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目的研究荷瘤小鼠脾脏中髓源抑制细胞(myeloid derived suppresser-cell,MDSCs)的分选与鉴定方法。方法常规培养小鼠肾癌细胞(Renca细胞),于Balb/c小鼠皮下建立小鼠肾癌模型;分离小鼠脾脏制成单细胞悬液,磁珠分选Gr-1+CD11b+双阳性的MDSCs;台酚蓝染色检测细胞存活率;流式细胞仪(flow cytometry,FCM)测其细胞纯度;显微镜下观察细胞形态;免疫荧光测其表面Gr-1、CD11b荧光表达情况;RT-PCR检测Cox2和Arg-1的m RNA的表达情况;小鼠皮下成瘤实验观察MDSCs促肿瘤生长。结果成功建立小鼠肾癌模型,磁珠分选后经FCM检测Gr-1+CD11b+双阳性的MDSCs细胞可达92.3%,显著高于分选前(P<0.01);免疫荧光鉴定结果显示:分选后的细胞形态完整,Gr-1和CD11b的荧光表达于细胞膜且2种荧光可以融合;RT-PCR检测分选后的MDSCs细胞群的Cox2和Arg-1的m RNA相对表达量显著高于分选前(P<0.05);小鼠皮下成瘤观察到MDSCs组与实验对照组有显著差异(P<0.05)。结论用免疫磁珠从荷瘤小鼠脾脏中成功分选得到MDSCs,具有较高纯度和良好的生物活性,为后续实验提供了理想的细胞来源。
Objective To study the methods of sorting and identification of myeloid derived suppresser-cells (MDSCs) in spleen of tumor-bearing mice. Methods Mouse kidney cancer cells (Renca cells) were cultured routinely, and mouse kidney cancer model was established subcutaneously in Balb / c mice. Single spleen cells were isolated from the spleens of mice and screened for Gr-1 + CD11b + double positive MDSCs; cell viability was assayed by trypan blue staining; cell purity was measured by flow cytometry (FCM); cell morphology was observed under a microscope; fluorescence staining of Gr-1 and CD11b on the surface was detected by immunofluorescence; Cox2 and Arg-1 m RNA expression; subcutaneous tumor formation in mice MDSCs tumor growth. Results The mouse renal cell carcinoma model was established successfully. The number of Gr-1 + CD11b + double positive MDSCs after magnetic bead sorting was 92.3% which was significantly higher than that before sorting (P <0.01). Immunofluorescence results showed that: After sorting, the morphology of the cells was intact. The fluorescence of Gr-1 and CD11b was expressed on the cell membrane and the two fluorescences could be fused. The mRNA expression of Cox2 and Arg-1 in MDSCs were significantly higher after RT-PCR Before sorting (P <0.05), there was significant difference between MDSCs group and experimental control group (P <0.05). Conclusion MDSCs were successfully sorted from the spleens of tumor-bearing mice by immunomagnetic beads, with high purity and good biological activity, which provided an ideal cell source for subsequent experiments.