自噬对粒细胞样髓源性抑制细胞生存与功能的影响

目的: 初步探索自噬对Lewis肺癌移植瘤小鼠脾脏中粒细胞样髓源性抑制细胞（granulocytic myeloid derived suppressor cells，G-MDSCs）体外存活能力与抑制性功能分子的影响。方法: Lewis肺癌细胞系体外培养后，构建小鼠Lewis肺癌皮下移植瘤模型，通过免疫磁珠分选方法获得脾脏G-MDSCs，与荧光抗体（Ly-6C-FITC/Ly-6G-PE/CD11b-PE/CY5）孵育后，流式细胞术检测G-MDSCs细胞纯度；瑞吉染色后观察其形态特征；采用雷帕霉素和氯喹处理后，通过蛋白质印迹法和流式细胞术检测自噬标志性分子微管相关蛋白1-轻链3 Ⅱ（microtubule-associated protein 1 light chain 3 Ⅱ，LC3-Ⅱ）蛋白的表达水平。培养体系中加入雷帕霉素和自噬抑制剂3-甲基腺嘌呤（3-methy-ladenine，3-MA），7-AAD染色检测自噬对G-MDSCs生存的影响；提取细胞总蛋白后检测胞内精氨酸酶的水平。结果： 通过磁珠分选的方法可从小鼠脾脏中获得纯度较高的G-MDSCs，其细胞核呈环状或分叶状。雷帕霉素处理G-MDSCs后LC3-Ⅱ表达水平上调，3-MA抑制自噬作用后，G-MDSCs存活率降低, 精氨酸酶活性下降。结论： 自噬能够促进G-MDSCs体外存活并上调抑制性效应分子精氨酸酶的表达，提示自噬是调控G-MDSCs生物学功能的重要因素。

Effect of autophagy on survival and function of granulocytic myeloid derived suppressor cells

Objective: To investigate the effect of autophagy on the survival and suppressive function of granulocytic myeloid derived suppressor cells (G-MDSCs) obtained from the spleen of Lewis lung carcinoma-bearing mice. Methods: Subcutaneous transplant tumor in mice was constructed with cultured Lewis lung carcinoma cells. G-MDSCs were obtained through MACS sorting method from the spleen of tumor-bearing mice, after CD11b and Ly-6G/Ly-6C tagged, the purity of cells was detected by flow cytometry. Cells Morphological characteristics of G-MDSCs were identified with optical microscope after stained with Wright-Giemsa stain. G-MDSCs were cultured within Rapamycin and chloroquine, and then collected to detect the level of autophagosomes formation marker-microtubule associated protein 1light chain 3-Ⅱ (LC3-Ⅱ) by Western blotting and flow cytometry. 7-amino-actinomycin D(7-ADD) staining was used to detect the effect of autophagy on the survival of G-MDSCs cultured with or without autophagy inhibitor 3-methyladenine (3-MA); the activity of arginase was measured with enzyme-linked immunosorbent assay(ELISA)in G-MDSCs. Results: G-MDSCs with high purity were obtained, and these cells have a typical polymorphonuclear morphology. The level of LC3-Ⅱ was up-regulated in G-MDSCs after treated with Rapamycin. Proportion of living cells and arginase activity were decreased in G-MDSCs with 3-MA treatment. Conclusion: Autophagy facilitates the survival and enhances the level of arginase in G-MDSCs in vitro. It may be implied that autophagy is involved in regulating the biological function of G-MDSCs. ［Key words］granulocytic myeloid derived suppressor cells; autophagy; Lewis lung carcinoma; 3 methyladenine; arginase; immunotherapy