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

Enhanced Cell Surviving against Oxidative Stress and Treg-inducing Ability of Dedifferentiated Human Umbilical Cord Mesenchymal Stem Cells Modified by IDO Gene

【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+CD127low 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+CD127lowTreg 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.