缺氧条件下人卵巢癌细胞对顺铂耐药与TLR9的关系研究  

Study on the Relationship between Cisplatin-resistance of Human Ovarian Cancer Cell and TLR9 under Hypoxia

作　　者：李玲[1] 李斌[1] 幸海燕[1] 陈剑鸿[1] 

机构地区：[1]第三军医大学第三附属医院药剂科,重庆400042

出　　处：《中国药房》2017年第13期1744-1747,共4页China Pharmacy

基　　金：国家自然科学基金面上项目(No.30973493)

摘　　要：目的:研究缺氧条件下人卵巢癌细胞(SKOV3)对顺铂耐药与Toll样受体9(TLR9)的关系。方法:采用免疫荧光法检测破膜和未破膜SKOV3中TLR9的表达。取SKOV3,加入TLR9特异性激动剂Cp G-ODN 2006(A组)及其同型对照Cp G-ODN 2006control(B组)作用6、12、24 h后,加入顺铂,以CCK-8法检测细胞抑制率。取SKOV3或经0(未加)、1、10、102、103μmol/L TLR9特异性拮抗剂氯喹预处理3 h后的SKOV3,加入SKOV3常氧(21%O2)、缺氧(1%O2)孵育6、12、24 h的细胞上清作用24 h后,加入顺铂,检测细胞增殖抑制率,计算预处理细胞缺氧时药物抑制率的降低倍数(HICR);并采用Western blot法检测常氧24 h细胞上清(C组)、缺氧24 h细胞上清(D组)、缺氧24 h细胞上清+10μmol/L氯喹(E组)条件下SKOV3中多药耐药相关蛋白(MRP)的表达。结果:TLR9在SKOV3的细胞膜和细胞质中均有表达。与A组比较,B组细胞增殖抑制率降低(P<0.01)。与常氧细胞上清比较,缺氧细胞上清作用后细胞增殖抑制率降低(P<0.05)。与未加氯喹比较,加入10、102μmol/L氯喹能明显降低HICR(P<0.01),其中缺氧24 h细胞上清+10μmol/L氯喹降低最明显。与C组比较,D组细胞中MRP表达增强(P<0.01);与D组比较,E组细胞中MRP表达减弱(P<0.01)。结论:TLR9受体在缺氧下可被激活,并可导致SKOV3对顺铂耐药,该作用可能与上调MRP表达有关。OBJECTIVE：To study the relationship between cisplatin-resistant of human ovarian cancer cells （SKOV3） and Toll-like receptor 9 （TLR9） under hypoxia. METHODS：Immunofluorescence method was used to detect the TLR9 expression in rupture and intact membranes SKOV3. SKOV3 was taken,adding cisplatin,then CCK-8 was used to detect cell inhibition rate af-ter 6,12,24 h of added into TLR9 specific agonists CpG-ODN 2006 （group A） and its isotype control CpG-ODN 2006 control （group B）. SKOV3 or SKOV3 pretreated by 0（not added）,1,10,102,103 μmol/L TLR9 specific antagonist chloroquine for 3 h were taken,adding cisplatin,cell inhibition rate was detected after 24 h of added into SKOV3 normoxia（21% O2）,hypoxia（1%O2）incubating 6,12,24 h,HICR under hypoxia in pretreatment was calculated. Western blot method was used to detect the multi-drug resistance associated protein（MRP）expression in SKOV3 under the conditions of normoxia 24 h cell supernatant（group C）, hypoxia 24 h cell supernatant（group D）,hypoxia 24 h cell supernatant＋10 μmol/L chloroquine（group E）. RESULTS：TLR9 was expressed in both cell membrane and cytoplasm of SKOV3. Compared with group A,cell inhibition rate in group B was decreased （P〈0.01）. Compared with normoxia cell supernatant,cell inhibition rate was decreased after hypoxia cell supernatant （P〈0.05）. Compared with not added chloroquine,adding 10,102 μmol/L chloroquine can obviously decrease HICR（P〈0.01）,and the most significant decrease was showed when using hypoxia 24 h cell supernatant＋10 μmol/L chloroquine. Compared with group C,MRP expression in group D was strengthened （P〈0.01）;compared with group D,MRP expression in group E was weakened （P〈0.01）. CONCLUSIONS：TLR9 receptors can be activated under hypoxia,and cause SKOV3 to cisplatin-resistance,the effect may be related with up-regulating MRP expression.

关 键 词：人卵巢细胞 缺氧 化疗敏感性 TOLL样受体9 顺铂 

分 类 号：R361.3[医药卫生—病理学]