肝素结合表皮生长因子在紫杉醇耐药卵巢上皮性癌细胞和组织中的表达及意义  

Expression and significance of heparin binding-epidermal growth factor-like growth factor in paclitaxel-resistant ovarian cancer

作　　者：汤小晗[1] 卢美松[1] 李翠萍 邓锁[1] 李萌[1] 

机构地区：[1]哈尔滨医科大学附属第一医院妇产科,150001 [2]齐齐哈尔市中医医院妇产科

出　　处：《中华妇产科杂志》2014年第7期517-522,共6页Chinese Journal of Obstetrics and Gynecology

基　　金：黑龙江省自然科学基金（ZD200906）

摘　　要：目的：检测肝素结合表皮生长因子（HB-EGF）在紫杉醇耐药卵巢上皮性癌（卵巢癌）细胞和组织中的表达水平，并探讨HB-EGF与卵巢癌紫杉醇耐药性的关系。方法（1）体外实验：蛋白印迹法检测卵巢癌细胞株A2780（紫杉醇敏感）和A2780/Taxol（紫杉醇耐药）细胞中HB-EGF蛋白的表达。体外细胞实验分为4组，A2780＋CRM197组、A2780/Taxol＋CRM197组、A2780组、A2780/Taxol组，采用四甲基偶氮唑蓝（MTT）比色法检测4组细胞的增殖能力[以吸光度（A）值表示]，流式细胞仪检测4组细胞的细胞周期比例，酶标仪检测4组细胞中半胱氨酸天冬氨酸蛋白酶3（caspase-3）蛋白的活性[以4硝基苯胺（pNa）浓度表示]。（2）体内实验：将A2780和A2780/Taxol细胞分别接种于裸鼠腋下，建立卵巢癌裸鼠移植瘤模型；免疫组化SP法检测裸鼠移植瘤组织中HB-EGF蛋白的表达。体内动物实验分为4组，方法同体外细胞实验分组，每组5只裸鼠，观察4组裸鼠移植瘤的生长情况，并计算抑瘤率。结果（1）体外实验结果：A2780/Taxol细胞中HB-EGF蛋白的表达水平为2.11±0.41，明显高于A2780细胞的0.75±0.20（P〈0.01）。A2780＋CRM197组、A2780/Taxol＋CRM197组细胞增殖的抑制作用随CRM197浓度的增加而增强，呈明显的浓度依赖性（P〈0.01），且当CRM197浓度≥1μg/ml时，A2780/Taxol＋CRM197组细胞增殖的抑制作用明显高于A2780＋CRM197组细胞（P〈0.05）；A2780＋CRM197组细胞G0/G1期比例[（67±4）%]高于A2780组[（54±6）%]，A2780/Taxol＋CRM197组细胞G0/G1期比例[（72±4）%]高于A2780/Taxol组[（24±8）%]，分别比较，差异均有统计学意义（P〈0.01）；A2780＋CRM197组细胞中pNa浓度[（40±6）μmol/L，即caspase-3蛋白活性]高于A2780组[（6±6）μmol/L]，A2780/Taxol＋CRM197组细胞中pNa浓度[（66±12）μmol/L]高于A2780/Taxol组[（9±6）μmol/L]，分别比较，差异均有统计学意义（P〈0Objective To examine the expression of heparin binding-epidermal growth factor-like growth factor （HB-EGF） in paclitaxel-resistant ovarian cancer and elucidate the relationship between HB-EGF and the resistance of ovarian cancer to paclitaxel. Methods The human ovarian carcinoma cell line A2780 and the paclitaxel-resistant human ovarian carcinoma cell line A2780/Taxol were cultured in vitro. Western blot was used to dectect the expression of HB-EGF protein in A2780 and A2780/Taxol groups. The A2780 cells were treated with cross-reacting material 197 （CRM197 and A2780 ＋ CRM197 group） or dimethyl sulphoxide （DMSO;A2780 group）, while the A2780/Taxol cells were treated with CRM197 （A2780/Taxol＋CRM197 group） or DMSO （A2780/Taxol group）. The effects of CRM197 on growth and proliferation was tested by methyl thiazolyl tetrazolium（ MTT） and the results were showed as absorbance （A）.The effects of CRM197 on cell cycles was tested by flow cytometry, while the effects of CRM197 on apoptosis was examined by caspase-3 activity assay and the results were showed as p-nitroaniline（pNa）. In animal experiment, four groups of cells were inoculated to BALB/c nude mouse subcutaneously to observe tumor formation ability following CRM197 treatment. Immunohistochemistry was used to determine the expression of HB-EGF protein in A2780 and A2780/Taxol group. Results The expression level of HB-EGF protein in A2780/Taxol group （2.11±0.41） was significantly higher than that of A2780 group （0.75±0.20;P〈0.01）. The inhibition effect of CRM197 on the cell growth of A2780＋CRM197 and A2780/Taxol＋CRM197 group was accompanied by the acceleration of CRM197 concentration（P〈0.01）. When CRM197≥1 μg/ml, the inhibition effect of CRM197 on the cell growth of A2780/Taxol＋CRM197 group was significantly higher than that in A2780/Taxol group（P〈0.05）. In cell cycle experiment, CRM197 induced the cell-cycle arrest at the G0/G1 phase in A2780＋CRM197 cells[（67 ± 4）%] compared with A2780 cel

关 键 词：卵巢肿瘤 胞间信号肽类和蛋白质类 细菌蛋白质类 紫杉酚 抗药性 肿瘤 细胞系 肿瘤 

分 类 号：R737.31[医药卫生—肿瘤]