γ-分泌酶抑制剂3,5-二氟苯乙酰基-L-丙氨酰基-L-2-苯基甘氨酸叔丁酯与氯喹的交叉对话作用对胃癌MKN-45细胞增殖、凋亡的影响  

Effect ofγ-secretase inhibitor notch signaling pathway inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester and chloroquine on the proliferation and apoptosis of gastric cancer MKN-45 cells

作　　者：李良庆[1] 方祥建 林淳 潘敦[1] 郑光威[2] Li Liangqing;Fang Xiangjian;Lin Chun;Pan Dun;Zheng Guangwei(Department of Gastrointestinal Surgery,the First Affiliated Hospital of Fujian Medical University,Fuzhou 350005,China;Department of Emergency Surgery,the First Affiliated Hospital of Fujian Medical University,Fuzhou 350005,China)

机构地区：[1]福建医科大学附属第一医院胃肠外科,福州350005 [2]福建医科大学附属第一医院急诊外科,福州350005

出　　处：《中华实验外科杂志》2021年第3期409-412,共4页Chinese Journal of Experimental Surgery

基　　金：福建省医学创新课题(2019-CX-22);福建省自然科学基金重点项目(2020J02050)。

摘　　要：目的观察胃癌MKN-45细胞中Notch信号通路抑制剂3,5-二氟苯乙酰基-L-丙氨酰基-L-2-苯基甘氨酸叔丁酯(DAPT)对自噬的影响及自噬抑制剂氯喹(CQ)对Notch信号通道的影响,探讨DAPT联合CQ对MKN-45细胞增殖、凋亡的影响。方法将MKN-45细胞分成4个组,对照组(Control组,RPMI 1640完全培养基培养);氯喹组(CQ组,含40μmol/L氯喹的RPMI 1640完全培养基培养);γ-分泌酶抑制剂组(DAPT组,含80μmol/L DAPT的RPMI 1640完全培养基培养);联合用药组(Comb组,含40μmol/L CQ和80μmol/L DAPT的RPMI 1640完全培养基培养)。应用噻唑蓝(MTT)法、流式细胞仪检测细胞增殖、凋亡;应用透射电镜、蛋白质印迹法(Western blot)、反转录-聚合酶链反应(RT-PCR)检测细胞自噬水平;应用Western blot检测Notch信号通路表达。两组间比较采用独立样本t检验。结果MTT结果显示,Comb组[(55.09±1.38)%]细胞增殖能力明显低于DAPT组[(78.83±1.74)%]和CQ组[(64.82±1.89)%],差异均有统计学意义(t=18.514、7.185,P<0.01)。流式细胞结果显示,Comb组[(43.43±2.30)%]凋亡率明显高于DAPT组[(27.26±3.02)%]和CQ组[(22.16±2.24)%],差异均有统计学意义(t=7.339、11.415,P<0.01)。Western blot、RT-PCR结果显示,DAPT组自噬相关的微管相关蛋白1轻链3Ⅱ(LC3Ⅱ)、苄氯素1(Beclin1)、自噬相关基因7(ATG7)信使核糖核酸(mRNA)、自噬相关基因12(ATG12)mRNA的相对表达量(0.587±0.030、1.503±0.066、1.797±0.110、2.433±0.111)明显高于Control组(0.333±0.002、1.137±0.068、1.000±0.000、1.000±0.000),差异均有统计学意义(t=14.893、6.695、12.527、22.446,P<0.01)。CQ组Notch1蛋白的相对表达量(0.932±0.061)明显高于Control组(0.477±0.031),差异有统计学意义(t=11.590,P<0.01)。结论胃癌MKN-45细胞中自噬与Notch信号通路存在交叉对话,两者相互调节,DAPT联合CQ可以使胃癌MKN-45细胞增殖能力下降、凋亡率提高。Objective To explore the effects of Notch signaling pathway inhibitor N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester(DAPT)on autophagic signaling pathway and the effects of autophagy inhibitor chloroquine(CQ)on Notch signaling pathway in MKN-45,and preliminarily investigate the effects of DAPT combined with CQ on the proliferation and apoptosis of MKN-45.Methods The cells were divided into 4 groups:the control group(cultured in complete medium of RPMI 1640 without intervention drugs);CQ group(cultured on RPMI 1640 complete medium containing 40μmol/L CQ);DAPT group roswell park memorial institute(RPMI)1640 complete medium containing 80μmol/L DAPT);combined treatment group(RPMI 1640 complete medium containing 40μmol/L CQ and 80μmol/L DAPT).Methyl thiazolyl tetrazolium(MTT)assay and flow cytometry were used to detect cell proliferation and apoptosis.Transmission electron microscopy,Western blotting and reverse transcriptase-polymerase chain reaction(RT-PCR)were performed to detect the level of autophagy.Western blotting was used to detect the expression of Notch signaling pathway.comparison between two groups was done with independent sample T-test.Results MTT and flow cytometry results showed that cell proliferation in combined treatment group[(55.09±1.38)%]was significantly lower than that in DAPT group[(78.83±1.74)%]and CQ group[(64.82±1.89)%,t=18.514,7.185,P<0.01],and apoptosis rate in combined treatment group[(43.43±2.30)%]was significantly higher than that in DAPT group[(27.26±3.02)%]and CQ group[(22.16±2.24)%,t=7.339,11.415,P<0.01].Western blotting and RT-PCR indicated that the relative expression of microtubuler-associated protein 1 light chain 3Ⅱ(LC3Ⅱ),Beclin1,autophagy-related gene 7(ATG7)mRNA and ATG12 mRNA in DAPT group(0.587±0.030,1.503±0.066,1.797±0.110,2.433±0.111)was significantly higher than that in control group(0.333±0.002,1.137±0.068,1.000±0.000,1.000±0.000,t=14.893,6.695,12.527,22.446,P<0.01).The relative expression of Notch 1 protein in CQ group(0.93

关 键 词：胃癌 自噬 氯喹 

分 类 号：R735.2[医药卫生—肿瘤]