Ca2+通道抑制剂对雌激素作用下子宫内膜癌HEC-1A细胞生物学特性的影响
Ca2+통도억제제대자격소작용하자궁내막암HEC-1A세포생물학특성적영향
Impact of calcium channel antagonists for estrogen action on the endometrial carcinoma HEC-1A cells
  • 万方数据
    中华妇产科杂志 중화부산과잡지
    CHINESE JOUNAL OF OBSTETRICS AND GYNECOLOGY
    2012年 3期 212-217 ,共6页

    包晓霞%王建六%魏丽惠

    포효하%왕건륙%위려혜

    子宫内膜肿瘤%硝苯地平%咪拉地尔%钙通道阻滞药%雌激素类%肿瘤细胞%培养的 子宮內膜腫瘤%硝苯地平%咪拉地爾%鈣通道阻滯藥%雌激素類%腫瘤細胞%培養的
    자궁내막종류%초분지평%미랍지이%개통도조체약%자격소류%종류세포%배양적
    Endometrial neoplasms%Nifedipine%Mibefradil%Calcium channel blockers%Estrogens%Tumor cells,cultured
    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.
    목적 탐토L형Ca2+통도아형( Cav1.3)억제제——초분지평(nifedipine)화T형Ca2+통도아형( Cav3.1)억제제——미패랍지이(mibefradil)대자격소작용하자궁내막암세포계HEC-1A세포생물학특성적영향.방법 (1)이10μmol/L적nifedipine화mibefradil분별예처리HEC-1 A세포15 min,연후여10 μmol/L적17β자이순(17β-E2)화100 μmol/L적자이순우련우혈청백단백(E2-BSA)분별처리HEC-1A세포0min、5min、15 min、30 min、1h、2h,채용역전록(RT) -PCR기술검측각시간점HEC-1A세포중Cav1.3화Cav3.1 mRNA적표체,단백인적법검측HEC-1A세포중Cav1.3화Cav3.1단백적표체.(2)이불동농도(균분별위1.25、2.5、5、10、20、40、80、100 μmol/L)적nifedipine 화mibefradil분별처리HEC-1A세포24、48、96h후,채용사갑기우담서람(MTT)비색법검측세포적증식정황.(3)이10 μmol/L적nifedipine화mibefradil분별처리HEC-1A세포0min、30 min、1h、6h、24h후,채용막련단백V(annexin V)-전화병정(PI)쌍염색법검측세포적조망솔.(4)이10 μmol/L적nifedipine화mibefradil처리HEC-1A세포36 h,체외침습실험검측세포적체외천이능력(이천막세포수표시).결과(1) nifedipine예처리후:①가입17β-E2:HEC-1A세포중Cav1.3 mRNA적표체재15 min시강지최저,30 min시회복；Cav1.3단백재30 min시강지최저,1h시승고.Cav3.1 mRNA적표체재5 min시강지최저,30 min시최고,이후축점회복；Cav3.1단백적표체재각시간점무명현변화.②가입E2-BSA:HEC-1A세포중Cav1.3 mRNA적표체재5min시강지최저,15 min시회복；Cav1.3단백재15 min지내축점증가,15 min후축점강저.Cav3.1 mRNA적표체각시간점균명현강저；Cav3.1단백재5 min시강지최저,15 min후회복.mibefrdial예처리후:①가입17β-E2:HEC-1A세포중Cav1.3 mRNA적표체재각시간점무명현변화；Cav1.3단백적표체분별재15 min화1h시승고.Cav3.1 mRNA적표체재각시간점균명현하강；Cav3.1단백재30 min시초승고.②가입E2-BSA:HEC-1A세포중Cav1.3 mRNA적표체재15 min후강저；Cav1.3단백재15 min후명현하강.Cav3.1 mRNA적표체재각시간점균명현하강；Cav3.1단백재1h시하강지최저.(2)이불동농도적nifedipine화mibefradil분별처리HEC-1A세포24、48、96h후,HEC-1A세포증식적억제작용정명현적농도화시간의뢰성(P＜0.05).(3) nifedipine처리30 min후,HEC-1A세포적조기조망솔교처리시간위0min시명현하강지최저수평,이만기조망솔교처리시간위0 min시명현승고체최고수평,분별비교,차이균유통계학의의(P＜0.05)；이mibefradil처리24h후,HEC-1A세포적만기조망솔(위8.41±0.07)교0 min시(위3.74±0.18)증가근1배,량자비교,차이유통계학의의(P＜0.05).(4)nifedipine처리후HEC-1A세포적천막세포수위(94.0±8.2)개,명현소우미경처리자적(160.0±9.5)개(P=0.020)；이mibefradil처리후HEC-1A세포적천막세포수위(12.0±1.6)개,야명현소우미처리자(P=0.018).결론(1)nifedipine화mibefradil균능억제자격소적촉진Cav1.3화Cav3.1 mRNA화단백표체승고적작용,차mibefradil적작용경지속.(2)nifedipine화mibefradil균능억제HEC-1A세포증식、조망화체외천이능력,이mibefradil교nifedipine적작용경명현.
    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.
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