摘要
目的:通过体外实验观察自然杀伤细胞(NK细胞)联合他莫昔芬(TAM)对乳腺癌细胞(BCC)的协同杀伤作用,初步探讨其作用机制。方法:选用3种受体表达程度不同的BCC,设置空白对照组及TAM各浓度/时间梯度实验组,MTT法检测各组细胞增殖抑制率,并确定最终实验浓度为5μmol·L-1。设自然释放组、最大释放组、TAM组、NK细胞组和联合实验组(BCC+NK细胞+TAM),钙黄绿素-AM释放法观察不同效靶比下两者的联合杀伤作用。设空白对照组(NK细胞)、NK细胞+TAM组、NK细胞+BCC组和联合实验组,ELISA法检测各组NK细胞中TNF-α和IFN-γ水平。设空白对照组(NK细胞)、NK细胞+TAM组、NK细胞+BCC组和联合实验组,流式分析法分别检测NK活化性受体(NKp46)、NK抑制性受体(CD158a、CD158b、CD158b2和CD158e)表达水平。设空白对照组(BCC)、BCC+TAM组、BCC+NK细胞组和联合实验组,流式细胞术检测各组细胞中活化性配体(MICA、ULBP1和ULBP2)的表达水平。结果:MTT法检测,TAM对3种BCC的增殖抑制率具有明显的时间和浓度依赖性(P<0.05)。钙黄绿素-AM释放法检测,随着效靶比的增加,与NK细胞组比较TAM组BCC杀伤率明显升高(P<0.01),且联合组杀伤率明显高于NK细胞组和TAM组(P<0.05)。ELISA法检测,与空白对照组比较,各实验组无论有无BCC存在,NK细胞中分泌TFN-α和IFN-γ水平均升高(P<0.05或P<0.01),且与TAM联合后TFN-α和IFN-γ水平明显升高(P<0.05)。流式细胞术检测,与空白对照组比较,各实验组NK细胞中NKp46表达水平均升高(P<0.05);各实验组NK细胞中CD158a、CD158b、CD158b2和CD158e表达水平均明显下降(P<0.05);各实验组BCC中MICA、ULBP1和ULBP2表达水平均明显升高(P<0.05)。结论:NK细胞与TAM联合体外杀伤BCC具有协同作用,其协同作用机制可能为TAM能够通过促进NK细胞分泌TNF-α和IFN-γ而增强其杀伤能力;通过增加NK细胞活化性受体和活化性配体表达水平、降低NK细胞抑制性受体表达水平,进而增加NK细胞的杀伤能力。
Objective:To investigate the synergistic killer effect of natural killer cells(NK cells)combined with tamoxifen(TAM)on breast cancer cells(BCC)through the experiment in vitro,and to explore its mechanism.Methods:Three kinds of BCC with different receptor expression levels were selected for the experiment.Blank control group,different concentrations of TAM groups and different time groups were set up.MTT assay was used to detect the inhibitory rates of proliferation of cells,and the final experiment concentration of 5μmol·L-1 was determined.The cells were divided into natural-release group,largest-release group,TAM group,NK cells group,and combined-experimental group(BCC+NK cells+TAM),and the synergistic killer effect of NK cells combined with TAM in different effector-target ratios were detected with Calcein-AM release assay.In ELISA assay the cells were divided into blank control group(NK cells),NK cells+TAM group,NK cells+BCC group and combined-experimental group,and the levels of IFN-γand TNF-αin the NK cells in various groups were measured.In flow cytometry detection the cells were divided into blank control group(NK cells),NK cells+TAM group,NK cells+ BCC group,and combined-experimental group,and the expression levels of NKp46,CD158 a,CD158b,CD158b2,and CD158 ewere determined;while the cells were divided into blank control group(BCC),BCC+TAM group,BCC + NK cells group,and combined-experimental group,and the expression levels of the MICA,ULBP1 and ULBP2 were detected.Results:The MTT assay results showed that the inhibitory rates of proliferation of 3kinds of BCC had obvious time-and concentration-dependence(P〈0.05).The Calcein-AM release assay results showed that the killing-rates of BCC in TAM groups were increased with the increase effectortarget ratios of compared with NK cells group;and the killing-rate in combined experimental group was obviously higher than those in NK cells and TAM groups(P〈0.05).The ELISA assay results showed that the levels of TNF-αand IFN-γof NK cells in various experimental groups with BCC or not were increased compared with blank control group(P〈0.05 or P〈0.01);the levels were significantly increased when combined with TAM(P〈0.05).The flow cytometry results showed that the NKp46 expression levels in various experimental groups were elevated compared with blank control group(P〈0.05);the expression levels of CD158 a,CD158b,CD158b2,and CD158 ewere significantly decreased(P〈0.05);the expression levels MICA,ULBP1,and ULBP2 in BCC were significantly increased(P〈0.05).Conclusion:The NK cells combined with TAM has the synergistic killer effect on the BCCin vitro.The synergetic mechanism may be as follows:TAM could increase the secretion of TNF-αand IFN-γof NK cells to enhance their cytotoxicity;TAM also could up-regulate the expression levels of activating receptors and activating ligands,and down-regulate the expression levels of inhibitory receptors to increase the killing ability of NK cells.
作者
王迦南
冯辉
王倩
何悦铭
宋艳秋
朱继红
WANG Jianan FENG Hui WANG Qian HE Yueming SONG Yanqiu ZHU Jihong(Tumor Center, First Hospital, Jilin University, Changchun 130021, China Reproductive Medicine Center, First Hospital, Jilin University, Changchun 130021, China)
出处
《吉林大学学报(医学版)》
CAS
CSCD
北大核心
2017年第2期281-287,共7页
Journal of Jilin University:Medicine Edition
基金
吉林省科技厅自然科学基金资助课题(20160101028JC)