复合CTLA4的脱钙骨基质抑制T淋巴细胞免疫能力和增强骨髓间充质干细胞迁移能力的体外作用研究

In vitro effects of demineralized bone matrix compounded with CTLA4 on inhibiting immune ability of T lymphocyte and enhancing migration ability of bone marrow mesenchymal stem cells

目的分析复合细胞毒性T淋巴细胞相关蛋白4(CTLA4)的脱钙骨基质(DBM)对T淋巴细胞免疫能力和骨髓间充质干细胞(BMMSCs)迁移能力的体外调控作用。方法通过特殊负压灌注装置将纤维蛋白凝胶复合CTLA4溶液灌注到DBM,构建复合CTLA4的DBM,并通过扫描电镜观察其微观形态。分离外周血单核细胞(PBMCs),通过植物血凝素(PHA)激活T淋巴细胞来构建免疫激活微环境。将复合CTLA4的DBM在培养基中孵育5、10、20、30、40 d,ELISA检测CTLA4的浓度,由此计算CTLA4的累积释放率。将PHA预处理的PBMCs与复合CTLA4的DBM进行Transwell共培养,ELISA检测培养基中IL-2和IFN-γ的含量。分离BMMSCs,Transwell共培养法分析复合CTLA4的DBM对BMMSCs增殖和迁移能力的影响。结果复合CTLA4的DBM结构完整有序,具有良好的三维网络结构。复合材料中CTLA4在5、10、20、30、40 d的累积释放率分别为(11.3%±1.9%)、(27.9%±3.7%)、(48.4%±3.6%)、(62.8%±3.8%)和(83.0%±2.5%)。与单纯的DBM相比,与复合CTLA4的DBM共培养可显著减少培养基中IL-2(P=0.0004)和IFN-γ(P=0.0007)的含量,增强BMMSCs的增殖能力(P=0.0006)和迁移能力(P=0.0004)。结论复合CTLA4的DBM在体外环境下具有抑制T淋巴细胞免疫能力和增强BMMSCs迁移能力的作用。

Objective To analyze the regulatory effects of demineralized bone matrix(DBM)compounded with cytotoxic T lymphocyte-associated protein 4(CTLA4)on the immune ability of T lymphocyte and migration ability of bone marrow mesenchymal stem cells(BMMSCs)in vitro.Methods The fibrin gel composite CTLA4 solution was poured into the DBM by a special negative pressure perfusion device to construct a DBM compounded with CTLA4,and its microscopic morphology was observed by a scanning electron microscope.The peripheral blood mononuclear cells(PBMCs)were isolated,and the T lymphocyte was activated by phytohaemagglutinin(PHA)to construct an immune-activated microenvironment.The DBM compounded with CTLA4 was incubated in the medium for 5,10,20,30,and 40 days,and the concentration of CTLA4 was determined by ELISA to calculate the cumulative release rate of CTLA4.PBMCs pretreated with PHA were co-cultured with DBM compounded with CTLA4 by Transwell,and the contents of IL-2 and IFN-γin the medium were detected by ELISA.The BMMSCs were isolated,and the Transwell co-culture method was used to analyze the effects of DBM compounded with CTLA4 on the proliferation and migration ability of BMMSCs.Results The structure of DBM compounded with CTLA4 was complete and orderly,with a good three-dimensional network structure.The cumulative release rates of CTLA4 in the composite material 5,10,20,30,and 40 days were(11.3%±1.9%),(27.9%±3.7%),(48.4%±3.6%),(62.8%±3.8%),and(83.0%±2.5%),respectively.Compared with DBM alone,co-culture with DBM compounded with CTLA4 could significantly reduce the contents of IL-2(P=0.0004)and IFN-γ(P=0.0007)in the medium,enhance the proliferation ability(P=0.0006)and migration ability(P=0.0004)of BMMSCs.ConclusionThe DBM compounded with CTLA4 can inhibit the immune ability of T lymphocytes and enhance the migration ability of BMMSCs in vitro.