雷帕霉素对γδT细胞体外增殖和细胞毒活性的影响  

Effects of Rapamycin on proliferation and cytotoxicity ofγδT cells in vitro

作　　者：曾雪娇 张瑞[1] 谢仁古丽·阿力木 曲建华[1] ZENG Xue-jiao;ZHANG Rui;XIERENGULI Alimu;QU Jian-hua(Center of Hematology,The First Affiliated Hospital of Xinjiang Medical University(Hematology Institute of Xinjiang Uygur Autonomous Region),ürümqi 830054,Xinjiang,China)

机构地区：[1]新疆医科大学第一附属医院血液病中心(新疆维吾尔自治区血液病研究所),新疆乌鲁木齐830054

出　　处：《生物医学工程与临床》2024年第1期1-8,共8页Biomedical Engineering and Clinical Medicine

基　　金：国家自然科学基金资助项目(82160034,81860033);新疆维吾尔自治区科技计划项目(2022D14008)。

摘　　要：目的探讨雷帕霉素与Torin2对人γδT细胞体外培养增殖和细胞毒活性的影响及相关机制。方法选择10例健康志愿者,其中男性5例,女性5例;平均年龄68.0岁(标准差4.7岁)。选择人慢性淋巴细胞白血病(CLL)肿瘤细胞株MEC-1。从志愿者外周静脉血获得外周血单个核细胞(PBMC),经处理获得γδT细胞。体外增殖培养的人γδT细胞在第10天观察细胞形态特征,流式细胞术检测人γδT细胞百分率。γδT细胞分别用100 nmol/L雷帕霉素(雷帕霉素组)和Troin2处理(Troin2组),同时用RPMI 1640培养液作为空白对照(对照组),分别培养24、48 h,计算活细胞数。采用不同效靶比(1∶1、3∶1、9∶1、18∶1、36∶1),乳酸脱氢酶(LDH)释放法检测人γδT细胞对MEC-1细胞的杀伤作用。流式细胞术检测各组白细胞介素(IL)-17的表达。Western blot检测磷脂酰肌醇3-激酶(PI3K)、丝苏氨酸激酶(AKT)、磷酸化AKT(p-AKT)、哺乳动物雷帕霉素靶蛋白(mTOR)、磷酸化mTOR(p-mTOR)、起始因子4E结合蛋白1(4EBP-1)的表达。结果扩增前人γδT细胞百分含量为4.70%±1.17%,经10 d扩增且纯化后γδT细胞百分含量为94.20%±2.18%。雷帕霉素组24 h扩增倍数为1.45±0.20,48 h扩增倍数为2.71±0.06,与对照组间差异无统计学意义(P>0.05);Torin2组24 h扩增倍数为1.14±0.05,48 h扩增倍数为2.09±0.06,均显著低于雷帕霉素组及对照组(P<0.05)。雷帕霉素组γδT细胞的杀伤活性分别为0.05%±0.01%(1∶1)、10.84%±0.88%(3∶1)、23.02%±0.65%(9∶1)、50.74%±2.96%(18∶1)、77.75%±0.55%(36∶1),均显著高于对照组(P<0.05);Torin2组γδT细胞的杀伤活性分别为0.06%±0.01%(1∶1)、4.06%±0.84%(3∶1)、10.72%±2.97%(9∶1)、18.20%±2.83%(18∶1)、36.18%±2.19%(36∶1),均低于雷帕霉素组及对照组(P<0.05)。雷帕霉素组IL-17表达为(32.7±1.7)%,Troin2组IL-17表达为(12.2±1.4)%,均显著低于对照组(73.1±2.7)%(均P<0.05)。且Troin2组抑制γδT分泌IL-17程度更明显,差异有�Objective To investigate the effects of Rapamycin and Torin2 on the proliferation and cytotoxicity of humanγδT cells in vitro and the related mechanisms.Methods A total of 10 healthy volunteers were enrolled,which included 5 males and 5 females with mean age of 68.0 years old(standard deviation 4.7 years old).The human chronic lymphocytic leukemia(CLL)tumor cell line MEC-1 was selected,the peripheral blood mononuclear cells(PBMC)were obtained from peripheral venous blood of volunteers,andγδT cells were obtained after treatment.The morphological characteristics of humanγδT cells cultured in vitro were observed on the 10th day,and the percentage of humanγδT cells was detected by flow cytometry.TheγδT cells were treated with 100 nmol/L Rapamycin(Rapamycin group)and Troin2(Troin2 group),respectively,and RPMI 1640 culture medium was used as blank control(control group).The cells were cultured for 24-hour and 48-hour,and live cell counts was calculated.The killing effect of humanγδT cells on MEC-1 cells was detected by lactate dehydrogenase(LDH)release method with different effector-target ratios(1∶1,3∶1,9∶1,18∶1,36∶1).The expression of interleukin(IL)-17 in each group was detected by flow cytometry,and Western blot was used to detect the expression of phosphatidylinositol 3-kinase(PI3K),serine threonine kinase(AKT),phosphorylated AKT(p-AKT),mammalian rapamycin target protein(mTOR),phosphorylated mTOR(p-mTOR)and initiation factor 4E binding protein 1(4EBP-1)protein.Results The percentage ofγδT cells was 4.70%±1.17%,and the percentage ofγδT cells was 94.20%±2.18%after 10-day amplification and purification.In Rapamycin group,the 24-hour amplification factor was 1.45±0.2,and 48-hour amplification factor was 2.71±0.06,and there was no significant difference between Rapamycin group and control group(P>0.05).The amplification folds of Torin2 group at 24-hour and 48-hour were 1.14±0.05 and 2.09±0.06,respectively,which were significantly lower than those of Rapamycin group and control group(P<0.

关 键 词：雷帕霉素 ΓΔT细胞 MEC-1 MTOR 白细胞介素(IL)-17 细胞增殖 细胞活性 细胞毒活性 

分 类 号：R96[医药卫生—药理学]