雌激素和紫杉醇对子宫内膜癌Ishikawa细胞中Bub1 mRNA表达的影响

摘要

目的 探讨雌激素和紫杉醇对子宫内膜癌Ishikawa细胞中Bub1 mRNA表达的影响.方法 (1)Ishikawa细胞中加入10 nmol/L的17β雌二醇(17β-E2)作用24、48和72 h,采用活细胞计数(CCK-8)法检测其增殖活力[以吸光度(A)值表示],以17β-E2浓度0 nmol/L者为空白对照.(2)按如下方法处理Ishikawa细胞:①加入不同浓度(0.1、10、1000 nmol/L)的17β-E2作用不同时间(5、15、30 min及1、2、4、8、12、16、24、30 h);②经同步化处理(即先期予无血清培养基培养),然后加入不同浓度(10、100 nmol/L)的紫杉醇作用不同时间(8、24 h);③未经同步化处理,直接加入100 nmol/L的紫杉醇作用4、8、16、24、48 h;均以作用时间0 h者为空白对照.采用实时定量PCR技术检测经上述方法处理后的细胞中Bub1 mRNA的表达水平(设空白对照为1,以倍数表示Bub1 mRNA的表达水平).结果 (1)17β-E2作用后Ishikawa细胞的增殖活力随着作用时间(24、48和72 h)的延长逐渐增加,其A值分别为0.86±0.10、1.66 ±0.10、2.32±0.24,呈时间依赖性(P＜0.01).(2)不同浓度(0.1、10、1000 nmol/L)的17β-E2作用不同时间(5、15、30 min及1、2、4、8、12、16、24、30 h)后,Ishikawa细胞中Bub1 mRNA的表达水平(与空白对照比较)随着17β-E2浓度的升高呈下降趋势,而随着作用时间的延长无明显变化.当17β-E2浓度为10 nmol/L时,细胞中Bub1 mRNA的表达水平最低,与空白对照比较,差异则有统计学意义(P=0.020);而当浓度为0.1、1000 nmol/L时,与空白对照比较,差异则无统计学意义(P=0.746,P=0.197).经同步化处理的Ishikawa细胞,予10 nmol/L的紫杉醇作用8、24 h后,Ishikawa细胞中Bub1 mRNA的表达水平下降,分别为(0.403±0.008)、(0.775±0.144)倍,两者比较,差异无统计学意义(P=0.251);紫杉醇作用8 h时与空白对照比较,差异有统计学意义(P=0.009);而紫杉醇作用24 h时与空白对照比较,差异则无统计学意义(P=0.396).100nmol/L的紫杉醇作用8、24 h后,Ishikawa细胞中Bub1 mRNA的表达水平下降,分别为(0.697±0.017)、(0.850±0.004)倍,两者比较,差异无统计学意义(P=0.061);而分别与空白对照比较,差异则均有统计学意义(P=0.038,P=0.019).未经同步化处理的Ishikawa细胞,予100 nmol/L紫杉醇作用4、8、16、24、48 h后,细胞中Bub1 mRNA的表达水平呈上升趋势,分别为(1.127±0.105)、(1.614±0.154)、(2.092±0.179)、(1.381±0.061)、(1.519±0.182)倍,各时间点间比较,差异有统计学意义(P＜0.01),其中作用16 h时表达最强.结论 雌激素可降调Ishikawa细胞中Bub1 mRNA的表达;紫杉醇对先期无血清培养的Ishikawa细胞可下调其Bub1 mRNA的表达,而对正常培养的细胞则上调其Bub1 mRNA的表达,提示紫杉醇在不同条件下杀伤子宫内膜癌细胞的效力与Bub1mRNA的表达失调有关.%Objective To explore the regulation of Bub1 mRNA expression in endometrial carcinoma cells by estrogen and paclitaxel. Methods The high differentiated endometrial adenocarcinoma cells ( Ishikawa cell line) were cultured in DMED/F12 supplemented with 10% fetal bovine serum(FBS) or phenol red-free DMED/F12 supplemented with 5% dextran-charcoal FBS (dcFBS). Firstly, the cells were stimulated by 10 nmol/L estradiol (17β-E2 ) or no hormonal stimulation as control group, and the cell proliferation was quantified at 24, 48 and 72 hours using cell counting kit-8 (CCK-8) method. Then the cells were stimulated with different concentrations of 17β-E2 (0.1, 10, 1000 nmol/L) at different periods (5,15,30 minites and 2,4,8,12,16,24,30 hours), the expression of Bub1 mRNA was detected by real-time quantitative PCR. Ishikawa cells were cultured with non-serum DMEM/F12 to be synchronized, and then were treated with different concentrations of paclitaxel( 10,100 nmol/L) for 8 and 24 hours. While, nonsynchronized Ishikawa cells were exposed to 100 nmoL/L paclitaxel for different periods(4, 8, 16, 24,48 hours), and real-time quantitative PCR was also used to detect the expression levels of Bub1 mRNA.Data were presented as folds change relative to control group, in which values ＜ 1 were down-regulated, and those ＞ 1 were up regulated. Results The proliferation rate of cells in the presence of 17β-E2 was significantly highter than that of the control group after treated 24 hours (A value: 0. 70 ±0. 08 vs. 0. 86 ±0.10, P = 0.049). Time-dependent experiments revealed that addition of 17β-E2 could increase cell numbers during 72 hours period, while the expression level of Bub1 mRNA was decreased in Ishikawa cell.Dose-dependent experiments revealed maximal estradiol stimulation effects at 10 nmol/L( P = 0. 020). After being treated with serum-free culture, Ishikawa cells were exposed to 10 nmol/L paclitaxel for 8 and 24 hours, and the expression of Bubl mRNA decreased (0. 403 ± 0. 008 vs. 0. 775 ± 0. 144, P = 0. 251 ).Compared to the control cells, the mRNA expression levels of Bub1 in cells treated by paclitaxel for 8 hours was significantly decreased ( P = 0. 009 ), while there was not significantly decreased at 24 hours ( P =0. 396). When exposed to 100 nmol/L paclitaxel for 8 and 24 hours, the expression of Bubl mRNA was also decreased(0. 697 ±0. 017 vs. 0. 850 ±0. 004, P =0. 061 ). Compared to the control cells, Bub1 mRNA expression was also significantly decreased (P = 0. 038 and P= 0. 019, respectively). While with serum freetreatment culture, when Ishikawa cells exposed to 100 nmoL/L paclitxel for 4, 8, 16, 24 or 48 hours, the expression of Bub1 mRNA significantly increased ( 1. 127 ± 0. 105 vs. 1. 614 ± 0. 154 vs. 2. 092 ± 0. 179vs. 1. 381 ± 0. 061 vs. 1. 519 ± 0. 182, P = 0. 002 ), of which was signicantaly increased at 16 hours treatment. Conclusion Bub1 exrpession could be regulated by estradiol and paclitaxel, in which deregulated Bubl expression may contribute to chemotherapeutic efficacy of paclitaxel.