肝素结合表皮生长因子调控核因子κB诱导卵巢上皮性癌紫杉醇耐药的作用及机制  

Role and mechanism of the regulation of nuclear factor-κB by heparin binding-epidermal growth factor-like growth factor in the induction of paclitaxel resistance of ovarian cancer

作　　者：汤小晗[1] 卢美松[1] 邓锁[1] 李萌[1] Tang Xiaohan;Lu Meisong;Deng Suo;Li Meng(Department of Obstetrics and Gynecology, the First Affiliated Hospital of Harbin Medical University, Harbin 150001, China)

机构地区：[1]哈尔滨医科大学附属第一医院妇产科,150001

出　　处：《中华妇产科杂志》2019年第4期255-261,共7页Chinese Journal of Obstetrics and Gynecology

基　　金：国家自然科学基金(81572551).

摘　　要：目的探讨肝素结合表皮生长因子(HB-EGF)通过上调核因子κB(NF-κB)的表达从而诱导卵巢上皮性癌(卵巢癌)对紫杉醇耐药的作用及可能的机制。方法(1)NF-κB表达的检测:将卵巢癌亲本细胞系A2780细胞和紫杉醇耐药细胞系A2780/Taxol细胞及其对应的裸鼠分成4组,即A2780组、A2780+交叉反应物质197(CRM197,为HB-EGF抑制剂)组、A2780/Taxol组和A2780/Taxol+CRM197组,采用双重免疫荧光染色法检测4组卵巢癌细胞中HB-EGF和表皮生长因子受体(EGFR)蛋白的表达,蛋白印迹法(western blot法)检测4组卵巢癌细胞中NF-κB的表达,免疫组化SP法检测4组裸鼠移植瘤组织中NF-κB蛋白的表达。(2)NF-κB功能的检测:将A2780/Taxol细胞分4组,即空载体组、NF-κB小分子干扰RNA(siRNA)组、空载体+CRM197组、NF-κB siRNA+CRM197组,四甲基偶氮唑蓝(MTT)比色法检测4组A2780/Taxol细胞对紫杉醇的耐药性[以50%抑制浓度(IC50)表示],western blot法检测4组A2780/Taxol细胞中P-糖蛋白(P-gp)的表达,流式细胞仪检测4组A2780/Taxol细胞中P-gp的功能[以细胞内累积的若丹明(Rh123)的荧光强度表示]。结果(1)NF-κB表达的检测:A2780组、A2780+CRM197组、A2780/Taxol组和A2780/Taxol+CRM197组细胞中HB-EGF蛋白的表达水平分别为(5.6±1.3)、(2.1±1.2)、(11.7±3.5)、(6.2±1.4)分,EGFR蛋白的表达水平分别为(5.1±1.6)、(2.8±0.6)、(10.4±3.1)、(5.6±1.9)分,NF-κB蛋白的表达水平分别为1.89±0.23、0.74±0.12、3.45±0.16、1.31±0.08;4组裸鼠移植瘤组织中NF-κB蛋白的表达水平分别为(3.3±1.1)、(1.4±0.4)、(8.7±2.3)、(3.6±1.2)分。其中,A2780组和A2780/Taxol组细胞中HB-EGF、EGFR、NF-κB蛋白的表达水平及裸鼠移植瘤组织中NF-κB蛋白的表达水平分别高于A2780+CRM197组和A2780/Taxol+CRM197组,而A2780组细胞中HB-EGF、EGFR、NF-κB蛋白的表达水平及裸鼠移植瘤组织中NF-κB蛋白的表达水平低于A2780/Taxol组,分别比较,差异均有统计学意义(P<0.05)。(2)NF-κBObjective To investigate the role and mechanism of the regulation of nuclear factor-κB (NF-κB) by heparin binding-epidermal growth factor-like growth factor (HB-EGF) in paclitaxel resistance of ovarian cancer in vitro and in vivo. Methods (1) The detection of NF-κB expression: parental (A2780) and paclitaxel-resistant (A2780/Taxol) ovarian carcinoma cells were divided into four groups, named A2780 group, A2780+cross-reacting material 197 (CRM197, HB-EGF inhibitor) group, A2780/Taxol group and A2780/Taxol+CRM197 group. Among four groups, the expression level HB-EGF and epidermal growth factor receptor (EGFR) were examined by immunofluorescence double staining on confocal microscopy. Western blot was used to detect the expression level of NF-κB. In vivo, A2780 and A2780/Taxol cells were injected intraperitoneally to nude mouse to determine the expression level of NF-κB of the tumors from these four groups by immunohistochemistry method.(2) The detection on the function of NF-κB: A2780/Taxol cells were divided into four groups, named transfected with empty vector+saline group, NF-κB small interference RNA (siRNA)+saline group, empty vector+CRM197 group and NF-κB siRNA+CRM197 group respectively. Among four groups, the 50% inhibitory concentrations (IC50) of A2780/Taxol cells to paclitaxel, the expression level of plasma membrane glycoprotein (P-gp) and the effect of intracellular rhodomine123 (Rh123) accumulation were detected. Results (1) The detection of NF-κB expression: the expression scores of HB-EGF protein among four groups were 5.6±1.3, 2.1±1.2, 11.7±3.5 and 6.2±1.4;the expression scores of EGFR protein were 5.1±1.6, 2.8±0.6, 10.4±3.1 and 5.6±1.9, respectively. The expression levels of NF-κB protein in the cells of the group named A2780, A2780+CRM197, A2780/Taxol and A2780/Taxol+CRM197 group were 1.89±0.23, 0.74±0.12, 3.45±0.16 and 1.31±0.08, respectively;the expression scores of NF-κB protein in the tissue tumors from four groups were 3.3±1.1, 1.4±0.4, 8.7±2.3 and 3.6±1.2, respecti

关 键 词：肝素结合表皮生长因子 核因子ΚB 卵巢肿瘤 紫杉酚 

分 类 号：R737.31[医药卫生—肿瘤]