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目的:建立正常CD33~+细胞与肿瘤细胞共培养系统,模拟肿瘤局部髓系来源抑制细胞的诱导过程,观察吲哚胺2,3双加氧酶在MDSCs中表达及其免疫抑制作用,并探讨MDSCs中IDO表达相关机制。方法:免疫磁珠分选健康供者CD33~+细胞,体外与乳腺癌细胞系MDA-MB-231共孵育诱导MDSCs,以单独培养CD33~+细胞为对照组,培养2天后收获实验组MDSCs及对照组CD33~+细胞,流式细胞仪检测细胞表型;实时定量PCR和Western Blotting方法检测TDO和STAT3的表达与活化情况;AnnexinV-FITC的方法检测MDSCs对T细胞促凋亡作用,并观察IDO在MDSCs诱导T细胞凋亡中的作用。结果:正常CD33~+细胞与MDA-MB-231共孵育2天后,出现一群表型为Lin~-CD33~+13~+CD14~- CD15~-的MDSCs,体外诱导的MDSCs中IDOmRNA及蛋白表达增加、STAT3蛋白磷酸化增强,经过STAT3磷酸化抑制剂JSI-124预处理后,MDSCs中IDO蛋白表达明显降低并可诱导高百分比T细胞凋亡,经IDO抑制剂1-MT处理后T细胞凋亡显著降低。结论:体外将乳腺癌细胞系与正常CD33~+细胞共孵育可以诱导出具有独特表型特征和免疫抑制活性的MDSCs,而STAT3磷酸化导致的IDO表达升高可能是MDSCs抑制T细胞功能的主要机制之一。
OBJECTIVE: To establish a co-culture system of normal CD33 + cells and tumor cells, to simulate the induction process of inhibitory cells derived from local myeloid origin and to observe the expression and immunosuppressive effects of indoleamine 2,3 dioxygenase in MDSCs Mechanism of IDO expression in MDSCs. Methods: CD33 ~ + cells were sorted by immunomagnetic beads and incubated with breast cancer cell line MDA-MB-231 in vitro to induce MDSCs. CD33 ~ + cells were cultured in vitro as control group. The control group CD33 ~ + cells, flow cytometry cell phenotype; real-time quantitative PCR and Western Blotting method to detect TDO and STAT3 expression and activation; AnnexinV-FITC method to detect MDSCs on T cells apoptosis and observed Role of IDO in the induction of T cell apoptosis by MDSCs. Results: After incubated with normal CD33 + cells and MDA-MB-231 for two days, a group of MDSCs with phenotypes of Lin ~ -CD33 ~ +13 ~ + CD14 ~ ~ CD15 ~ ~ appeared. IDO mRNA and protein expression in MDSCs induced in vitro STAT3 protein phosphorylation was enhanced. After pretreatment with STAT3 phosphorylation inhibitor JSI-124, the expression of IDO protein in MDSCs was significantly decreased and a high percentage of T cell apoptosis was induced. After IDO inhibitor 1-MT treatment, T cell apoptosis Death significantly reduced. CONCLUSION: MDSCs with unique phenotypic characteristics and immunosuppressive activity can be induced by co-incubation of breast cancer cell lines with normal CD33 + cells in vitro. The increased expression of IDO induced by STAT3 phosphorylation may be the main inhibitory effect of MDSCs on T cell function One of the mechanisms.