沉默IFN-γR表达对大鼠BMMSCs免疫调节能力的影响
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摘要
目的:通过沉默大鼠骨髓间充质干细胞(BMMSCs)干扰素γ受体(IFN-γR)基因表达,研究抑制IFN-γ信号通路对大鼠BMMSCs免疫调节能力的影响。方法:根据大鼠IFN-γR基因序列,构建沉默IFN-γR基因的重组慢病毒shIFNγR-LV,并转染大鼠BMMSCs,沉默大鼠BMMSCs的IFN-γR基因表达;混合淋巴细胞培养实验分别检测对照组、BMMSCs阴性对照(BMMSC-NC)组和BMMSC-shIFNγR组淋巴细胞的活力和炎症因子浓度。结果:构建的慢病毒shIFNγR-LV能显著降低大鼠BMMSCs的IFN-γR mRNA表达;混合淋巴细胞培养实验显示,相对BMMSC-NC组,BMMSC-shIFNγR组抑制淋巴细胞活力的作用减弱(P<0.05)。混合淋巴细胞培养体系中,BMMSC-NC组肿瘤坏死因子α浓度低于BMMSC-shIFNγR组,而白细胞介素10浓度高于BMMSC-shIFNγR组(P<0.05)。结论:利用RNA干扰技术能有效沉默大鼠BMMSCs的IFN-γR基因表达,并能显著降低BMMSCs的调节免疫功能。
AIM: To investigate the effect of inhibiting interferon-γ signaling pathway on the immune regulation of rat bone marrow mesenchymal stem cells(BMMSCs) by silencing the expression of interferon-γ receptor(IFN-γR) gene. METHODS: According to the rat IFN-γR gene sequence, lentivirus carrying INF-γR shRNA(shIFNγR-LV) was constructed and transfected into the rat BMMSCs to silence the expression of IFN-γR gene. The lymphocyte proliferation ability and the concentrations of inflammatory factors in control group, BMMSCs negative control(BMMSC-NC) group and BMMSC-shIFNγR group were detected by mixed lymphocyte culture experiments. RESULTS: The constructed shIFNγR-LV significantly decreased the mRNA expression of IFN-γR in the rat BMMSCs. Mixed lymphocyte culture experiments showed that the inhibition of lymphocyte viability was weakened in BMMSC-shIFNγR group compared with BMMSC-NC group(P<0.05). In the mixed lymphocyte culture system, the concentration of tumor necrosis factor-α in BMMSC-NC group was lower than that in BMMSC-shIFNγR group, and the concentration of interleukin-10 was higher than that in BMMSC-shIFNγR group(P<0.05). CONCLUSION: RNA interference technique effectively silences the expression of IFN-γR gene in the rat BMMSCs and significantly reduces the immune function of BMMSCs.
AIM: To investigate the effect of inhibiting interferon-γ signaling pathway on the immune regulation of rat bone marrow mesenchymal stem cells(BMMSCs) by silencing the expression of interferon-γ receptor(IFN-γR) gene. METHODS: According to the rat IFN-γR gene sequence, lentivirus carrying INF-γR shRNA(shIFNγR-LV) was constructed and transfected into the rat BMMSCs to silence the expression of IFN-γR gene. The lymphocyte proliferation ability and the concentrations of inflammatory factors in control group, BMMSCs negative control(BMMSC-NC) group and BMMSC-shIFNγR group were detected by mixed lymphocyte culture experiments. RESULTS: The constructed shIFNγR-LV significantly decreased the mRNA expression of IFN-γR in the rat BMMSCs. Mixed lymphocyte culture experiments showed that the inhibition of lymphocyte viability was weakened in BMMSC-shIFNγR group compared with BMMSC-NC group(P<0.05). In the mixed lymphocyte culture system, the concentration of tumor necrosis factor-α in BMMSC-NC group was lower than that in BMMSC-shIFNγR group, and the concentration of interleukin-10 was higher than that in BMMSC-shIFNγR group(P<0.05). CONCLUSION: RNA interference technique effectively silences the expression of IFN-γR gene in the rat BMMSCs and significantly reduces the immune function of BMMSCs.
引文
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[12] Li D, Han Y, Zhuang Y, et al. Overexpression of COX-2 but not indoleamine 2,3-dioxygenase-1 enhances the immunosuppressive ability of human umbilical cord-derived mesenchymal stem cells[J]. Int J Mol Med, 2015, 35(5):1309-1316.
[13] 陈书尚, 吴承耀, 朱凌峰, 等. 骨髓间充质干细胞抑制T淋巴细胞增殖和miRNA-155表达的体外研究[J]. 福建医科大学学报, 2014, 48(4):217-221.
[14] Hermankova B, Zajicova A, Javorkova E, et al. Suppression of IL-10 production by activated B cells via a cell contact-dependent cyclooxygenase-2 pathway upregulated in IFN-γ-treated mesenchymal stem cells[J]. Immunobiology, 2016, 221(2):129-136.
[2] Lee KC, Lin HC, Huang YH, et al. Allo-transplantation of mesenchymal stem cells attenuates hepatic injury through IL1Ra dependent macrophage switch in a mouse model of liver disease[J]. J Hepatol, 2015, 63(6):1405-1412.
[3] 黄雪琼, 檀卫平, 吴葆菁, 等. 骨髓间充质干细胞对重症哮喘患儿外周血Th17/Treg的免疫调节作用[J]. 中国病理生理杂志, 2014, 30(9):1694-1697.
[4] 李鹏, 李恒平, 周东, 等. PDL1Ig基因修饰的骨髓间充质干细胞诱导大鼠肝移植免疫耐受[J].中国病理生理杂志, 2016, 32(7):1279-1284.
[5] Ge Xh, Bai C, Yang JM, et al. Intratracheal transplantation of bone marrow-derived mesenchymal stem cells reduced airway inflammation and up-regulated CD4+CD25+ regulatory T cells in asthmatic mouse[J]. Cell Biol Int, 2013, 37(7):675-686.
[6] Polchert D, Sobinsky J, Douglas GW, et al. IFN-γ activation of mesenchymal stem cells for treatment and prevention of graft versus host disease[J]. Eur J Immunol, 2008, 38(6):1745-1755.
[7] Kim DS, Jang IK, Lee MW, et al. Enhanced immunosuppressive properties of human mesenchymal stem cells primed by interferon-γ[J]. EBioMedicine, 2018, 28:261-273.
[8] Kota DJ, Prabhakara KS, Toledano-Furman N, et al. Prostaglandin E2 indicates therapeutic efficacy of mesenchymal stem cells in experimental traumatic brain injury[J]. Stem Cells, 2017, 35(5):1416-1430.
[9] Daneshmandi S, Karimi MH, Pourfathollah AA, et al. TGF-β1 transduced mesenchymal stem cells have profound modulatory effects on DCs and T cells[J]. Iran J Immunol, 2017, 14(1):13-23.
[10] 杜晶晶, 孙轶华. 慢病毒载体介导的RNA干扰及其在T淋巴细胞的应用[J]. 中国病理生理杂志, 2014, 30(10):1905-1909.
[11] Blouin CM, Lamaze C. Interferon gamma receptor: the beginning of the journey[J]. Front Immunol, 2013, 4:267.
[12] Li D, Han Y, Zhuang Y, et al. Overexpression of COX-2 but not indoleamine 2,3-dioxygenase-1 enhances the immunosuppressive ability of human umbilical cord-derived mesenchymal stem cells[J]. Int J Mol Med, 2015, 35(5):1309-1316.
[13] 陈书尚, 吴承耀, 朱凌峰, 等. 骨髓间充质干细胞抑制T淋巴细胞增殖和miRNA-155表达的体外研究[J]. 福建医科大学学报, 2014, 48(4):217-221.
[14] Hermankova B, Zajicova A, Javorkova E, et al. Suppression of IL-10 production by activated B cells via a cell contact-dependent cyclooxygenase-2 pathway upregulated in IFN-γ-treated mesenchymal stem cells[J]. Immunobiology, 2016, 221(2):129-136.