摘要
目的探讨塞来昔布在自然杀伤(NK)细胞对食管癌细胞的杀伤作用中的影响及其机制。方法选择人食管癌细胞系EC-1(所有细胞均来源于中国科学院上海细胞库)按实验转染方案随机分组,分为实验组(si-ULBP1)、对照组(si-ULBP1 NC)。采用实时定量反转录聚合酶链反应(qPCR)检测EC-1细胞中ULBP1、ULBP2、ULBP3、MICB、PD-L1、4-IBBL在30μmol/L塞来昔布处理0、6、12、24、36、48、72 h后的表达;小干扰RNA(siRNA)敲除48 h后,实验组和对照组EC-1细胞ULBP1表达量;采用细胞计数试剂盒(CCK-8)检测10、20、30、40、50、60、80、100μmol/L浓度下,塞来昔布处理24、36、48 h对EC-1细胞生长的影响;采用流式细胞术分析体外培养14 d后NK细胞、CD107a阳性细胞、干扰素α(IFN-α)表达阳性细胞群的比例;在1∶1、5∶1、10∶1效靶比下,NK细胞对塞来昔布处理EC-1细胞的杀伤效应;siRNA敲除后,NK细胞对实验组和对照组EC-1细胞的杀伤效应。采用t检验比较两组间差异。结果CCK-8结果显示,在36 h不同浓度塞来昔布处理条件下,50μmol/L组细胞存活率低于10μmol/L组[(78.82±1.93)%比(91.66±1.50)%,t=9.12,P<0.05],60μmol/L组细胞存活率低于20μmol/L组[(57.65±6.42)%比(91.87±1.07)%,t=9.11,P<0.05],80μmol/L组细胞存活率低于30μmol/L组[(43.53±1.93)%比(90.16±1.50)%,t=33.12,P<0.05],100μmol/L组细胞存活率低于40μmol/L组[(19.79±1.50)%比(88.88±2.57)%,t=40.27,P<0.05]。qPCR结果显示ULBP1组表达量高于ULBP2、ULBP3、MICB、PD-L1、4-IBBL组(ULBP1组μ=6.37比ULBP2组μ=3.76,t=7.95,P<0.01,ULBP3组μ=2.57,t=12.14,P<0.01,MICA组μ=2.32,t=12.27,P<0.01,MICB组μ=1.55,t=12.13,P<0.01,PDL1组μ=0.41,t=17.06,P<0.01,4-IBBL组μ=0.70,t=13.53,P<0.01);流式细胞术结果显示,NK细胞对塞来昔布处理组EC-1细胞杀伤效应高于对照组[1∶1(12.23±2.70)%比(24.06±1.58)%,t=6.48,P<0.01、2∶1(28.57±2.93)%比(45.49±3.38)%,t=6.54,P<0.01、4∶1(61.50±3.39)%比(74.36±2.94)%,t=4.97,P<0.01、8∶1(88.12±2
Objective This study aims to investigate the mechanism of the cytotoxic effect of Celecoxib on esophageal cancer cells by natural killer(NK)cells.Methods All cells were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences.Randomized grouping was conducted for the selection of the human esophageal cancer cell line EC-1.The cells were divided into an experimental group(si-ULBP1)and a control group(si-ULBP1 NC).Real-time quantitative reverse transcription polymerase chain reaction(qPCR)was employed to detect the expression of ULBP1,ULBP2,ULBP3,MICB,PD-L1,and 4-IBBL in EC-1 cells after treatment with 30μmol/L of celecoxib for 0,6,12,24,36,48,and 72 hours.The expression levels of ULBP1 in the experimental and control groups of EC-1 cells were measured after small interfering RNA(siRNA)knockdown for 48 hours.Cell counting kit-8(CCK-8)was used to evaluate the effect of celecoxib treatment at concentrations of 10,20,30,40,50,60,80,and 100μmol/L for 24,36,and 48 hours on the growth of EC-1 cells.Flow cytometry analysis was performed to assess the proportions of NK cells,CD107a-positive cells,and interferon-alpha(IFN-α)expressing cells in the in vitro cultured cells after 14 days.The cytotoxic effect of NK cells against celecoxib-treated EC-1 cells was evaluated at effector-to-target ratios of 1∶1,5∶1,and 10∶1.The cytotoxicity of NK cells against the experimental and control groups of EC-1 cells was assessed after siRNA knockdown.T-test was used to compare the differences between the two groups.Results The CCK-8 results showed that under different concentrations of Selpercatinib treatment for 36 hours,the cell viability in the 50μmol/L group was lower than that in the 10μmol/L group[(78.82±1.93)%vs.(91.66±1.50)%,t=9.12,P<0.05],60μmol/L group was lower than 20μmol/L group[(57.65±6.42)%vs.(91.87±1.07)%,t=9.11,P<0.05],80μmol/L group was lower than 30μmol/L group[(43.53±1.93)%vs.(90.16±1.50)%,t=33.12,P<0.05],and 100μmol/L group was lower than 40μmol/L group[(19.79±1.50)%vs.(88.88±2.5
作者
李博文
徐一帆
黄岚
李子豪
刘亚飞
齐宇
Li Bowen;Xu Yifan;Huang Lan;Li Zihao;Liu Yafei;Qi Yu(Department of Thoracic Surgery,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China;Translational Medicine Center of the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)
出处
《中华实验外科杂志》
CAS
北大核心
2023年第10期2035-2038,共4页
Chinese Journal of Experimental Surgery
基金
河南省科学技术厅科技攻关项目(222102310239)
河南省卫生健康委员会中青年卫生健康科技创新人才培养领军人才项目(YXKC2022014)。
关键词
食管癌
塞来昔布
自然杀伤细胞
Esophageal cancer
Celecoxib
Natural killer cells
