细胞周期调控基因p21~(WAF1/CIP1)与宫颈癌

p21WAF1/CIP1基因是1993年发现并克隆的细胞周期依赖性蛋白激酶抑制因子(Cyclin-dependent kinas inhibitor,CDKI)的家族成员,与p21ras不同,它参与细胞的多种功能活动。近年的许多研究表明,p21WAF1/CIP1基因与宫颈癌的发生、发展关系密切,从而引起学者的关注。1p21WAF1/CIP1基因的发现与命名p21WAF1/CIP1基因是在不同实验室用不同的方法克隆出来的。Harper等〔1〕从细胞周期出发寻找CDK调节蛋白时,发现一种可与细胞周期依赖性蛋白激酶(cyclin-depedent kinase,CDK)相互作用的蛋白,并将其克隆,命名为CDK相互作用蛋白(cyclin-…     

大蒜素对子宫颈癌Hela细胞增殖的影响

目的:探讨大蒜素对宫颈癌Hela细胞增殖的影响。方法:不同浓度的大蒜素作用于体外培养的Hela细胞,倒置显微镜下观察Hela细胞的形态学变化;采用MTT法检测大蒜素对Hela细胞增殖抑制能力,采用流式细胞术分析大蒜素对Hela细胞凋亡的影响。结果:MTT显示大蒜素能抑制人宫颈癌Hela细胞的增殖,并具有时间-剂量依赖性;流式细胞术测定结果显示50μg/ml浓度的大蒜素可明显诱导Hela细胞凋亡,并将细胞周期阻滞在G2/M期。结论:大蒜素对人宫颈癌Hela细胞的生长有抑制作用,其抑制作用与诱导Hela细胞凋亡和阻抑细胞周期有关。     

大蒜素影响子宫颈癌Hela细胞增殖的实验研究

目的探讨大蒜素对宫颈癌Hela细胞增殖的影响。方法不同浓度的大蒜素作用于体外培养的Hela细胞,倒置显微镜下观察Hela细胞的形态学变化,采用MTT法检测大蒜素对Hela细胞增殖抑制能力,流式细胞术分析大蒜素对Hela细胞凋亡的影响。结果 MTT显示大蒜素能抑制人宫颈癌Hela细胞的增殖,并具有时间-剂量依赖性;流式细胞术测定结果显示50μg/ml浓度大蒜素可明显诱导Hela细胞凋亡,并将细胞周期阻滞在G2/M期。结论大蒜素对人宫颈癌Hela细胞增殖有抑制作用,其抑制作用与诱导Hela细胞凋亡和阻抑细胞周期有关。     

顺铂诱导人宫颈癌Hela细胞凋亡及其对细胞周期影响的研究

目的:观察顺铂对人宫颈癌Hela细胞凋亡及细胞周期的影响,以揭示顺铂治疗宫颈癌的机理。方法:运用体外细胞培养技术,以不同浓度的顺铂作用于培养的人Hela细胞72 h,用流式细胞仪分析其凋亡率及细胞周期。结果:顺铂能以浓度依赖的方式诱导Hela细胞凋亡,并呈现出G0/G1期阻滞作用。结论:顺铂能诱导Hela细胞凋亡并改变其细胞周期分布,阻滞Hela细胞于G0/G1期,这可能是顺铂抗宫颈癌作用的重要机制之一。     

人IL-21基因联合γ射线照射对子宫颈癌Hela细胞生长的影响

目的 研究含人IL-21基因的重组腺病毒(Ad-IL-21)联合γ射线照射对子宫颈癌Hela细胞生长的抑制作用。方法 重组腺病毒Ad-IL-21于体外转染子宫颈癌Hela细胞,转染后6 h进行6Gy ¹³⁷Cs γ射线照射,用MTT方法和流式细胞仪测定Hela细胞的生长曲线、抑制率及细胞周期。结果 Ad-IL-21基因组、照射组和基因联合照射组对Hela细胞生长均具有抑制作用,联合照射组的抑制效应最强(44%),明显高于Ad-IL-21组和照射组。联合照射组Hela细胞出现明显的G1期阻滞,G1期细胞所占比例最高,达到88.9%,S期细胞最少(1%)。结论 IL-21基因联合γ射线照射对子宫颈癌的抑瘤作用具有协同效应,有效地抑制肿瘤细胞的生长。     

茶多酚和茶色素对肝癌细胞株HepG2细胞周期的影响

为探讨茶对肝癌细胞周期的影响 ,体外培养人肝癌细胞株HepG2 ,加入终浓度为 50mg L和 1 0 0mg L茶多酚和茶色素作用 48h后 ,流式细胞仪分析DNA含量 ,Westernblot观察对细胞周期蛋白P2 1 WAF1 CIP1 和细胞周期素D1 (cyclinD1 ) ,RT PCR检测细胞周期素依赖激酶 4 (Cdk4)在mRNA水平上的表达情况。结果表明 ,茶多酚和茶色素引起了细胞周期G1 期阻滞 (G1 arrest) ,抑制了cyclinD1蛋白的表达 ,诱导了P2 1 WAF1 CIP1 蛋白的表达增加 ,并且显著抑制了Cdk4在mRNA水平上的表达。因此 ,诱导细胞周期阻滞可能是茶预防肿瘤的一个重要机制     

米非司酮对人宫颈癌Hela细胞及其NF-κBp65蛋白表达的影响

目的:探讨米非司酮(MIF)对人子宫颈癌Hela细胞的凋亡诱导作用。方法:体外培养人子宫颈癌Hela细胞,采用流式细胞术分析不同浓度MIF对Hela细胞的凋亡率及细胞周期分布的影响;采用Westernblot法检测MIF对Hela细胞中NF-κBp65蛋白表达的影响。结果:3个浓度的MIF均能使Hela细胞凋亡率明显增加,使S期、G2/M期细胞比例减少,G0/G1期细胞比例显著增加,且作用呈剂量依赖性;随着MIF浓度的增加,Hela细胞中NF-κBp65蛋白表达水平逐渐下降。结论:MIF可能通过NF-κB信号通路降调NF-κBp65蛋白表达,从而调节细胞周期、诱导Hela细胞凋亡。     

芬太尼对MCF-7细胞增殖和细胞周期影响研究

目的　探讨芬太尼对MCF -7细胞增殖和细胞周期的影响及其机制。方法　MCF -7细胞在不同浓度芬太尼、纳洛酮和两药联合干预后 ,采用MTT法检测细胞增殖活性 ;流式细胞术检测细胞周期 ;免疫细胞化学染色SP法检测p5 3、p2 1/WAF1的表达。结果　芬太尼浓度≥ 0 1μmol/L时 ,细胞增殖活性较对照组显著下降 (P <0 0 5 ) ,其效应与时间和浓度均呈正相关 (P <0 0 1) ,72h半数抑制浓度 (IC50 )为 0 81± 0 0 2 μmol/L。随着芬太尼浓度增加 ,G0 /G1期比例逐渐增加 ,(S +G2 /M )期比例逐渐减少。芬太尼组浓度≥ 0 1μmol/L时 ,细胞增殖活性较对照组显著下降 (P <0 0 5 ) ,较纳洛酮和芬太尼联合给药组差异无显著性。给予芬太尼 10 μmol/L后胞核内p5 3、p2 1/WAF1AOD显著增加 (P <0 0 5 )。结论　芬太尼浓度和时间依赖性抑制MCF -7细胞增殖 ,主要是将细胞阻滞在G1期 ;其机制可能是上调野生型p5 3和p2 1/WAF1的表达。     

siRNA抑制IKCa1基因表达对宫颈癌HeLa细胞增殖的影响

目的:研究siRNA(small interfering RNA)抑制钙激活性中电导钾离子通道(intermediate-conductance Ca2+-activated K+channels,IKCa1)表达对宫颈癌HeLa细胞增殖的影响。方法:构建含有IKCa1基因的siRNA表达载体,脂质体法转染HeLa细胞,通过RT-PCR和Western Blot检测HeLa细胞IKCa1 mRNA和蛋白表达变化;WST-1检测细胞增殖能力;流式细胞仪分析HeLa细胞周期变化。结果:成功构建针对IKCa1基因的真核表达载体,所获表达载体转染HeLa细胞可使IKCa1mRNA和蛋白表达水平显著降低(P<0.05),并能有效抑制HeLa细胞增殖(P<0.05),使细胞周期停留在G0～G1期,S期细胞明显减少。结论:应用siRNA干扰技术能有效抑制IKCa1基因的表达,同时也可有效抑制宫颈癌HeLa细胞体外增殖活性。     

Pin1抑制剂对宫颈癌SiHa细胞增殖和凋亡的影响

目的:研究Pin1抑制剂(Juglone)对宫颈癌SiHa细胞增殖和凋亡的影响,探讨Juglone的抗肿瘤作用。方法:体外培养人宫颈癌细胞SiHa,采用MTT试验观察细胞生长抑制状况,流式细胞仪检测细胞周期阻滞以及Hoechst33258检测细胞凋亡情况。结果:MTT试验表明,Juglone对宫颈癌SiHa细胞生长有明显的抑制作用,且随着作用浓度和作用时间的增加,抑制作用增强。流式细胞仪检测表明,Juglone药物(50μmol/L)培养12、24、48 h后,G2/M期细胞比例数明显增加,而S期细胞比例数却较对照组降低。Hoechst33258实验组可见凋亡细胞。结论:Juglone可能通过体外抑制细胞增殖,诱导细胞凋亡途径抑制宫颈癌的发展。     

5-氮杂胞苷对宫颈癌细胞FHIT基因去甲基化及抑制增殖作用

目的:探讨5-氮杂胞苷对Hela宫颈癌细胞脆性组氨酸三联基因(Fragile histidine triad gene,FHIT)启动子去甲基化作用及对其增殖的影响。方法:采用低浓度和高浓度5-氮杂胞苷作用Hela宫颈癌细胞,分别采用BSP和RT-PCR检测处理前后FHIT基因启动子甲基化状态和mRNA表达,应用MTT法检测细胞增殖改变。结果:Hela宫颈癌细胞FHIT基因启动子表现高度甲基化状态(CG位点甲基化率为80%～100%),mRNA表达完全受到抑制,细胞呈指数增殖状态;低浓度5-氮杂胞苷作用后的甲基化率显著降低(10%～40%),细胞增殖速度降低;高浓度组FHIT基因启动子甲基化状态被完全逆转,mRNA表达明显高于低浓度组,细胞增殖速度显著低于正常组和低浓度组。结论:5-氮杂胞苷可逆转Hela宫颈癌细胞FHIT基因甲基化状态,使基因恢复表达而抑制细胞增殖,去甲基化和抑制增殖作用随5-氮杂胞苷剂量增加而增加。     

The ethanol extract from Artemisia princeps Pampanini induces p53-mediated G1 phase arrest in A172 human neuroblastoma cells

In the present study, the antiproliferative effects of the ethanol extract of Artemisia princeps Pampanini (EAPP) and the mechanism involved were investigated. Of the various cancer cells examined, human neuroblastoma A172 cells were most sensitive to EAPP, and their proliferation was dose- and time-dependently inhibited by EAPP. DNA flow cytometry analysis indicated that EAPP notably induced the G(1) phase arrest in A172 cells. Of the G(1) phase cycle-related proteins examined, the expressions of cyclin-dependent kinase (CDK) 2, CDK4, and CDK6 and of cyclin D(1), D(2), and D(3) were found to be markedly reduced by EAPP, whereas cyclin E was unaffected. Moreover, the protein and mRNA levels of the CDK inhibitors p16(INK4a), p21(CIP1/WAF1), and p27(KIP1) were increased, and the activities of CDK2, CDK4, and CDK6 were reduced. Furthermore, the expressions of E2F-1 and of phosphorylated pRb were also decreased, and the protein levels of p53 and pp53 (Ser15) were increased. Up-regulation of p21(CIP1/WAF1) was found to be mediated by a p53-dependent pathway in EAPP-induced G(1)-arrested A172 cells. When these data are taken together, the EAPP was found to potently inhibit the proliferation of human neuroblastoma A172 cells via G(1) phase cell cycle arrest.     

NaHS抑制宫颈癌细胞增殖的初步机制

目的:探讨NaHS对子宫颈癌细胞增殖的影响及其机制。方法:首先将子宫颈癌HeLa细胞分成对照组和不同剂量的NaHS处理组,并采用MTT法检测细胞存活率和流式细胞仪检测细胞凋亡率。使用不同浓度的格列本脲预先处理HeLa细胞30 min,再用400μmol/L的NaHS处理24 h,探讨NaHS抑制子宫颈癌细胞增殖的初步机制。结果:不同剂量的NaHS处理组能明显抑制HeLa细胞增殖而促进HeLa细胞凋亡;格列本脲预先处理可以部分阻断这种作用。结论:NaHS抑制子宫颈癌细胞增殖可能与ATP敏感性钾通道有关。     

Trans-10,cis-12, not cis-9,trans-11, conjugated linoleic acid inhibits G1-S progression in HT-29 human colon cancer cells

Commercial preparations of conjugated linoleic acid (CLA) contain both positional and geometric isomers of octadecadienoic acid, with cis-9,trans-11 CLA (c9t11) and trans-10,cis-12 CLA (t10c12) as the principal isomers. We showed previously that CLA reduced the incidence of colon tumors in rats treated with 1,2-dimethylhydrazine. In addition, our previous in vitro studies showed that t10c12 inhibited the growth of HT-29 and Caco-2 human colon cancer cells, whereas c9t11 had no effect on cell growth. In the present study, to examine the effects of the CLA isomers on cell cycle and cell cycle regulatory proteins, we treated HT-29 cells with various concentrations (0-4 micromol/L) of the individual CLA isomers. A DNA flow cytometric analysis revealed that t10c12 induced a G1 arrest, whereas c9t11 had no effect on the cell cycle. Western blot analysis of total cell lysates revealed no alteration in the protein expression of cyclin A, cyclin D, cyclin E, cyclin-dependent kinase (CDK) 2, or CDK4 due to t10c12 treatment. However, t10c12 substantially increased the protein expression and mRNA accumulation of the CDK inhibitor p21(CIP1/WAF1). The t10c12 isomer increased the association of p21(CIP1/WAF1) with CDK2 and proliferating cell nuclear antigen, but decreased the levels of phosphorylated retinoblastoma protein (Rb), with an increase in the levels of hypophosphorylated Rb protein. An in vitro kinase assay using histone H1 as a substrate showed that the activities of CDK2 were significantly decreased by t10c12. These results indicate that t10c12 exerts its growth inhibitory effects in colon cancer cells through the induction of G1 cell cycle arrest. The induction of p21(CIP1/WAF1) may be one of the mechanisms by which t10c12 inhibits cell cycle progression in HT-29 cells.     

GRIM-19对子宫颈癌细胞株HeLa细胞Survivin基因表达的影响

目的:探讨稳定转染GRIM-19对子宫颈癌细胞株HeLa细胞凋亡抑制基因Survivin的表达以及细胞增殖的影响。方法:采用脂质体转染法将GRIM-19重组质粒转染子宫颈癌细胞株HeLa细胞,建立稳定表达GRIM-19的HeLa细胞株,采用RT-PCR检测HeLa细胞转染GRIM-19后mRNA水平表达情况,采用WesternBlot检测稳定转染后GRIM-19与Survivin的表达情况,应用细胞计数法观察各组细胞增殖水平。结果:建立稳定的GRIM-19基因表达的HeLa细胞株,命名为HeLa/G19细胞,对照组细胞命名为HeLa/Con细胞。RT-PCR检测显示GRIM-19mRNA表达明显增加,WesternBlot检测显示GRIM-19的蛋白表达显著升高、Survivin蛋白的表达水平显著降低。细胞计数法实验显示HeLa/GRIM-19细胞增殖较HeLa/Con细胞明显减慢(P<0.05)。结论:GRIM-19可以抑制子宫颈癌细胞Survivin的表达,阻滞细胞的生长,可能是子宫颈癌治疗的新靶点。     

trans-10,cis-12 conjugated linoleic acid inhibits the G1-S cell cycle progression in DU145 human prostate carcinoma cells

Conjugated linoleic acid (CLA) is a group of positional and geometric isomers of linoleic acid, and has evidenced anti-cancer activities in experimental animal cancer models and in vitro studies. The two predominant isomers of CLA are cis-9,trans-11 CLA (c9t11) and trans-10,cis-12 CLA (t10c12). The present study was performed to study the effect of the individual CLA isomers on DU145 cell growth. The cells were incubated in serum-free medium with different concentrations of the fatty acids. Treatment of cells with t10c12 (at 2.5-10 micromol/L) resulted in a dose-dependent reduction in the numbers of viable cells, whereas c9t11 CLA at a concentration of 5 micromol/L slightly increased viable cell numbers at 3 days (P < .05). DNA flow cytometric analysis revealed that the treatment of DU145 cells with t10c12 for 24 hours induced a small but significant increase in the number of cells in the G1 phase, accompanied by a complementary decrease in cells in the S phase. c9t, however, had no effect on cell cycle progression. To determine the molecular mechanisms underlying t10c12-induced G1 arrest, the levels of cell cycle regulatory proteins were estimated by western blot analyses. t10c12 induced a marked increase in p21(CIP1/WAF1) protein levels in a dose-dependent manner. p27(KIP1) was not affected by t10c12. t10c12 moderately decreased cyclin A and cyclin D1 protein levels (P > .05). However, t10c12 did not affect the expression of cyclin-dependent kinase (CDK) 2, CDK4, or cyclin E. t10c12 increased p21(CIP1/WAF1) bound to CDK2 and attenuated CDK2 activity. These results indicate that t10c12-induced p21(CIP1/WAF1) binds to CDK, and inhibits the activity of this enzyme, which results in the observed decrease in the G1-S progression in DU145 cells.     

Sodium butyrate inhibits cell growth and stimulates p21WAF1/CIP1 protein in human colonic adenocarcinoma cells independently of p53 status

Butyric acid, one of the short-chain fatty acids produced by microbial fermentation in the colon, exhibits antiproliferative activities in various cancer cell lines. The initial objective of the study was to assess whether the effect of sodium butyrate (NaB) on cell growth differed by p53 status of the cells. Four human colorectal adenocarcinoma cell lines were used: HT29 (p53 point mutation), Caco2 (p53 truncation), LS513 (p53 wild type), and Lovo (p53 wild type). NaB significantly inhibited cell growth in all four cell lines. NaB arrested HT29 and LS513 cells in G0/G1 and Caco2 and Lovo in G2-phase. A second objective was to determine whether NaB similarly affected the cyclin-dependent kinase inhibitor, p21WAF1/CIP1. In all cell lines, p21 mRNA levels were immediately elevated after NaB exposure, and p21 protein levels were increased within 6 h. NaB increased p21 promoter activity in both Caco2 and Lovo, suggesting p53 independence. NaB did not influence p21 mRNA stability. Although three DNase I hypersensitivity sites were identified in the region of the p21 gene, induction of p21 mRNA by NaB was not accompanied by relaxation of the chromatin in the region of the p21 gene.     

奈达铂对宫颈癌Hela及Siha细胞的作用

目的:比较奈达铂(NDP)体外对人宫颈腺癌细胞Hela细胞及鳞癌细胞Siha的抑制作用并探讨其作用机制。方法:以2.5～40μg/ml NDP分别作用于Hela及Siha细胞24 h,48 h,72 h,MTT法检测抑制率,计算半数抑制浓度(IC50);利用流式细胞术(FCM)观察细胞凋亡率及周期变化;半定量PCR法分析基因半胱氨酸天门冬氨酸蛋白酶3(Caspase 3,Caspase 9)及基质金属蛋白酶2(MMP-2)的表达变化。结果:NDP对宫颈癌Hela细胞及Siha细胞均有抑制作用,呈时间-剂量依赖性,NDP对Hela细胞的抑制率大于Siha细胞;FCM显示,随时间、浓度增加,Hela及Siha细胞凋亡率增加,相同浓度和作用时间下,Hela细胞的凋亡率大于Siha细胞(P<0.05),S期细胞的比例增加,G0/G1期比例减少,G2/M期比例变化不明显;与对照组相比,Caspase 3,Caspase 9表达增加,MMP-2表达减少。结论:NDP可抑制宫颈腺癌细胞Hela及鳞癌细胞Si-ha的增殖,NDP体外对Hela细胞的抑制作用更强。NDP的作用机制与诱导细胞凋亡、细胞周期阻滞、上调Caspase3和Caspase9的表达有关;下调MMP-2的表达可能有利于降低宫颈癌细胞的侵袭力。     

Inotodiol对宫颈癌HeLa细胞增殖的影响

目的:研究桦褐孔菌单体(Inotodiol)对宫颈癌HeLa细胞增殖的影响。方法:采用MTT法检测Inotodiol对HeLa细胞的增殖抑制率;通过倒置显微镜、HE染色观察HeLa细胞形态学改变;通过免疫细胞化学法检测核增殖抗原Ki-67表达。结果:随着Inotodiol浓度增加及作用时间延长,抑制率明显增强,差异有统计学意义(P<0.05)。Inotodiol作用于Hela细胞后,形态学观察示细胞凋亡。Inotodiol组Ki-67表达明显降低,与对照组相比差异有统计学意义(P<0.01)。结论:Inoto-diol对宫颈癌HeLa细胞有明显的抑制增殖和诱导凋亡作用。