二十二碳六烯酸联合选择性环氧合酶-2抑制剂NS-398对人胆管癌QBC939细胞凋亡的影响  

The effect of docosahexaenoic acid combined with cyclooxygenase-2 selective inhibitor NS-398 on the apoptosis of bile duct cancer cells QBC939

作　　者：姜海涛[1] 周惟[2] 毛联钢[3] 陈云杰[1] 曹景玉[4] Jiang Haitao;Zhou Wei;Mao Liangang;Chen Yanjie;Cao Jingyu.(Department of General Surgery, Ningbo No. 2 Hospital, Ningbo 315010, Zhejiang Province, China;Department of Hepatobiliary Surgery, The Affiliated Hospital of Qingdao University, Qingdao 266003, Shandong Province, China)

机构地区：[1]宁波市第二医院外二科,315010 [2]宁波市第二医院科教部,315010 [3]宁波市第二医院检验科,315010 [4]青岛大学附属医院肝胆外科

出　　处：《中华肝胆外科杂志》2018年第5期336-340,共5页Chinese Journal of Hepatobiliary Surgery

基　　金：华美研究基金（2017HMKY11）

摘　　要：目的研究二十二碳六烯酸（DHA）联合选择性环氧合酶-2（COX-2）抑制剂NS-398对人胆管癌细胞QBC939凋亡的影响及其作用机制。方法体外培养人胆管癌QBC939细胞株，分别设立DHA组、NS-398组、二者联合组和空白对照组。将0、15、30、45、60、75μg/ml的DHA和0、25、50、100、150、200μmol／L的NS-398分别联合作用于胆管癌QBC939细胞；应用CCK8法检测其对QBC939细胞生长的相对抑制率；采用流式细胞术检测QBC939细胞的凋亡情况；应用实时荧光定量PCR（RT—PCR）检测QBC939细胞中β-连环蛋白（catenin）和COX-2两种因子的基因表达情况；应用SP免疫细胞化学法测定作用前后QBC939细胞浆中β-catenin和COX-2两种蛋白的表达情况；应用ELISA法测定QBC939细胞上清中两种蛋白分泌情况。结果DHA和NS-398在体外均能单独抑制QBC939细胞生长。DHA浓度为45μg／m1，联合NS-398浓度为100μmol／L时，作用24小时后，细胞生长抑制率达到90．0％，实验组与DHA组、NS-398组和空白组差异均有统计学意义（P〈0．05）。继续增加两种药物浓度，细胞生长抑制率变化不明显。流式细胞术显示，DHA联合NS-398能明显促进QBC939细胞早期凋亡；实时PCR显示二者联合能明显抑制QBC939细胞中6-catenin和COX-2基因表达；SP免疫细胞化学实验显示，二者联合能抑制QBC939细胞浆中β-catenin和COX-2两种蛋白表达；ELISA实验显示，二者联合能抑制QBC939细胞中两种蛋白的细胞外分泌。结论DHA联合NS-398能明显抑制胆管癌QBC939细胞生长，促进胆管癌细胞早期凋亡。二者联合对胆管癌QBC939细胞生长的抑制作用可能是通过抑制β-catenin和COX-2两种因子基因及蛋白水平的表达，进而促进胆管癌QBC939细朐的早期凋亡实现的．Objective To investigate the effect of docosahexaenoic acid （DHA） combined with cyclooxygenase-2 （COX-2） selective inhibitor NS-398 on the apoptosis of cholangiocarcinoma QBC939 cells and the mechanism. Methods In vitro, cholangiocarcinoma QBC939 cells were treated with 0, 15, 30, 45, 60 and 75 μg/ml DHA with 0, 25, 50, 100, 150 and 200 μmol/L NS-398, respectively. The absor- bances of the QBC939 cells were measured by CCK8 and its growth inhibition ratios were analyzed. Flow cy- tometry was applied to detect cell apoptosis. The level of β-catenin and COX-2 mRNA and protein were measured by real-time PCR, immunocytochemistry and enzyme-linked immunoadsordent assay, respectively. Results DHA combined with NS-398 could significantly suppress the growth of QBC939 cells （ P 〈0. 05 ）. When the concentration of DHA went up to 45 μg/ml and NS-398 was 100 μmol/L, the relative growth inhi- bition rate of QBC939 cells was 90.0%. If the concentrations were increased, the result showed no signifi- cant differences. Furthermore, flow cytometry analysis indicated that DHA combined with NS-398 could in-duce QBC939 cells apoptosis at the early stage, and the apoptosis rate was significantly different between the experimental and control groups （P 〈 0.01 ）. Real-time PCR showed low β-catenin and COX-2 expression in QBC939 cells disposed by DHA combined with NS-398, and their expression were significantly different be- tween the experimental and control groups （P 〈 0. 01 ）. Immunocytochemistry and ELISA demonstrated that DHA combined with NS-398 could decrease β-catenin and COX-2 protein expression in QBC939 cells. Con- elusion DHA combined with NS-398 induced apoptosis and inhibited proliferation of cholangiocarcinoma cells QBC939 in vitro through targeting β-catenin and COX-2.

关 键 词：二十二碳六烯酸 NS-398 胆管癌 凋亡 Β-连环蛋白 环氧合酶-2 

分 类 号：R735.8[医药卫生—肿瘤]