沉默hMSH2基因表达对紫杉醇耐药的卵巢上皮性癌OC3/TAX300细胞耐药性的影响  

Study on RNA interference silencing hMSH2 induces the reversal chemo-resistance of ovarian carcinoma cell line OC3/TAX300

作　　者：王爱新[1] 李健[2] 李红霞[2] 

机构地区：[1]北京市房山区妇幼保健院妇产科, 102488 [2]首都医科大学附属北京世纪坛医院妇产科, 100038

出　　处：《中华妇产科杂志》2014年第3期213-217,共5页Chinese Journal of Obstetrics and Gynecology

基　　金：国家“十一五”863计划（2006AA02A245）

摘　　要：目的 应用RNA干扰（RNAi）技术沉默hMSH2基因后,观察其对紫杉醇耐药的卵巢上皮性癌（卵巢癌）细胞系OC3/TAX300细胞耐药性的影响.方法 设计靶向沉默耐药基因hMSH2的小分子干扰RNA（siRNA）序列,转染OC3/TAX300细胞（实验组）,以转染随机无关序列的细胞为阴性对照组,未转染的细胞为空白对照组.（1）采用实时定量逆转录（RT）-PCR技术和蛋白印迹法分别检测3组细胞中hMSH2 mRNA和蛋白的表达.（2）采用四甲基偶氮唑蓝（MTT）比色法检测紫杉醇（2 μg/ml,下同）作用12、24、48、72 h后3组细胞的增殖能力（以细胞生长抑制率表示）,流式细胞术检测紫杉醇作用24、48 h后3组细胞的细胞凋亡率和细胞周期比例,透射电镜观察紫杉醇作用48 h后3组细胞的形态学改变.结果 （1）实验组细胞中hMSH2mRNA的相对表达水平为0.004±0.000,低于阴性对照组的0.053±0.006和空白对照组的0.057±0.012（P＜0.05）;hMSH2蛋白的相对表达水平为0.19±0.04,低于阴性对照组的1.00±0.07和空白对照组的0.95±0.03（P ＜0.05）.（2）紫杉醇作用24、48、72 h后,实验组细胞生长抑制率均明显高于阴性对照组和空白对照组（P＜0.05）;紫杉醇作用24、48 h后,实验组细胞的凋亡率和G2/M期细胞比例均明显高于阴性对照组和空白对照组（P＜0.05）;而阴性对照组细胞的生长抑制率、凋亡率、G2/M期细胞比例分别与空白对照组,差异均无统计学意义（P＞0.05）.透射电镜下观察,紫杉醇作用48 h后,实验组大多数细胞可见细胞固缩,部分出现核碎裂和凋亡小体,核仁消失,线粒体明显肿胀,胞质内可见大量空泡状结构;而阴性对照组和空白对照组大多数细胞形态较规则,细胞核位于细胞中央,核仁清晰,线粒体轻度肿胀.结论 RNAi技术沉默紫杉醇耐药的卵巢癌细胞系OC3/TAX300细胞中hMSH2基因的表达后,可逆转OC3/TAX300细胞对紫杉醇的耐药性.Objective To investigate the reversal effect of hMSH2 small interference RNA（siRNA） on chemo-resistance of ovarian carcinoma cell line OC3/TAX300,explore the clinical significance.Methods The specific hMSH2 siRNA （experimental group） and non-specific hMSH2 siRNA （negative control group） was designed,synthesized and transfected into ovarian carcinoma cell line OC3/TAX300.The expression mRNA and protein levels of hMSH2 were detected by real-time reverse transcription （RT）-PCR and western blot.The cell proliferation was detected by methyl thiazolyl tetrazolium（MTT） method after 12,24,48,72 hours of 2 μg/ml taxol,the apoptosis rate after 24,48 hours of 2 μg/ml taxol was analyzed by flow cytometry.Morphological changes and ultramicrostructure of cells after 48 hours of 2 μg/ml taxol were observed with transmission electron microscope.Results （1） The mRNA levels of hMSH2 were 0.004 ± 0.000,0.053 ±0.006 and 0.057 ± 0.012 in experimental group,negative control group and non-infected group,respectively.The protein levels of hMSH2 were 0.19 ± 0.04,1.00 ± 0.07 and 0.95 ± 0.03 in experimental group,negative control group and non-infected group,respectively.（2） Compared with the noninfected group and the negative control group.The cell proliferation was effectively inhibited after 12,24,48,72 hours of 2 μg/ml taxol（P 〈0.05）.The cell cycle was arrested at G2/M phase,the apoptotic rate was significantly increased after 24,48 hours of 2 μg/ml taxol （P 〈 0.05）.The experimental group after 48 hours of 2 μg/ml taxol was found to have more visible cell shrinkage,more serious chromatin margination,nucleus condensation,fragmentation and apoptotic body formation,nucleolus disappeared,markedly swollen mitochondria,mitochondrial cristae disappeared and other signs of apoptosis.While the nucleus was located in the cells of the central and nucleolus is clear,only mild chromatin pyknosis and marginalized,mild swelling of mitochondria in the control group and blank group.Conclusion siRNA ta

关 键 词：卵巢肿瘤 MutS同源蛋白2 RNA 小分子干扰 紫杉酚 抗药性 肿瘤 细胞系 肿瘤 

分 类 号：R737.31[医药卫生—肿瘤]