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目的探讨再生障碍性贫血(aplastic anemia,AA)患者骨髓间充质干细胞(bone marrow mesenchymal stem cells,BM-MSCs)对正常CD4-细胞功能的影响。方法采集和分离15例AA患者和10例正常人BM-MSCs进行体外培养。倒置显微镜结合荧光共聚焦显微镜观察BM-MSCs的形态和生长情况,流式细胞术检测免疫表型,诱导BM-MSCs成骨和成脂,综合进行BM-MSCs鉴定。BM-MSCs与免疫磁珠负集的外周血CD4-细胞共培养观察其对CD4-细胞功能的影响,采用倒置显微镜和BrdU方法分析CD4-细胞的克隆形成和增殖能力,酶联免疫吸附实验(ELISA)检测CD4-细胞分泌的细胞因子水平。结果正常人和AA患者骨髓间充质干细胞均呈长梭形,辐射状或旋涡状贴壁生长;诱导后均向成骨细胞和脂肪细胞分化;均表达CD105、CD73、CD90、CD29、CD44、CD49e、CD166、CD13和HLA-ABC,不表达CD34、CD45和HLA-DR。与正常对照组比较,AA患者BM-MSCs抑制CD4-细胞克隆形成和增殖能力减弱(P<0.05)。同时,AA患者BM-MSCs抑制CD4-细胞分泌肿瘤坏死因子α(TNF-α)和干扰素γ(IFN-γ)的能力明显低于正常对照组(P<0.05)。结论 AA患者骨髓间充质干细胞在调节CD4-细胞免疫功能方面存在缺陷,可能进一步加重骨髓免疫损伤。
Objective To investigate the effect of bone marrow mesenchymal stem cells (BM-MSCs) on normal CD4-cell function in patients with aplastic anemia (AA). Methods Fifteen cases of AA and 10 normal BM-MSCs were collected and cultured in vitro. The morphology and growth of BM-MSCs were observed by inverted microscope and confocal microscopy. The immunophenotypes were detected by flow cytometry. The BM-MSCs were induced to osteogenesis and adipogenesis. BM-MSCs were identified comprehensively. The effect of CD4-cell co-culture on peripheral blood mononuclear cells (PBMCs) of BM-MSCs and immunomagnetic beads negative cells was observed. The function of CD4-cell was analyzed by inverted microscope and BrdU method. The results of enzyme-linked immunosorbent assay ELISA) to detect CD4-cell-secreted cytokine levels. Results Bone marrow mesenchymal stem cells of normal and AA patients all showed long fusiform, radial or swirling adherent growth. All of them differentiated into osteoblasts and adipocytes after induction; all expressed CD105, CD73, CD90, CD29, CD44, CD49e, CD166, CD13 and HLA-ABC, but not CD34, CD45 and HLA-DR. Compared with the normal control group, the BM-MSCs in AA patients inhibited the formation of CD4-cell clone and the ability of proliferation decreased (P <0.05). Meanwhile, the ability of BM-MSCs in patients with AA to inhibit the secretion of tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) from CD4 cells was significantly lower than that of the normal control group (P <0.05). Conclusion AA patients with bone marrow mesenchymal stem cells in the regulation of immune function of CD4 cells deficiencies, may further aggravate bone marrow immune damage.