四君子汤调节NKG2A表达影响NK细胞抗结肠癌作用  

Si Junzitang Regulates NKG2A Expression to Improve Anti-colon Cancer Function of NK Cells

作　　者：陈良燕 朱月伊 王鑫鑫 石晓兰[1] CHEN Liangyan;ZHU Yueyi;WANG Xinxin;SHI Xiaolan(Putuo Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 200062,China)

机构地区：[1]上海中医药大学附属普陀医院,上海200062

出　　处：《中国实验方剂学杂志》2022年第15期28-34,共7页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：上海市普陀区卫生系统自主创新科研资助项目(重点项目)(ptkwws201804);全国(上海市)名中医普陀传承工作室建设项目(ptzygzs2101)。

摘　　要：目的:探讨中药复方四君子汤通过调节NKG2A的表达影响自然杀伤(NK)细胞抗结肠癌的作用。方法:分离、纯化来自健康人外周血NK细胞进行培养,分别构建人NK细胞单培、HCT116细胞单培和NK细胞与HCT116细胞共培模型,实时荧光定量聚合酶链式反应(Real-time PCR)检测NK细胞中自然杀伤细胞2族成员A(NKG2A)、白细胞介素(IL)-15及结肠癌细胞中组织相容性白细胞抗原E(HLA-E)mRNA的表达,酶联免疫吸附测定法(ELISA)检测NK细胞中IL-15分泌;噻唑蓝(MTT)比色法筛选IL-15的浓度,检测四君子汤和IL-15对NK细胞活性和共培模型中HCT116细胞活性的影响,Real-time PCR检测四君子汤和IL-15对NK细胞中NKG2A和HCT116中HLA-E mRNA表达的影响;使用Anti-NKG2A抗体(M)阻断NKG2A/HLA-E通路,MTT比色法检测共培模型中HCT116细胞增殖情况。结果:与单培组比较,NK细胞和HCT116细胞相互作用引起NK细胞的NKG2A和HCT116中的HLA-E mRNA表达均上调(P<0.05);IL-15 mRNA表达和分泌也增加(P<0.05);IL-15可增强NK细胞活性和抗结肠癌作用(P<0.01),也可上调NKG2A表达(P<0.05);与空白组比较,四君子汤组、四君子汤+IL-15组的NK细胞活性和抗结肠癌作用进一步增加(P<0.01),Real-time PCR结果显示,与空白组比较,四君子汤组和四君子汤+IL-15组中NK细胞NKG2A表达下调(P<0.05),共培模型中四君子汤组HCT116中HLA-E表达下调(P<0.01);与相应未加M组比较,M组与IL-15+M组HCT116细胞增殖被抑制(P<0.01)。结论:NK细胞与结肠癌细胞相互作用可引起NKG2A-HLAE通路活化导致NK功能受损,四君子汤可抑制该信号激活从而恢复NK细胞抗结肠癌作用。Objective:To explore the mechanism of Si Junzitang in regulating the expression of NKG2A to affect the anti-colon cancer function of natural killer(NK)cells.Method:NK cells isolated from healthy honors were cultured and used to construct the three incubation models of NK cells,human colon cancer HCT116 cells,and NK cells + HCT116 cells(co-incubation).real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)was conducted to determine the mRNA levels of natural killer group 2 member A(NKG2A)and interleukin(IL)-15 in NK cells,as well as the mRNA level of histocompatibility leucocyte antigen E(HLA-E)in HCT116 cells.The secretion of IL-15 was detected by enzyme-linked immunosorbent assay(ELISA).Methyl thiazolyl tetrazolium(MTT) assay was employed to determine the applicable concentration of IL-15 and test the effects of Si Junzitang and IL-15 on the activities of NK cells and the HCT116cells in the co-incubation model.The effects of Si Junzitang and IL-15 on the mRNA levels of NKG2A in NK cells and HLA-E in HCT116 cells were detected by Real-time PCR.Monalizumab(M,anti-NKG2A mab)was used to block the NKG2A-HLA-E pathway in co-incubation model,and then the proliferation of HCT116 cells was detected by MTT assay.Result:The interaction of NK cells and HCT116 cells up-regulated the mRNA levels of NKG2A in NK cells and HLA-E in HCT116 cells(P<0.05),as well as the expression level and secretion of IL-15(P<0.05).Compared with the blank group,Si Junzitang and Si Junzitang + IL-15 promoted the proliferation and improved the anti-colon cancer function of NK cells(P<0.01).Furthermore,they downregulated the mRNA levels of NKG2A in NK cells and HLA-E in the HCT116 cells co-incubated with NK cells(P<0.01).M and IL-15 + M inhibited the proliferation of HCT116 cells compared with the groups without M(P<0.01).Conclusion:The interaction of NK cells and HCT116 cells can induce activation of NKG2A-HLA-E pathway to impair NK cell function.Si Junzitang can inhibit the activation of NKG2A-HLA-E pathway to restore the anti

关 键 词：NK细胞 自然杀伤细胞2族成员A(NKG2A) 组织相容性白细胞抗原E(HLA-E) 白细胞介素(IL)-15 结肠癌 四君子汤 

分 类 号：R22[医药卫生—中医基础理论] R242[医药卫生—中医学] R2-031R285.5