mTOR信号通路在调控卵巢癌CD4^(+)Treg糖代谢中的作用

Role of mTOR signaling pathway in regulating CD4^(+)Treg glucose metabolism in ovarian cancer

目的:初步探讨mTOR信号通路在卵巢癌患者CD4^(+)Treg(CD4^(+)CD25^(+)CD127low调节性T细胞)糖代谢过程中的作用。方法:流式细胞术检测卵巢癌患者(ovarian cancer,OC)、卵巢良性肿瘤患者(benign ovarian tumor,BOT)以及健康对照者(healthy controls,HC)外周血CD4^(+)Treg中mTOR^(+)细胞的百分比;建立CD4^(+)Treg与SKOV3共培养体系,检测共培养前后mTOR^(+)的CD4^(+)Treg百分比,使用雷帕霉素抑制mTOR信号后,采用比色法检测CD4^(+)Treg细胞糖摄取和糖酵解水平,采用实时荧光定量PCR(real-time fluorescence quantitative PCR,RT-PCR)和Western blot检测CD4^(+)Treg细胞糖代谢相关基因和蛋白的表达水平。结果:卵巢癌患者外周血中mTOR^(+)的CD4^(+)Treg百分比显著高于健康对照组[(77.4±8.12)%vs.(64.19±9.7)%,P<0.01],与SKOV3共培养后CD4^(+)Treg中mTOR^(+)细胞百分比明显升高(P<0.05),mTOR信号通路相关基因和蛋白表达水平也升高;抑制SKOV3生长环境中CD4^(+)Treg的mTOR信号后其糖代谢相关基因和蛋白表达水平显著下降,糖摄取(193.49±13.28 vs.174.05±14.58,P<0.01)和糖酵解水平(21.97±0.87 vs.4.85±1.54,P<0.001)也明显下降。结论:卵巢癌患者CD4^(+)Treg中mTOR及其下游信号通路明显活化,mTOR信号通路可通过调节CD4^(+)Treg的糖摄取和糖酵解水平,并调控糖代谢相关基因和蛋白的表达水平,影响CD4^(+)Treg的糖代谢进程。

Objective:To explore the role of mTOR signaling pathway in CD4^(+)regulatory T cell(Treg)glucose metabolism in ovarian cancer patients.Methods:Flow cytometry was used to detect the percentage of mTOR^(+)CD4^(+)Treg in peripheral blood with ovarian cancer(OC)patients,benign ovarian tumor(BOT)patients and healthy controls(HC).We established a coculture system of human CD4^(+)Treg with ovarian cancer cell SKOV3,and detected the percentage of mTOR^(+)CD4^(+)Treg before and after co⁃culture.After mTOR signal was inhibited by rapamycin,the glucose uptake and glycolysis levels of CD4^(+)Treg were detected by colorimetry,and the expression levels of genes and proteins related to glucose metabolism in CD4^(+)Treg were detected by real⁃time quantitative PCR and Western blot.Results:The percentage of mTOR^(+)CD4^(+)Treg in peripheral blood of ovarian cancer patients was significantly higher than that of healthy controls[(77.4±8.12)%vs.(64.19±9.7)%,P<0.01].After co⁃culture with SKOV3,the percentage of mTOR^(+)CD4^(+)Treg were elevated(P<0.05),and the levels of mTOR signaling pathway related genes and proteins also increased.After inhibition of mTOR signal of CD4^(+)Treg in SKOV3 growth environment,the glucose uptake(193.49±13.28 vs.174.05±14.58,P<0.01)and glycolysis level(21.97±0.87 vs.4.85±1.54,P<0.001)were decreased significantly.Glucose metabolism⁃related genes and proteins were also significantly reduced.Conclusion:mTOR and its downstream signaling pathway are significantly activated in peripheral blood CD4^(+)Treg of ovarian cancer patients,and mTOR signaling pathway can affect glucose metabolism of CD4^(+)Treg by regulating glucose metabolist⁃related genes and protein levels.