Objective To study the effects of nifedipine and mibefradil on the cell proliferation,apoptosis,migration on the HEC-1 A in vitro and also study the mRNA and protein expression levels of the calcium channel alpha1 D( Cav1.3 ) and calcium channel alpha1G (Cav3.1 ) to discuss the effects of the calcium antagonists on the mechanisms of the endometrial carcinoma.Methods ( 1 ) Add 10 μmol/L nifedipine and mibefradil at 15 minutes before adding 10 μmol/L 17β-estradiol( 17β-E2 ) and 100 μmol/L b-estradiol-6-(O-carboxymethyl) oxime(E2-BSA) to the HEC-1A in different time including 0,5,15,30,60,120 minutes.Then the changes of mRNA and protein were detected by reverse transcripiton( RT)-PCR and western-blot.(2)Add 1.25,2.5,5,10,20,40,80,100 μmol/L nifedipine and mibefradil to the HEC-1 A at 24,48,96 hours to detect the cell proliferation by 3-( 4,5 )-dimethylthiahiazo (-z-y1)-3,5-diphenytetrazoliumromide (MTT) method.(3) Add 10 μmol/L nifedipine and mibefradil to the HEC-1A,then detect the apoptosis at 0 minute,30 minutes,1 hour,6 hours,24 hours and migration in vitro at 36 hours with transwell methods.Results ( 1 ) After the pretreated effect of the nifedipine before 17β-E2,the mRNA express of Cav1.3 genes was lowest at 15 minutes,and returned to the control level after 30 minutes.The protein level didn't change very much in 30 minutes,but rose after 60 minutes.The Cav3.1 genes mRNA express was lowest at 5 minutes,rose at 30 minutes and returned to the 0 minute level gradually.(2) After the pretreated effect of the nifedipine before E2-BSA,the Cav1.3 genes mRNA was lowest at 5 minutes and returned at 15 minutes.The protein level rose gradually in 15 minutes but reduced after 15 minutes.The Cav3.1 mRNA and protein level were reduced at every time point.(3) After the pretreated effect of the mibefradil before 17β-E2,there was no change of mRNA expression of Cav1.3 genes.The protein level rose at 15 and 60 minutes,there was no change in any other time.The Cav3.1 genes mRNA were gradually reduced and the protein level rose at 15 minutes,and there was no change in any other times.(4) After the pretreated effect of the mibefradil before E2-BSA,the mRNA and protein of Cav1.3 levels were reduced after 15 minutes.There was no mRNA expression of Cav3.1,while the protein level was lowest at 15 minutes.(5)Nifedipine and mibefradil affected HEC-1A proliferation depended on the different concentration and interval time points.There was significant difference than those in control group ( P ＜ 0.05 ).( 6 ) There were statistical differences in apoptosis rate after adding nifedipine ( P ＜ 0.05 ),while rose at mibefradil treated the same time ( 24 hours:8.41 ± 0.07,0 minute:3.74 ± 0.18 ; P ＜ 0.05 ).(7) The numbers of stained cells after both nifedipine and mibefradil treated reduced more than control group.Conclusions ( 1 ) Nifedipine and mibefradil could inhibit both the effect of the estrogen on the L-type and T-type calcium channel in short time,meanwhile the mibefradil effects last long time. (2) The inhibited effect of the mibefradil on the proliferation,apoptosis and migration of HEC-1A cells in vitro is more significant than that by nifedidipine.%目的 探讨L型Ca2+通道亚型( Cav1.3)抑制剂——硝苯地平(nifedipine)和T型Ca2+通道亚型( Cav3.1)抑制剂——米贝拉地尔(mibefradil)对雌激素作用下子宫内膜癌细胞系HEC-1A细胞生物学特性的影响.方法 (1)以10μmol/L的nifedipine和mibefradil分别预处理HEC-1 A细胞15 min,然后予10 μmol/L的17β雌二醇(17β-E2)和100 μmol/L的雌二醇偶联牛血清白蛋白(E2-BSA)分别处理HEC-1A细胞0min、5min、15 min、30 min、1h、2h,采用逆转录(RT) -PCR技术检测各时间点HEC-1A细胞中Cav1.3和Cav3.1 mRNA的表达,蛋白印迹法检测HEC-1A细胞中Cav1.3和Cav3.1蛋白的表达.(2)以不同浓度(均分别为1.25、2.5、5、10、20、40、80、100 μmol/L)的nifedipine 和mibefradil分别处理HEC-1A细胞24、48、96h后,采用四甲基偶氮唑蓝(MTT)比色法检测细胞的增殖情况.(3)以10 μmol/L的nifedipine和mibefradil分别处理HEC-1A细胞0min、30 min、1h、6h、24h后,采用膜联蛋白V(annexin V)-碘化丙啶(PI)双染色法检测细胞的凋亡率.(4)以10 μmol/L的nifedipine和mibefradil处理HEC-1A细胞36 h,体外侵袭实验检测细胞的体外迁移能力(以穿膜细胞数表示).结果(1) nifedipine预处理后:①加入17β-E2:HEC-1A细胞中Cav1.3 mRNA的表达在15 min时降至最低,30 min时恢复；Cav1.3蛋白在30 min时降至最低,1h时升高.Cav3.1 mRNA的表达在5 min时降至最低,30 min时最高,以后逐渐恢复；Cav3.1蛋白的表达在各时间点无明显变化.②加入E2-BSA:HEC-1A细胞中Cav1.3 mRNA的表达在5min时降至最低,15 min时恢复；Cav1.3蛋白在15 min之内逐渐增加,15 min后逐渐降低.Cav3.1 mRNA的表达各时间点均明显降低；Cav3.1蛋白在5 min时降至最低,15 min后恢复.mibefrdial预处理后:①加入17β-E2:HEC-1A细胞中Cav1.3 mRNA的表达在各时间点无明显变化；Cav1.3蛋白的表达分别在15 min和1h时升高.Cav3.1 mRNA的表达在各时间点均明显下降；Cav3.1蛋白在30 min时稍升高.②加入E2-BSA:HEC-1A细胞中Cav1.3 mRNA的表达在15 min后降低；Cav1.3蛋白在15 min后明显下降.Cav3.1 mRNA的表达在各时间点均明显下降；Cav3.1蛋白在1h时下降至最低.(2)以不同浓度的nifedipine和mibefradil分别处理HEC-1A细胞24、48、96h后,HEC-1A细胞增殖的抑制作用呈明显的浓度和时间依赖性(P＜0.05).(3) nifedipine处理30 min后,HEC-1A细胞的早期凋亡率较处理时间为0min时明显下降至最低水平,而晚期凋亡率较处理时间为0 min时明显升高达最高水平,分别比较,差异均有统计学意义(P＜0.05)；而mibefradil处理24h后,HEC-1A细胞的晚期凋亡率(为8.41±0.07)较0 min时(为3.74±0.18)增加近1倍,两者比较,差异有统计学意义(P＜0.05).(4)nifedipine处理后HEC-1A细胞的穿膜细胞数为(94.0±8.2)个,明显少于未经处理者的(160.0±9.5)个(P=0.020)；而mibefradil处理后HEC-1A细胞的穿膜细胞数为(12.0±1.6)个,也明显少于未处理者(P=0.018).结论(1)nifedipine和mibefradil均能抑制雌激素的促进Cav1.3和Cav3.1 mRNA和蛋白表达升高的作用,且mibefradil的作用更持续.(2)nifedipine和mibefradil均能抑制HEC-1A细胞增殖、凋亡和体外迁移能力,而mibefradil较nifedipine的作用更明显.
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