去铁胺联合干扰素γ预处理对人牙髓干细胞的免疫调节作用

Immunomodulatory effects of deferoxamine and interferon gamma on human dental pulp stem cells

背景:免疫排斥是组织和器官移植面临的巨大挑战,免疫抑制剂的使用常给患者带来严重的不良反应,间充质干细胞的免疫抑制作用为免疫排斥的治疗带来新的希望。目的:探讨去铁胺和干扰素γ对人牙髓干细胞免疫调节能力的影响。方法:用去铁胺、干扰素γ以及二者联合预处理人牙髓干细胞,CCK-8检测细胞增殖能力,Q-PCR检测相关基因mRNA的表达,ELISA检测培养上清中炎性因子水平;预处理后的人牙髓干细胞与CFSE标记的小鼠脾淋巴细胞共培养,流式细胞术检测脾淋巴细胞的增殖能力。建立小鼠异体皮肤移植模型,经尾静脉注射PBS或不同预处理的人牙髓干细胞,记录移植皮肤存活时间,进行组织病理学和免疫组化分析,检测移植小鼠脾脏Treg细胞比例。结果与结论:①去铁胺和干扰素γ预处理均抑制了人牙髓干细胞的生长,去铁胺生长抑制作用更为明显;②去铁胺联合干扰素γ预处理明显提高了血管内皮生长因子、白细胞介素6、转化生长因子β1、环氧合酶2、人类白细胞抗原G、肿瘤坏死因子Α诱导蛋白6和白细胞介素10基因表达,促进了白细胞介素6、转化生长因子β1和前列腺素E2的分泌;③体内实验结果证实,去铁胺联合干扰素γ预处理的人牙髓干细胞可以延长小鼠异体移植皮肤存活时间,减轻了移植皮片处CD4^(+)T、CD8^(+)T以及巨噬细胞的浸润,促进了受体小鼠脾脏中Treg细胞增加;④研究结果表明,经去铁胺联合干扰素γ预处理后,人牙髓干细胞的免疫调节作用明显增强,减轻了皮肤移植免疫排斥反应。

BACKGROUND:Immune rejection is a great challenge for tissue or organ transplantation,and immunosuppressors often bring serious side effects to patients.The immunosuppressive effect of mesenchymal stem cells brings new hope for the treatment of immune rejection.OBJECTIVE:To explore the effect of combined action of deferoxamine and interferon-γ on the immunomodulatory ability of human dental pulp stem cells.METHODS:After pretreatment of human dental pulp stem cells with deferoxamine or interferon-γ or their combination,the proliferation of cells was detected with cell counting kit-8.The mRNA expression of some gene mRNA was examined by Q-PCR.The secretion of some immune regulatory factors was determined using ELISA.Thereafter,pretreated human dental pulp stem cells were co-cultured with mouse spleen lymphocytes,and then the proliferation of lymphocytes was detected by CFSE labeling by flow cytometry.After allogenic skin transplantation,mice were injected with PBS,human dental pulp stem cells or pretreated human dental pulp stem cells into tail vein immediately.The survival time of skin grafts was recorded.The pathology and immunohistochemistry of skin grafts were examined.Moreover,the proportion of Treg cells in spleen of transplanted mice was measured.RESULTS AND CONCLUSION:(1)Deferoxamine or interferon-γpretreatment inhibited the growth of human dental pulp stem cells,and the inhibition of deferoxamine was more significant than interferon-γ.(2)Pretreatment of deferoxamine combined with interferon-γsignificantly increased the gene expression of vascular endothelial growth factor,interleukin 6,transforming growth factor β1,cyclooxygenase 2,human leukocyte antigen G,tumor necrosis factor A inducible protein 6 and interleukin 10,and promote secretion of interleukin 6,transforming growth factor β1 and prostaglandin E2.(3)The results of in vivo experiments confirmed that human dental pulp stem cells pretreated with deferoxamine combined with interferon-γ prolonged the survival time of mouse allograft skin,reduced the infiltration of CD4^(+)T,CD8^(+)T and macrophages at the grafted skin,and increased the proportion of Treg cells in the spleen of recipient mice.(4)It is concluded that deferoxamine combined with interferon-γ pretreatment enhanced the immunomodulatory effect of human dental pulp stem cells,and inhibited the immune rejection of skin allografts.