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目的 优化小鼠Lewis肺癌(LLC)皮下肿瘤组织中髓源性抑制细胞(MDSC)的流式分选流程，提升单细胞制备效率和分选纯度，为后续功能研究提供高质量细胞来源。方法 取C57BL/6小鼠皮下LLC肿瘤组织，首先比较2种酶学消化体系对单细胞制备效果的影响。经死活染料(Live/Dead)及抗小鼠CD45、CD11b、Gr-1抗体标记后，上样至Beckman CytoFLEX流式细胞分选仪。其次采用常规与优化2种设门策略，优化策略以CD45+细胞群为起始门直接圈定MDSC富集区域，后续筛选CD11b~+Gr-1+细胞。最后通过流式细胞术检测纯度，台盼蓝染色检测活率，并以RT-qPCR分析MDSC特征基因Arg1、Nos2、IL-10、S100A8/A9的表达。结果 优化的消化体系显著提高单细胞数量及活率。优化圈门策略后，分选阳性率显著提升。Arg1、Nos2、IL-10及S100A8/A9基因在分选细胞中高表达，提示分选后细胞保持MDSC特征性功能。结论 该研究通过优化“组织消化体系和流式设门策略”，建立了高效分离肿瘤组织MDSC的流程，在保证抗原结构完整的同时提高了单细胞制备得率与纯度，为后续MDSC功能与代谢研究提供可靠的方法学基础。
Abstract：Objective To optimize the flow cytometric sorting workflow for myeloid-derived suppressor cells(MDSCs) from murine Lewis lung carcinoma(LLC) subcutaneous tumor tissues, improving single-cell suspension preparation efficiency and sorting purity, thereby providing high-quality cells for subsequent functional studies.Methods Subcutaneous LLC tumor tissues were harvested from C57BL/6 mice, and two enzymatic digestion protocols were first compared to evaluate their effects on single-cell suspension preparation. After staining with a viability dye(Live/Dead) and fluorochrome-conjugated anti-mouse antibodies against CD45, CD11b, and Gr-1, cells were loaded onto a Beckman CytoFLEX cell sorter. Next, conventional and optimized gating strategies were applied. In the optimized strategy, the CD45+ population was used as the initial gate to directly define an MDSCenriched region, followed by selection of CD11b~+Gr-1+ cells. Finally, purity was assessed by flow cytometry, viability was determined by trypan blue staining, and RT-qPCR was performed to measure the expression of MDSC signature genes, including Arg1, Nos2, IL-10, and S100A8/A9.Results The optimized enzymatic digestion protocol significantly increased the yield and viability of single cells. After optimization of the gating strategy, the postsort positivity rate(purity) was markedly improved. Arg1, Nos2, IL-10 and S100A8/A9 were highly expressed in the sorted cells, indicating that the isolated cells retained characteristic MDSC features and functional signatures.Conclusion By optimizing both the tissue digestion protocol and the flow cytometric gating strategy, we establish an efficient workflow for isolating MDSCs from tumor tissues. This method improves the yield and purity of singlecell preparations while preserving antigen integrity, providing a reliable methodological foundation for subsequent studies on MDSC function and metabolism.
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基本信息:
DOI：10.19405/j.cnki.issn1000-1492.2026.07.001
中图分类号:R730.3
引用信息:
[1]杜佳,饶路路,侯昕,等.流式细胞术分选小鼠肿瘤髓源性抑制细胞的方案优化及鉴定[J].安徽医科大学学报,2026,61(07):1163-1169.DOI:10.19405/j.cnki.issn1000-1492.2026.07.001.
基金信息:
国家自然科学基金项目(编号：82170603、81502123); 宁波市自然科学基金重点项目(编号：2024J033)~~
2026-05-13
2026-05-13
2026-05-13