大鼠树突状细胞的体外分离、纯化、扩增及鉴定

Isolation, purification, proliferation and identification of rat bone marrow-derived dendritic cells

目的 探讨诱导及纯化大鼠树突状细胞 (Dendriticcells ,DC)的方法 ,为进一步研究树突状细胞的功能、特性及临床应用提供技术方法。方法 应用重组大鼠rGM CSF、IL 4在体外培养大鼠骨髓前体细胞获得大量DCs,经标抗大鼠OX62单抗免疫磁珠分离纯化后的DCs经形态学观察、表型检测、功能学实验鉴定。结果 大鼠骨髓来源的DC在体外培养 12～ 14d后完全成熟 ,99 3 6%培养纯化后DC表达大鼠特异性表面标志OX62 ;典型的成熟大鼠DC形态上类似于小鼠和人类的DC ,表型为MHCⅡ + + ,OX62 + + ,FcγR(CD3 2 ) + / -,CD80 + / -,CD86+ + ,体外功能分析显示该DC能够强烈刺激MLR并有效递呈可溶性抗原OVA刺激初始型T细胞的增殖。结论 成功地建立了体外大量扩增大鼠骨髓DC的方法 。

Objective To explore methods for inducing rat bone marrow cells to differentiate to dendritic cells (DCs), and for purification. Methods DCs obtained from SD rat bone marrow were propagated in vitro under the condition of GM CSF and IL 4. DCs were purified by monoclonal antibody OX62 and magnetic beads. The DCs harvested 14 d later was identified with morphology, transmission electron microscopy, phenotype and mixed lymphocyte reaction stimulating activity. Results Cultured DCs expressed OX62 (the marker of rat DCs) by 99.36%. Most mature DCs were obtained in 12～14 d cultivation as determined with morphology and high level expression of major histocompatibility complex (MHC) class II, CD80 and CD86, and they effectively stimulated MLR and present soluble ovalbumin to naive T cells. Conclusion A method to generate large number of DCs from rat bone marrow in vitro is established, and may contribute to further study on the role of DCs in xenotransplantation and tolerance.