胰岛素和睾酮对Ishikawa细胞葡萄糖转运蛋白4表达的影响

目的探讨胰岛素(INS)和睾酮(T)对多囊卵巢综合征(PCOS)子宫内膜腺上皮细胞生长的影响和葡萄糖转运蛋白4(GLUT4)表达的调节机制。方法体外培养Ishikawa细胞,予不同浓度INS(90、60、30、3、0.3 U/L)或T(10-3、10-4、10-5、10-6、10-7mmol/ml)刺激Ishikawa细胞48 h,MTT法检测INS、T对Ishikawa细胞生长的作用;免疫细胞化学检测GLUT4蛋白在Ishikawa细胞定位表达;分别以30 U/L INS和10-5mmol/ml T刺激Ishikawa细胞24和48 h,逆转录聚合酶链反应(RT-PCR)方法测定INS和T对Ishikawa细胞GLUT4 mRNA表达的影响。结果(1)不同浓度的INS均可促进Ishikawa细胞的生长,随着INS浓度的增加,INS促进Ishikawa细胞生长作用越强,INS浓度自0.3~30 U/L时,Ishikawa细胞生长依次加强,与对照组相比均有显著性差异(P<0.01)。INS浓度达60、90 U/L时,细胞生长状况与INS浓度为30 U/L相似。不同浓度的T均可抑制Ishikawa细胞的生长,随着T浓度的增加,T抑制Ishikawa细胞生长作用越明显。T浓度自10-7、10-6、10-5mmol/ml,Ishikawa细胞生长依次减弱,与对照组相比均有显著性差异(P<0.01,P<0.05),T浓度达10-4、10-3mmol/ml时,细胞生长抑制状况与T浓度10-5mg/ml相似。(2)GLUT4蛋白,定位表达于Ishikawa细胞的细胞浆内。(3)Ishikawa细胞中GLUT4 mRNA表达,在INS组和T组均较对照组减弱(P<0.01,P<0.05),INS组比T组减弱更明显(P<0.05),且INS和T作用24和48 h GLUT4 mRNA表达无显著性差异(P>0.05)。结论不同浓度INS和T均可影响Ishikawa细胞生长,并降低GLUT4 mRNA的表达,推测PCOS高胰岛素、高雄激素血症的病理生理特性有可能影响子宫内膜的代谢过程,与子宫内膜的病变相关。     

A New Model Supporting Stability Quality of Materials and Industrial Products

Stabilizing the quality of industrial product materials remains a challenge. This applies mainly to new or significantly modified materials. It also refers to special processes. The tests of product quality can stabilize the quality of industrial product materials. The popular method for this is using the non-destructive testing (NDT). The NDT identifies incompatibility but does not determine the cause of its occurrence. Hence, it was necessary to support the process of identifying causes of incompatibilities in products. The purpose of the article was to develop a model based on a new approach to determine the ranking of actions that are possible as part of the process of stabilizing the quality of industrial products. The model was developed to improve quality through sequential and systematic methods of identification (and reduce) and incompatibility. The quality management techniques and decision method were applied and combined in this model, i.e., SMART(-ER) the method, method of selecting a team of experts, brainstorming (BM), Ishikawa diagram with the 5M rule, Likert scale validation technique, arithmetic average, and Grey Relational Analysis (GRA). The test of this model was carried out to find cracks in the outer hull of 418 alloy four-point bearing (CPW-S 5616), which was identified by NDT (magnetic-powder method). As a result, a ranking of activities was obtained to stabilize the quality of the product and the main cause of incompatibility was indicated, i.e., the cause which can influence to the most degree influence on occurrence the incompatibility. The originality of the proposed model is an application in the right order of specially selected and combined qualitative methods and supporting decision methods. The finding of causes of incompatibility of products is the basis of product improvement in the area of stabilizing the quality of materials, mainly by the occurrence of special processes. The universality of the model refers to the possibility of its application for any material, processes of its formation, and processes of products, and any incompatibilities where the model can be integrated with quality control.     

疏肝益肾方剂对子宫内膜癌Ishikawa细胞株的作用研究

目的:研究疏肝益肾方剂对体外Ishikawa细胞的作用及意义.方法:采用中药血清药理学方法制备大鼠含疏肝益.肾方剂血清(含药血清组),并体外作用Ishikawa细胞,同时设正常细胞组(阴性对照组)、大鼠阴性血清对照组(阴性血清组)、顺铂组(阳性对照组)和疏肝益.肾方剂联合顺铂组(联合用药组),MTT检测各组对Ishikawa细胞株的抑制率、RT-PCR检测各组雌激素受体α(estrogen receptor α,ERα)、雌激素受体β(estrogen:recepto βERβ)的转录水平.结果:MTT结果显示含药血清组、联合用药组与阴性对照相比均对Ishikawa细胞株有抑制作用(P＜0.05),而与阳性对照组相比无统计学差异.RT-PCR结果显示含药血清组与阴性对照相比可轻微升高Ishikawa细胞株中ER卢的转录水平,对ERα的转录水平无明显影响;联合用药组则降低ERα的转录水平(P＜0.05)、降低ERβ作用不明显;而阳性对照组降低ERα,ERβ作用均显著(P＜0.05).结论:中药疏肝益肾方剂对体外Ishikawa细胞株有抑制作用,并轻微提升ERβ的表达,但几乎不影响ERα的表达.     

两株子宫内膜癌细胞中mTOR信号通路激活的研究

目的：探讨PTEN缺失Ishikawa和PTEN完整HEC-1A细胞中mTOR信号通路的激活。方法：激光共聚焦显微镜检测Ishikawa和HEC-1A细胞中PTEN蛋白表达;RT-PCR和western blot法从mRNA和蛋白水平分别检测mTOR mRNA和下游磷酸话靶蛋白S6K1和4E-BP1表达。结果：Ishikawa细胞中PTEN蛋白表达缺失,HEC-1A细胞PTEN蛋白表达显著 (P<0.01);两株细胞中均存在mTOR mRNA表达,而Ishikawa细胞中表达水平要高于HEC-1A细胞(P<0.05);Ishikawa细胞中S6K1和4E-BP1蛋白的磷酸化要明显高于HEC-1A细胞(P<0.05)。 结论：2种子宫内膜癌细胞中均存在mTOR信号通路的激活,这种激活水平与PTEN相关,PTEN缺失Ishikawa细胞的激活水平较高。 【关键词】子宫内膜癌;雷帕霉素靶蛋白;PTEN;Ishikawa细胞;HEC-1A细胞     

他莫昔芬对子宫内膜癌细胞株增殖的影响

目的 探讨他莫昔芬(Tamoxifen,TAM)对人子宫内膜癌Ishikawa细胞体外增殖的影响.方法 应用四甲基偶氮唑蓝(MTT)比色法研究他莫昔芬对Ishikawa细胞增殖的影响.结果 TAM浓度为1×10-8～1×10-6mol/L时,呈剂量依赖性促进Ishikawa细胞的增殖,TAM浓度为1×10-5mol/L时,可抑制细胞的增殖.而且这种促进作用呈现剂量-时间-效应关系.结论 低浓度的TAM具有促进子宫内膜癌细胞株Ishikawa细胞增殖的作用.     

雌激素调控子宫内膜癌Ishikawa细胞中LRP16基因表达及其意义

目的:探讨子宫内膜癌Ishikawa细胞中雌激素(E2)对LRP16基因表达的调控作用,LRP16基因过表达对Ishikawa细胞增殖及侵袭生长能力的影响以及可能的分子机制. 方法:采用Northern blot方法检测细胞中LRP16基因mRNA表达水平. 双荧光报告系统检测相对荧光素酶活性. 胎盘蓝死活细胞计数方法观察LRP16过表达对Ishikawa细胞增殖的影响,Matrigel包被的Transwell方法观察LRP16 过表达对Ishikawa细胞侵袭生长能力的影响. Western blot方法检测Ishikawa细胞中的蛋白水平. 结果:雌二醇诱导了Ishikawa细胞中LRP16基因mRNA水平表达上调;而ICI 182 780则Ishikawa细胞中LRP16 mRNA水平下调. 增加ERα表达量促使Ishikawa细胞中LRP16基因上调. pGL3-S5与ERα共转染Ishikawa细胞的相对荧光素酶活性较单纯pGL3-S5转染组细胞升高30倍. 我们没有观察到LRP16基因过表达对Ishikawa细胞的促增殖效应. Transwell结果显示LRP16基因过表达Ishikawa细胞的侵袭率较对照组细胞增加了30%. LRP16基因过表达的Ishikawa细胞中E-钙粘合素的mRNA以及蛋白水平较对照组细胞下调了3倍,而没有检测到MMP2,MMP9以及CD44蛋白水平的变化. 结论:我结果表明E2通过激活ERα上调子宫内膜癌Ishikawa细胞中LRP16基因mRNA水平,LRP16表达水平依赖于雌激素. LRP16基因表达上调没有促进子宫内膜癌细胞的增殖,但可能通过抑制E-钙粘合素表达水平增加了细胞的侵袭能力.     

磷脂酰肌醇3激酶抑制剂对17β-雌二醇作用下Ishikawa、HEC-1A细胞增殖和凋亡的影响

目的:观察磷脂酰肌醇3激酶(PI3K)抑制剂LY294002 对17β-雌二醇(E2)作用下子宫内膜癌细胞增殖、凋亡和细胞周期的影响,初步探讨阻断PI3K/Akt通路治疗子宫内膜癌的可能性. 方法:应用MTT法(10-10 mol/L、10-8 mol/L、10-6 mol/L、10-4 mol/L的E2或10-6 mol/L E2及0.1 μmol/L、1 μmol/L、10 μmol/L、50 μmol/L的LY294002)、流式细胞技术(0 mol/L、10-8 mol/L、10-6 mol/L E2或10-6 mol/L E2及0 μmol/L、1 μmol/L、50 μmol/L LY294002)观察LY294002对E2作用下子宫内膜癌细胞Ishikawa、HEC-1A增殖、凋亡和细胞周期的影响.结果:随着E2浓度的增加,Ishikawa细胞A(570 nm)值逐渐升高并呈时间依赖性(F=3.915,P=0.043),G0-G1期比例下降(F=50.926,P≤0.001),S期比例升高(F=17.836,P=0.001);而HEC-1A细胞周期各期比例无变化(P＞0.05).随着 LY294002浓度的增加,2种细胞A(570 nm)值逐渐下降并呈现时间依赖性(F=10.398,P=0.001;F=5.542,P=0.043),而且G0-G1期比例(F=32.024, P≤0.005;F=14.725,P≤0.017)升高,S期(F=30.132, P≤0.001;F=24.72,P≤0.01)比例下降.50 μmol/L和100 μmol/L LY294002作用2种细胞48 h后,凋亡细胞百分比均增加(P＜0.001).结论:LY294002可以抑制E2对子宫内膜癌细胞的增殖,促使其发生凋亡,抑制细胞周期进展,使细胞周期停滞在G1期.PI3K/Akt信号传导通路可作为治疗子宫内膜癌的新靶点.     

核心蛋白聚糖在Ishikawa细胞中的表达及意义

目的观察表皮生长因子受体（EGFR）信号通路抑制剂RG-14260（RG）单用及RG联合mTOR信号通路抑制剂亚罗奠司（RA）体外对子宫内膜癌Ishikawa细胞增殖及核心蛋白聚糖（DCN）表达的影响。方法子宫内膜癌Ishikawa细胞经RG及RG联合RA作用后,采用免疫组织化学法检测细胞增殖细胞核抗原（PCNA）的表达,反转录酶-聚合酶链式反应（RT-PCR）及免疫印迹法分别检测细胞内DCN基因和蛋白表达的变化。结果RG单用及联合RA均可降低Ishikawa细胞PCNA阳性细胞表达率,促进细胞DCN基因和蛋白的表达,但二者合用效果更为显著。结论单独和联合EGFR及mTOR信号通路抑制剂均可在基因和蛋白水平上上调Ishikawa细胞内DCN的表达,而升高的DCN反过来又可增强信号通路抑制剂对细胞增殖的抑制作用。     

Effects of levonorgestrel, medroxyprogesterone acetate, norethindrone, and 17beta-estradiol on vascular endothelial growth factor isomers 121 and 165 in Ishikawa cells

OBJECTIVE: To determine the effect of 17beta-E(2), levonorgestrel, medroxyprogesterone acetate, and norethindrone on the expression of vascular endothelial growth factor (VEGF) isoforms 121 and 165 in Ishikawa cells in vitro. DESIGN: Prospective basic research study. SETTING: Basic research laboratory. PATIENT(S): None. INTERVENTION(S): Ishikawa cells were cultured in vitro. After 24 hours' incubation in serum-free media, 1.0, 0.1, and 0.01 microM concentrations of E(2), levonorgestrel, medroxyprogesterone acetate, and norethindrone were added for a further 24 hours of incubation. MAIN OUTCOME MEASURE(S): Isolation and identification of VEGF isoforms 121 and 165 using semiquantitative polymerase chain reaction, gel electrophoresis, with beta-actin as an internal control. RESULT(S): Estradiol stimulated VEGF isoforms 121 and 165. The progestins studied increased mRNA for VEGF isoforms 121 and 165 at all doses. Medroxyprogesterone acetate resulted in the greatest increase in both VEGF 121 and 165 compared with norethindrone and levonorgestrel. CONCLUSION(S): Estradiol and progestins increased VEGF 121 and 165 isoform mRNA in Ishikawa cells in vitro. We hypothesize that differences in VEGF expression may be associated with the irregular bleeding during progestin use in clinical situations.     

大蒜素抑制人子宫内膜癌Ishikawa细胞生长的实验研究

目的 研究大蒜素抑制人子宫内膜癌Ishikawa细胞生长的作用,并探讨其可能的作用机制。方法 以不同浓度大蒜素（12.5、25、50μg/ml）和顺铂（40μg/ml）分别干预对数生长期Ishikawa细胞,采用MTT法检测Ishikawa细胞增殖抑制;通过流式细胞术检测细胞周期和细胞凋亡状况,采用Western blot法检测Ishikawa细胞中caspase-3蛋白表达,RT-PCR法检测Ishikawa细胞中Bax mRNA、bcl-2 mRNA表达并计算Bax/bcl-2表达比值。结果 大蒜素能够显著提高子宫内膜癌Ishikawa细胞增殖抑制率、阻滞Ishikawa细胞周期于G₂/M期,提高细胞凋亡率、上调caspase-3蛋白和Bax mRNA表达、下调bcl-2 mRNA表达、提高Bax/bcl-2表达比值,且上述作用均具有一定的剂量依赖性。结论 大蒜素对子宫内膜癌Ishikawa细胞增殖具有抑制作用以及促其凋亡的作用,从而表现出其抑制Ishikawa细胞生长的作用;作用机制可能与大蒜素能够改变Ishikawa细胞周期以及调节凋亡相关基因、蛋白表达有关。