modified by IDO gene still have the immunophenotype as well as the osteogenic/adipogenic differentiation abilities that are typical of mesenchymal stem cells.When challenged by 300μmol/L t-BHP,the number of viable cells in De-hMSC significantly elevated compared with hMSC(P<0.05),and the survival advantage of De-hMSC was not obviously affected by IDO gene modification(P>0.05).Conditioned medium containing the supernatant from IDO/De-hMSC dramatically up-regulated the percentage of CD4~+CD25~+CD127~(low)Treg cells in PBMC in contrast to the control groups(P<0.05).【Conclusions】IDO/De-hMSC have the same immunophenotype and differentiation capacity as the native hMSC,and can simultaneously enhance the ability of hMSC to survive against oxidative stress and to induce Treg cells,which may be a potential modification strategy of mesenchymal stem cells for immunosuppressive therapy.
引文
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[17]张慧,王贵超,韩兴龙,等.去分化间充质干细胞成骨再分化潜能的实验研究[J].南京医科大学学报(自然科学版),2013,33(8):1044-1048.Zhang H,Wang GC,Han XL,et al.Osteogenic redifferentiation of de-differentiated MSCs[J].J Nanjing Med Univ,2013,33(8):1044-1048.
[18]Tipnis S,Viswanathan C,Majumdar AS.Immunosuppressive properties of human umbilical cord-derived mesenchymal stem cells:role of B7-H1 and IDO[J].Immunol Cell Biol,2010,88(8):795-806.
[19]Palomares O,Akdis M,Akdis CA,et al.Mechanisms of immune regulation in allergic diseases:the role of regulatory T and B cells[J].Immunol Rev,2017,278(1):219-236.
[20]陈璇,袁茵,邵红伟,等.人脐带间充质干细胞培养上清对正常人淋巴细胞各亚群比例的影响[J].中国免疫学杂志,2014,30(5):577-581.Chen X,Yuan Y,Shao HW,et al.Influence of supernatant from human umbilical cord-derived mesenchymal stem cells on proportions of each human lymphoid subgroup[J].Chin J Immunol,2014,30(5):577-581.
[21]李贝贝,韩金秀,严丹,等.过表达IDO可增强大鼠骨髓间充质干细胞免疫抑制作用[J].现代免疫学,2018,38(3):191-196.Li BB,Han JX,Yan D,et al.Overexpression of IDO enhances the immunosuppressive effect of rat bone marrow mesenchymal stem cells[J].Modern Immunol,2018,38(3):191-196.
[22]He Y,Zhou S,Liu H,et al.Indoleamine 2,3-dioxgenase transfected mesenchymal stem cells induce kidney allograft tolerance by increasing the production and function of regulatory T cells[J].Transplantation,2015,99(9):1829-1838.
[2]Niu J,Yue W,Le-Le Z,et al.Mesenchymal stem cells inhibit T cell activation by releasing TGF-beta1 from TGF-beta1/GARP complex[J].Oncotarget,2017,8(59):99784-99800.
[3]赵一俏,曹东林,陈伟.间充质干细胞联合NK细胞输注抑制DC减轻aGVHD[J].中山大学学报(医学科学版),2013,34(2):200-206.Zhao YQ,Cao DL,Chen W.Alleviation of graftversus-host disease after infusion of mesenchymal stem cells combined with natural killer cells by reducing dendritic cells in mice[J].J Sun Yat-sen Univ(Med Sci),2013,34(2):200-206.
[4]Gebler A,Zabel O,Seliger B.The immunomodulatory capacity of mesenchymal stem cells[J].Trends Mol Med,2012,18(2):128-134.
[5]Xu H,Chen C,Hu L,et al.Gene-modified mesenchymal stem cell-based therapy in renal ischemiareperfusion injury[J].Curr Gene Ther,2017,17(6):453-460.
[6]Kalimuthu S,Zhu L,Oh JM,et al.Migration of mesenchymal stem cells to tumor xenograft models and in vitro drug delivery by doxorubicin[J].Int JMed Sci,2018,15(10):1051-1061.
[7]袁茵,王辉,鲁欣.银杏叶提取物对叔丁基过氧化氢损伤人脐带间充质干细胞的干预作用[J].广州中医药大学学报,2015,32(3):458-463.Yuan Y,Wang H,Lu X.Intervention of tert-butyl hydroperoxide-induced injury in human umbilical cord derived mesenchymal stem cells with ginkgo biloba extract[J].J Guangzhou Univ Tradit Chin Med,2015,32(3):458-463.
[8]Chen TL,Zhu GL,Wang JA,et al.Apoptosis of bone marrow mesenchymal stem cells caused by hypoxia/reoxygenation via multiple pathways[J].Int JClin Exp Med,2014,7(12):4686-4697.
[9]Kim DS,Jang IK,Lee MW,et al.Enhanced immunosuppressive properties of human mesenchymal stem cells primed by interferon-γ[J].E Biomed,2018,28:261-273.
[10]翟少峰.经去分化预处理脂肪间充质干细胞移植治疗大鼠急性心肌梗死的实验研究[D].新乡医学院,2013.Qu SF.Experimental study on dedifferentiationreprogrammed adipose-derived mesenchymal stem cells transplantation for treatment of acute myocardial infarction in rats[D].Xinxiang Med College,2013.
[11]Liu Y,Jiang X,Zhang X,et al.Dedifferentiationreprogrammed mesenchymal stem cells with improved therapeutic potential[J].Stem Cells,2011,29(12):2077-2089.
[12]Yuan Y,Lu X,Tao CL,et al.Forced expression of indoleamine-2,3-dioxygenase in human umbilical cord-derived mesenchymal stem cells abolishes their anti-apoptotic effect on leukemia cell lines in vitro[J].In Vitro Cell Dev Biol Abim,2013,49(10):752-758.
[13]Yuan Y,Lu X,Chen X,et al.Jagged1 contributes to the drug resistance of Jurkat cells in contact with human umbilical cord-derived mesenchymal stem cells[J].Oncol Lett,2013,6(4):1000-1006.
[14]Yuan Y,Zhou C,Chen X,et al.Suppression of tumor cell proliferation and migration by human umbilical cord mesenchymal stem cells:a possible role for apoptosis and Wnt signaling[J].Oncol Lett,2018,15:8536-8544.
[15]Jopling C,Boue S,Izpisua Belmonte JC.Dedifferentiation,transdifferentiation and reprogramming:three routes to regeneration[J].Nat Rev Mol Cell Biol,2011,12(2):79-89.
[16]Ullah M,Stich S,Notter M,et al.Transdifferentiation of mesenchymal stem cells-derived adipogenicdifferentiated cells into osteogenic-or chondrogenic-differentiated cells proceeds via dedifferentiation and have a correlation with cell cycle arresting and driving genes[J].Differentiation,2013,85(3):78-90.
[17]张慧,王贵超,韩兴龙,等.去分化间充质干细胞成骨再分化潜能的实验研究[J].南京医科大学学报(自然科学版),2013,33(8):1044-1048.Zhang H,Wang GC,Han XL,et al.Osteogenic redifferentiation of de-differentiated MSCs[J].J Nanjing Med Univ,2013,33(8):1044-1048.
[18]Tipnis S,Viswanathan C,Majumdar AS.Immunosuppressive properties of human umbilical cord-derived mesenchymal stem cells:role of B7-H1 and IDO[J].Immunol Cell Biol,2010,88(8):795-806.
[19]Palomares O,Akdis M,Akdis CA,et al.Mechanisms of immune regulation in allergic diseases:the role of regulatory T and B cells[J].Immunol Rev,2017,278(1):219-236.
[20]陈璇,袁茵,邵红伟,等.人脐带间充质干细胞培养上清对正常人淋巴细胞各亚群比例的影响[J].中国免疫学杂志,2014,30(5):577-581.Chen X,Yuan Y,Shao HW,et al.Influence of supernatant from human umbilical cord-derived mesenchymal stem cells on proportions of each human lymphoid subgroup[J].Chin J Immunol,2014,30(5):577-581.
[21]李贝贝,韩金秀,严丹,等.过表达IDO可增强大鼠骨髓间充质干细胞免疫抑制作用[J].现代免疫学,2018,38(3):191-196.Li BB,Han JX,Yan D,et al.Overexpression of IDO enhances the immunosuppressive effect of rat bone marrow mesenchymal stem cells[J].Modern Immunol,2018,38(3):191-196.
[22]He Y,Zhou S,Liu H,et al.Indoleamine 2,3-dioxgenase transfected mesenchymal stem cells induce kidney allograft tolerance by increasing the production and function of regulatory T cells[J].Transplantation,2015,99(9):1829-1838.