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为探讨一氧化氮(nitric oxide,NO)在间充质干细胞(mesenchymal stem cells,MSC)发挥免疫调节功能中的作用机制。通过全骨髓贴壁法分离培养得到高纯度的MSC,利用炎症因子(TNF-α,IL-1α)和Con A分别刺激MSC制备MSC上清培养液,3 H-TdR掺入法检测MSC及其培养上清液对脾细胞增殖的抑制效应;ELISA方法检测各组培养上清液中前列腺素E-2(prostaglandin E-2,PGE-2)的含量;通过NO抑制剂L-canavanine和NO的直接供体SNP分别作用于MSC,探讨NO对MSC免疫调节功能的影响。结果显示:全骨髓贴壁法可获得较高纯度的MSC;MSC及其上清培养液均能发挥免疫调节功能;MSC分泌大量的PGE-2;抑制NO产生可以下调MSC对PGE-2的分泌而增加NO量可以上调MSC对PGE-2的分泌。研究结果表明,NO通过刺激MSC分泌抗炎因子PGE-2,从而使MSC发挥长效免疫调节功能。本研究阐明了NO介导的MSC长效免疫调节功能的机制,为MSC的临床应用奠定了理论基础。
To investigate the role of nitric oxide (NO) in the immune regulatory function of mesenchymal stem cells (MSC). MSCs were isolated and cultured by whole bone marrow adherent method, MSCs were prepared by stimulating MSCs with inflammatory cytokines (TNF-α, IL-1α) and Con A, respectively. MSCs were harvested by 3 H-TdR incorporation The inhibitory effect of supernatant on the proliferation of splenocytes was detected. The content of prostaglandin E-2 (PGE-2) in culture supernatant of each group was detected by ELISA. The effects of NO inhibitor L-canavanine and NO Direct donor SNP were applied to MSC respectively to explore the effect of NO on immune function of MSC. The results showed that the whole bone marrow adherent method can obtain higher purity MSC; MSC and its supernatant culture medium can play an immunomodulatory function; MSC secrete a large amount of PGE-2; inhibiting the production of NO can down-regulate the secretion of PGE-2 However, increasing the amount of NO increased MSC secretion of PGE-2. The results show that NO stimulates MSC to secrete anti-inflammatory cytokine PGE-2, so that MSC can exert long-term immunomodulatory function. This study elucidated the mechanism of NO mediated long-term immune regulation of MSC, which laid the theoretical foundation for the clinical application of MSC.