子宫内膜间质细胞体外损伤模型的建立
자궁내막간질세포체외손상모형적건립
Establishment of damaged endometrial stromal cells model in vitro
  • 万方数据
    中华妇产科杂志 중화부산과잡지
    CHINESE JOUNAL OF OBSTETRICS AND GYNECOLOGY
    2012年 4期 274-280 ,共7页

    杨晓清%张沐%张玉泉%唐小军%李伟%徐云钊%杨兵

    양효청%장목%장옥천%당소군%리위%서운쇠%양병

    子宫内膜%间质细胞%米非司酮%细胞,培养的%模型,生物学 子宮內膜%間質細胞%米非司酮%細胞,培養的%模型,生物學
    자궁내막%간질세포%미비사동%세포,배양적%모형,생물학
    Endometrium%Stromal cells%Mifepristone%Cells,cultured%Models,biological
    目的 探讨子宫内膜间质细胞( ESC)体外损伤模型的建立方法.方法 选择2010年6月至12月在南通大学附属医院因子宫肌瘤或宫颈上皮内瘤变行子宫切除患者的子宫内膜组织标本16份,其中增生期8份,分泌期8份.(1)分离、原代培养增生期及分泌期ESC,并鉴定.(2)通过活细胞计数法(CCK-8)检测不同浓度米非司酮作用不同时间及米非司酮撤药后不同时间对ESC增殖抑制率的影响.(3)以米非司酮60 μmol/L为实验组,未加米非司酮为对照组,处理ESC 48 h后撤药,继续培养48 h.观察细胞形态变化,流式细胞仪检测增生期、分泌期ESC凋亡率.用荧光定量PCR技术和蛋白印迹法检测ESC中血管内皮生长因子(VEGF)及半胱氨酸天冬氨酸蛋白酶(caspase)3、8、9 mRNA和蛋白的相对表达水平.结果 (1)16份子宫内膜标本均成功分离并培养ESC.(2)ESC增殖抑制率与米非司酮作用的浓度及时间均呈正相关.撤药后,ESC在一定浓度范围内随撤药时间的延长增殖能力有所恢复,但当米非司酮浓度为100 μmol/L时,撤药后ESC增殖能力难以恢复.(3)实验组以米非司酮60 μmol/L处理ESC 48 h后撤药,继续培养48 h,可见ESC细胞间距增大,呈长梭形,胞质出现空泡化现象.实验组和对照组增生期ESC凋亡率分别为(52±12)％和(13±5)％,分泌期分别为(53±6)％和(32±3)％,分别比较,差异均有统计学意义(P＜0.05).增生期实验组和对照组VEGF mRNA相对表达水平为0.52 ±0.12、1.00±0.17,分泌期实验组与对照组分别为0.19±0.03、0.81 ±0.07,两组分别比较,差异有统计学意义(P＜0.05)；实验组增生期与分泌期VEGF蛋白相对表达水平均明显低于对照组(P＜0.05).增生期实验组与对照组caspase-3、8、9 mRNA的相对表达水平分别为5.62±0.65、1.00±0.44,5.41 ±0.53、1.00±0.21,7.22 ±0.51、1.00±0.32,分泌期实验组与对照组分别为10.22±0.72、1.42 ±0.14,25.30±1.72、1.14±0.28,9.48±1.89、1.16±0.12,两组分别比较,差异均有统计学意义(P＜0.05)；与对照组比较,实验组在增生期与分泌期caspase-3蛋白表达水平分别上调了2.04和1.60倍,caspase-8上调了4.23倍和1.49倍,caspase-9上调了2.65倍和3.50倍；分别比较,差异有统计学意义(P＜0.05).结论 60 μmol/L米非司酮作用于ESC48 h后撤药,继续培养48 h后可作为ESC的体外损伤模型.
    목적 탐토자궁내막간질세포( ESC)체외손상모형적건립방법.방법 선택2010년6월지12월재남통대학부속의원인자궁기류혹궁경상피내류변행자궁절제환자적자궁내막조직표본16빈,기중증생기8빈,분비기8빈.(1)분리、원대배양증생기급분비기ESC,병감정.(2)통과활세포계수법(CCK-8)검측불동농도미비사동작용불동시간급미비사동철약후불동시간대ESC증식억제솔적영향.(3)이미비사동60 μmol/L위실험조,미가미비사동위대조조,처리ESC 48 h후철약,계속배양48 h.관찰세포형태변화,류식세포의검측증생기、분비기ESC조망솔.용형광정량PCR기술화단백인적법검측ESC중혈관내피생장인자(VEGF)급반광안산천동안산단백매(caspase)3、8、9 mRNA화단백적상대표체수평.결과 (1)16빈자궁내막표본균성공분리병배양ESC.(2)ESC증식억제솔여미비사동작용적농도급시간균정정상관.철약후,ESC재일정농도범위내수철약시간적연장증식능력유소회복,단당미비사동농도위100 μmol/L시,철약후ESC증식능력난이회복.(3)실험조이미비사동60 μmol/L처리ESC 48 h후철약,계속배양48 h,가견ESC세포간거증대,정장사형,포질출현공포화현상.실험조화대조조증생기ESC조망솔분별위(52±12)％화(13±5)％,분비기분별위(53±6)％화(32±3)％,분별비교,차이균유통계학의의(P＜0.05).증생기실험조화대조조VEGF mRNA상대표체수평위0.52 ±0.12、1.00±0.17,분비기실험조여대조조분별위0.19±0.03、0.81 ±0.07,량조분별비교,차이유통계학의의(P＜0.05)；실험조증생기여분비기VEGF단백상대표체수평균명현저우대조조(P＜0.05).증생기실험조여대조조caspase-3、8、9 mRNA적상대표체수평분별위5.62±0.65、1.00±0.44,5.41 ±0.53、1.00±0.21,7.22 ±0.51、1.00±0.32,분비기실험조여대조조분별위10.22±0.72、1.42 ±0.14,25.30±1.72、1.14±0.28,9.48±1.89、1.16±0.12,량조분별비교,차이균유통계학의의(P＜0.05)；여대조조비교,실험조재증생기여분비기caspase-3단백표체수평분별상조료2.04화1.60배,caspase-8상조료4.23배화1.49배,caspase-9상조료2.65배화3.50배；분별비교,차이유통계학의의(P＜0.05).결론 60 μmol/L미비사동작용우ESC48 h후철약,계속배양48 h후가작위ESC적체외손상모형.
    Objective To investigate the method of establishing damaged endometrial stromal cells (ESC) model in vitro.Methods ( 1 ) From June to December 2011 ESC from normal endometrim at proliferation phase ( n =8 ) and secretory phase ( n =8 ) were isolated,cultured and identified in vitro.( 2 ) ESC was treated with different concentrations of mifepristone or withdrawal of mifepristone at different time point.The proliferation inhibition percent was measured by cell counting kit-8 ( CCK-8 ). ( 3 ) 0 μmol/L (control group)and 60 μmol/L(experimental group) concentration of mifepristone was added into ESC for 48 hours,then withdrew of mifepristone,continued to be cultured for 48 hours.The morphological changes were observed and apoptosis of ESC in different menstrual cycle were detected by flow cytometry.The mRNA and protein level of vascular endothelial growth factor ( VEGF),caspase-3,8,and 9 were determined by one-step quantitative real-time PCR (Q-PCR) and western blot.Results ( 1 ) ESC from 16 specimens of endometrium were all isolated and cultured successfully. (2) The proliferation inhibition rate of ESC was correlated with concentration and duration of mifepristone positively. The proliferation of ESC could be recovered at a range of time after withdrawal of mifepristone.However,when the concentration of mifepristone was 100 μmol/L,the growth of ESC recovered very hardly. (3) The damaged ESC spacing increased,the spindle shape and vacuolization in the cytoplasm were observed in experimental group; the rate of apoptosis of these damaged cells was significantly increased compared with control groups,which were (52 ± 12)％ vs.( 13 ± 5 ) ％ at the proliferative phase and (53 ± 6) ％ vs.( 32 ± 3 ) ％ at the secretory phase ( all P ＜0.05).The relative mRNA level of VEGF was 0.52 ± 0.12 in experimental group and 1.00 ± 0.17 in control group at proliferation phase (P ＜0.05).And the relative mRNA level of VEGF was 0.19 ±0.03 in experimental group and 0.81 ±0.07 in control group at secretory phase (P ＜ 0.05).The relative level of VEGF protein in the experimental group were both decreased 1.98 and 2.79 folds at the proliferation phase and the secretory phase when compared with those in control group,respectively ( P ＜ 0.05 ).While the relative levels of caspase-3,8,9 mRNA were 5.62 ± 0.65,5.41 ± 0.53,7.22 ± 0.51 in the experimental group and 1.00 ± 0.44,1.00 ± 0.21,1.00 ± 0.32 in control group at the proliferative phase.In the mean time,the relative levels of caspase-3,8,9 mRNA were 10.22 ± 0.72,25.3 ± 1.72,9.48 ± 1.89 in experimental group and 1.42 ± 0.14,1.14 ± 0.28,1.16 ± 0.12 in control group at the secretory phase,respectively (P ＜ 0.05).Compared with the control group,the levels of caspase protein in the experimental group were increased 2.04 and 1.60 folds in caspase-3,4.23 and 1.49 folds in caspase-8,2.65 and 3.5 folds in caspase-9 at the proliferative phase and at the secretory phase,respectively (P ＜ 0.05 ).Conclusion The damaged model of ESC can be established after 48 hours by the withdrawal of 60 μmol/L mifepristone in treatment of ESC for 48 hours.
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