从黑色素瘤荷瘤小鼠中分离纯化髓系抑制性细胞

Purification of myeloid derived suppressor cells from mice bearing transplanted melanoma

目的从黑色素瘤荷瘤小鼠脾脏中分离得到CD11b+Gr-1+的髓系抑制性细胞(Myeloid derived suppressor cells,MDSCs)。方法给C57BL/6小鼠注射LPS或者小鼠黑色素瘤细胞株B16BL6后,取小鼠的脾脏细胞,检测其中CD11b+Gr-1+的MDSCs细胞的比例。通过流式细胞仪(flow cytometry,FCM)分选出CD11b+Gr-1+的细胞,培养24小时后,CBA(cytometric bead array)法检测其分泌IL-10的情况;提取CD11b+Gr-1+细胞的总RNA,利用RT-PCR的方法检测其表达IFN-β、TGF-β、iNOS、ARG-1和IDO的情况。结果与注射LPS相比,注射B16BL6细胞株的小鼠脾脏中CD11b+Gr-1+的MDSCs细胞的比例更高;利用FCM分选得到CD11b+Gr-1+的MDSCs,进一步鉴定发现其表达IL-10、IFN-β、TGF-β、iNOS、ARG-1和IDO。与先前报道的MDSCs的特征相一致。结论可从B16BL6荷瘤小鼠脾脏中获得高纯度的MDSCs用于实验研究。

Objective To purify the CD11b＋Gr-1 ＋MDSCs from splenic of mice bearing transplanted melanoma.Methods Splenic cells were extracted from the C57BL/6 mice which were injected with lipopolysaccharide （LPS） or B16BL6 cells and were double-stained with antibodies to CD11b and Gr-1 for flow cytometry analysis.CD11b＋Gr-1 ＋MDSCs were sorted from C57BL/6 mice injected with B16BL6 cells, and the expression of IFN-β, TGF-β, iNOS, ARG-1, IDO and IL-10 in the purified MDSCs was checked by RT-PCR or CBA kits.Results C57BL/6 mice injected with B16BL6 cells could produce higher percentages of CD 11b＋Gr-1 ＋MDSCs than C57BL/6 mice injected with LPS.As previously described, FCM-sorted MDSCs expressed IFN-β,TGF-β,iNOS,ARG-1,IDO and IL-10.Conclusions MDSCs can be isolated from C57BL/6 mice injected with B16BL6 cells by FCM sorting.