顺铂耐药卵巢上皮性癌细胞株及其裸鼠移植瘤模型的建立及耐药特性探讨  

Establishment and biological characteristics of a platinum-resistance nude mouse model in epithelial ovarian cancer

作　　者：石丽君[1] 于红静[1] 张玮[1] 李力[1] 王琪[1] 

机构地区：[1]广西医科大学附属肿瘤医院区域高发肿瘤早诊早治教育部重点实验室,南宁530021

出　　处：《中华妇产科杂志》2014年第7期523-530,共8页Chinese Journal of Obstetrics and Gynecology

基　　金：国家高技术研究发展计划（2012AA02A507）;广西科技基础条件平台建设项目（10-108-19）;广西医学科学实验中心开放基金（KFJJ2010-3）

摘　　要：目的：建立顺铂耐药性稳定的卵巢上皮性癌（卵巢癌）细胞株及其裸鼠移植瘤模型并探讨其耐药特性，为研究体内微环境耐药机制及逆转靶基因筛选奠定基础。方法采用体内外交叉诱导和连续体内诱导两种方法，以绿色荧光蛋白（GFP）标记的卵巢癌细胞株SKOV3/GFP分别建立两株顺铂耐药的卵巢癌细胞株SKOV3/DDPⅠ（为耐药细胞的对照）和SKOV3/DDPⅡ及其裸鼠移植瘤模型。采用四甲基偶氮唑蓝（MTT）比色法和流式细胞仪检测细胞耐药指数，流式细胞仪检测细胞凋亡率和细胞周期比例，高效液相色谱仪检测细胞内顺铂积量；透射电镜观察移植瘤组织的超微结构，实时荧光定量PCR技术检测移植瘤组织中铂类耐药相关基因——PTEN、STAT5、XIAP、BRCA1和MDR1 mRNA的表达水平。结果成功构建顺铂耐药卵巢癌细胞株SKOV3/DDPⅠ和SKOV3/DDPⅡ及其裸鼠移植瘤模型。MTT比色法和流式细胞仪检测显示，SKOV3/DDPⅡ细胞的耐药指数分别为2.83±0.12和3.82±0.19，SKOV3/DDPⅠ细胞的耐药指数分别为2.20±0.16和3.40±0.20，两种检测方法分别比较，差异均有统计学意义（P〈0.05）。流式细胞仪检测显示，29.7和39.6μmol/L顺铂处理36 h后， SKOV3/DDPⅠ、SKOV3/DDPⅡ细胞的凋亡率[分别为（57.0±1.4）%、（74.4±2.3）%和（37.6±4.4）%、（50.5±3.4）%]均显著低于SKOV3/GFP细胞[（83.1±2.7）%和（87.4±4.0）%；P=0.024，P=0.001]，且SKOV3/DDPⅡ细胞的凋亡率明显低于SKOV3/DDPⅠ细胞（P=0.020）；SKOV3/DDPⅠ、SKOV3/DDPⅡ细胞G2/M期比例与顺铂处理时间（为0、12、24、36和48 h）呈正相关（R=0.800，P=0.104；R=0.915，P=0.029）。高效液相色谱仪检测显示，以9.9、19.8、29.7和39.6μmol/L的顺铂分别处理12、24、36和48 h后，SKOV3/DDPⅠ、SKOV3/DDPⅡ细胞内顺铂积量均显著低于SKOV3/GFP细胞（P〈0.05）。透射电镜观察，接种SKOV3/GFP细胞的裸鼠移植瘤组织中，在Objective To establish a platinum resistance nude mice model of epithelial ovarian cancer （EOC） and investigate its resistance to cisplatin （DDP） biological characteristics, so as to provide evidences for exploring chemoresistence mechanisms and screening for reversal targets in vivo micro-environment. Methods The resistance model was produced by repeating a crossover subcutaneous injection of human ovarian cancer SKOV3 cells labelled green fluorescent protein（GFP） and transplatation of tumor fragment into nude mice. Two kinds of cancer cell lines of SKOV3/DDPⅠand SKOV3/DDPⅡwere induced with acquired resistence to DDP. The chemosensitivities of EOC cells to DDP were tested and half maximal inhibitory concentration（IC50） was measured by methyl thiazolyl tetrazolium （MTT） and flow cytometry （FCS）. Dynamic analysis among the concentration of DDP treatment and cell apoptosis, cell cycle phase distribution and intracellular DDP concentration. The expression of PTEN, STAT5, XIAP, BRCA1 and MDR1 were examined by real time quantitative reverser transcription PCR （qRT-PCR） in vivo. Results IC50 value of cisplatin for SKOV3/DDPⅡ were 2.83 ± 0.12 and 3.82 ± 0.19 folds than those for SKOV3/GFP by MTT and flow cytometry, separately. SKOV3/DDPⅠwere 2.20±0.16 and 3.40±0.20 folds. The apoptosis rate of SKOV3/DDPⅡ and SKOV3/DDPⅠ were decreased significantly at 29.7 and 39.6μmol/L DDP when treatment for 36 hours,which were lower than that of SKOV3/GFP cells [（57.0±1.4）%vs （37.6 ± 4.36）%vs （83.1 ± 2.71）%,P=0.024;（74.4 ± 2.3）%vs （50.5 ± 3.4）%vs （87.4 ± 4.0）%,P=0.001]. SKOV3/DDPⅠ and SKOV3/DDPⅡ was positively related with cisplatin processing time. Intracellular DDP accumulation of SKOV3/DDPⅡand SKOV3/DDPⅠwere lower than SKOV3-GFP in dynamic processes（P〈0.05）. Besides intracellular DDP accumulation of SKOV3/DDPⅡ also lower than SKOV3/DDPⅠin dynamic processes （P〈0.05）. Transplanted tumor of SKOV3/GFP appeared organelle degradation and nu

关 键 词：卵巢肿瘤 顺铂 抗药性 肿瘤 细胞系 肿瘤 疾病模型 动物 

分 类 号：R737.31[医药卫生—肿瘤]