NS-398联合光动力疗法抑制环氧合酶-2、血管内皮生长因子-C对胆管癌QBC939细胞凋亡的影响  

Effect of NS-398 combined with photodynamic therapy on apoptosis of bile duct cancer cells QBC939 through inhibiting cyclooxygenase-2 and vascular endothelial growth factor-C

作　　者：马振华 姜海涛 张燕 石定 陈云杰 Ma Zhenhua;Jiang Haitao;Zhang Yan;Shi Ding;Chen Yunjie(HwaMei Hospital,University of Chinese Academy of Sciences,Ningbo Institute of Life and Health Industry,University of Chinese Academy of Sciences,Key Laboratory of Diagnosis and Treatment of Digestive System Tumors of Zhejiang Province,Ningbo 315010,China;Department of General Surgery,HwaMei Hospital,University of Chinese Academy of Sciences,Ningbo 315010,China;Department of Interventional Therapy,HwaMei Hospital,University of Chinese Academy of Sciences,Ningbo 315010,China)

机构地区：[1]中国科学院大学宁波华美医院,中国科学院大学宁波生命与健康产业研究院,浙江省消化系统肿瘤诊治及研究重点实验室,315010 [2]中国科学院大学宁波华美医院外二科,315010 [3]中国科学院大学宁波华美医院介入治疗科,315010

出　　处：《中华实验外科杂志》2020年第11期2062-2066,共5页Chinese Journal of Experimental Surgery

基　　金：浙江省消化系统肿瘤诊治及研究重点实验室(2019E10020);浙江省医药卫生科研项目(2018265085);宁波市消化系统肿瘤临床医学研究中心(2019A21003);华美重点研究基金(2020HMZD17);华美研究基金(2019HMKY67)。

摘　　要：目的探讨选择性环氧合酶-2(COX-2)抑制剂NS-398联合光动力疗法(PDT)对人胆管癌细胞QBC939凋亡的影响及其作用机制。方法将QBC939进行体外培养,人胆管癌QBC939细胞来源于上海中科院细胞库,采用随机化分组法进行分组,培养72 h后进行细胞计数盒-8(CCK-8)实验。实验分为4组,即单纯NS-398组、单纯PDT组、联合实验组和空白对照组,分别将0、25、50、100、200μmol/L浓度的NS-398和0、2、4、6、8、10 mg/L浓度的血卟啉衍生物(HPD)在0、5、10、15 J/cm^2的光照强度作用下,分别依次联合作用于QBC939细胞;运用CCK-8法检测各组的细胞生长抑制率(R);采用流式细胞法检测QBC939细胞的凋亡率(A);应用荧光定量聚合酶链反应(PCR)法检测QBC939细胞中COX-2和血管内皮生长因子-C(VEGF-C)的基因表达情况;采用免疫细胞化学链霉亲和素-过氧化物酶结合物(SP)法测定QBC939细胞质中COX-2和VEGF-C的蛋白表达情况;应用酶联免疫吸附实验(ELISA)测定QBC939细胞上清中COX-2和VEGF-C的蛋白分泌情况,组间比较进行单因素方差分析。结果NS-398和PDT两种措施在体外均能单独抑制QBC939细胞生长,当NS-398浓度为50μmol/L,HPD浓度为8 mg/L、光照强度为5 J/cm^2联合作用后,R可达95%,联合实验组与单纯NS-398组、单纯PDT组和空白对照组比较差异均有统计学意义,继续上调两者的强度,R变化不明显;流式细胞实验中联合实验组A为(55.19±1.14)%,与空白对照组(5.57±1.21)%、单纯NS-398组(19.55±1.06)%和单纯PDT组(18.62±1.18)%比较差异均有统计学意义(F=6153.000,P<0.05);荧光定量聚合酶链反应(Real-time PCR)结果可见COX-2在联合实验组(0.21±0.02)中表达量明显低于空白对照组(1.00±0.00)、单纯NS-398组(0.32±0.03)和单纯PDT组(0.35±0.02),差异均有统计学意义(F=8446.182,P<0.05),VEGF-C在联合实验组(0.23±0.01)中表达量明显低于空白对照组(1.00±0.00)、单纯NS-398组(0.36±0.02)和单纯PDT组(0.38±0.03),差�Objective To study the effect of NS-398,a selective cyclooxygenase-2(COX-2)inhibitor combined with photodynamic therapy(PDT)on apoptosis of cholangiocarcinoma QBC939 cells and its mechanism.Methods In vitro,QBC939 cells were collected from the Cell Bank of the Chinese Academy of Sciences(CAS)in Shanghai.QBC939 cells were cultured for 72 h and divided into four groups:dure NS-398 group,dure PDT group,combined experimental group and blank control group,which were exposed to 0,25,50,100,200μmol/L NS-398 and 0,2,4,6,8,10 mg/L Hematoporphyrin derivative(HPD)with 0,5,10,15 J/cm^2 light radiation respectively.The growth inhibition ratio(R)of the QBC939 cells was measured and calculated by cell counting kit-8(CCK-8)assay.Flow cytometry was used to detect cell apoptosis ratio(A).The mRNA and protein expression levels of COX-2 and vascular endothelial growth factor-C(VEGF-C)were detected by real-time quantitative polymerase chain reaction(Real-time PCR),immunocytochemistry and enzyme linked immune sorbent assay(ELISA)respectively.Univariate analysis of variance was used in the comparison between two groups,andP<0.05 was considered to be statistically significant.Results NS-398 and PDT could significantly suppress the growth of QBC939 cells in vitro.When NS-398 was 50μmol/L and the concentration of HPD was 8 mg/L,the light radiation was 5 J/cm^2,R was about 95%,and there were significant differences between combined experimental group and dure NS-398 group,dure PDT group and blank control group.If the intensity of both were increased,the results showed no statistically significant differences.The A value in combined experimental group[(55.19±1.14)%]was more than that in dure NS-398 group[(19.55±1.06)%],dure PDT group[(18.62±1.18)%]and blank control group[(5.57±1.21)%](F=6153.000,P<0.05).Real-time PCR showed that COX-2 in combined experimental group(0.21±0.02)was less than that in dure NS-398 group(0.32±0.03),dure PDT group(0.35±0.02)and blank control group(1.00±0.00,F=8446.182,P<0.05),and VEGF-C in combined experi

关 键 词：NS-398 光动力疗法 凋亡 选择性环氧合酶-2 血管内皮生长因子-C 

分 类 号：R735.8[医药卫生—肿瘤]