南方医科大学学报

南方医科大学学报 ›› 2019, Vol. 39 ›› Issue (12): 1427-1435.doi: 10.12122/j.issn.1673-4254.2019.12.06

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二甲双胍抑制酸性环境下的调节性T细胞增殖及功能

韦莉莉，罗周翔，李金龙，李红卫，梁 瑶，李金连，申玉婷，李天柏，宋 捷，胡志明   

  • 出版日期:2019-12-27 发布日期:2019-12-20
  • 基金资助:

Metformin inhibits proliferation and functions of regulatory T cells in acidic environment

  

  • Online:2019-12-27 Published:2019-12-20

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摘要： 目的 探讨二甲双胍对酸性环境下的调节性T细胞（Treg）的调控及二甲双胍的抗肿瘤作用。 方法 经磁珠分选得到CD4+CD25+ Treg细胞。在pH 7.4或pH 6.7环境加入二甲双胍干预的条件下，培养Treg或T细胞（Tcon）24~72 h后，采用流式细胞术检测细胞增殖、凋亡及Foxp3表达情况，采用Real-time PCR检测糖代谢相关基因水平。动物实验中，将32只C57BL/6雄性小鼠单次皮下注射RM-1细胞建立小鼠前列腺癌模型，随机分为PBS对照组、二甲双胍治疗组、疫苗治疗组及联合治疗组（各组n=8）。于接种后第4天开始，按100 mg· kg-1· d-1给予二甲双胍治疗组及联合治疗组小鼠腹腔注射二甲双胍，给予疫苗治疗组及联合治疗组小鼠每4 d一次肌注疫苗。PBS注射作为对照。连续监测肿瘤大小，在植瘤后第25天处死小鼠获取肿瘤及血液样本。采用流式细胞术检测瘤内及外周血内CD4+、CD8+、CD4+Foxp3+细胞亚群的变化。 结果 较中性缓冲条件，酸性环境下Treg细胞活性增强（P<0.05），而Tcon细胞增殖受到抑制（P<0.001）。QPCR结果显示，酸性环境较正常酸碱环境下，Treg细胞OXPHOS相关基因pgc1a（P<0.001）及cox5b（P<0.01）水平增高，而Tcon细胞内相关基因水平变化不显著。酸性环境下Treg细胞凋亡显著减少（P<0.01）、Foxp3+细胞比例增加（P<0.001）、胞内pH值偏碱性（P<0.001），加入二甲双胍，逆转了Treg细胞的酸性耐受。但是，二甲双胍对Tcon细胞影响不显著。动物实验中，二甲双胍（P<0.05）或疫苗（P<0.01）单独治疗均可减小肿瘤体积，而联合治疗组肿瘤体积减小最多（P<0.001）。二甲双胍单独治疗对肿瘤内CD4+细胞、CD8+细胞影响不明显，但可显著降低CD4+Foxp3+细胞比例（P<0.05），而疫苗单独治疗显著提高肿瘤内CD4+细胞、CD8+细胞（P<0.001），但CD4+Foxp3+细胞也升高（P<0.05）。联合治疗组肿瘤内CD4+细胞、CD8+细胞增高最多（P<0.01），CD4+Foxp3+细胞比例较疫苗单独治疗组下降（P<0.01）。结论 二甲双胍抑制酸性环境下的调节性T细胞增殖及功能，并且在体内通过减少Treg细胞比例提高肿瘤疫苗效果，发挥抗肿瘤功能。

Abstract: : Objective To investigate the regulatory effect of metformin on regulatory T cells (Treg) in acidic environment. Methods CD4 + CD25 + Treg cells were obtained by magnetic bead sorting. Treg and conventional T cells (Tcon) cells were cultured for 24-72 h in pH 7.4 or pH 6.7 medium, and the cell proliferation, apoptosis and Foxp3 expression were detected by flow cytometry. Real-time PCR was used to detect the expression levels of the genes related with glucose metabolism. Thirty-two C57BL/6 male mouse models bearing subcutaneous prostate cancer xenograft derived from RM-1 cells were randomized into 4 equal groups for treatment with PBS, metformin, tumor vaccine, or both metformin and the vaccine. The treatment started on the 4th day following tumor cell injection, and metformin (100 mg/kg) or PBS was administered by intraperitoneal injection on a daily basis; the vaccine was intramuscularly injected every 4 days. The tumor size was continuously monitored, and the mice were euthanized on day 25 after tumor implantation to obtain tumor and blood samples. Flow cytometry was used to detect the changes in CD4 + , CD8 +, CD4 + Foxp3 + cell subsets in the tumor tissue and peripheral blood. Results Treg cells showed significantly enhanced proliferation (P<0.05) while the proliferation of Tcon cells was suppressed in acidic medium (P<0.001). Treg cells cultured in acidic medium showed significantly increased expressions of OXPHOS-related genes pgc1a (P<0.001) and cox5b (P<0.01), which did not vary significantly in Tcon cells in acidic medium. Treg cells exhibited significantly decreased apoptosis in acidic medium (P<0.01) with increased Foxp3 + cells (P<0.001) and intracellular alkaline levels (P<0.01). Metformin obviously reversed the acid tolerance of Treg cells without producing significant effect on Tcon cells. In the animal experiment, both metformin (P<0.05) and vaccine (P<0.01) alone reduced the tumor volume, but their combined treatment more potently reduced the tumor volume (P<0.001). Metformin alone did not obviously affect CD4+ cells or CD8+ cells but significantly decreased the percentage of CD4+Foxp3+ (P<0.05); the vaccine alone significantly increased CD4+ cells and CD8+ cells (P<0.001) and also the percentage of CD4+Foxp3+ cells (P<0.05). The combined treatment, while reducing the percentage of CD4+Foxp3+cells to a level lower than that in the vaccine group (P<0.01), produced the strongest effect to increase CD4+ cells and CD8+ cells (P< 0.01). Conclusion Metformin can inhibit the proliferation and function of regulatory T cells in an acidic environment and enhance the effect of tumor vaccine by reducing the proportion of Treg cells in vivo to achieve the anti-tumor effect.