硒蛋氨酸诱导食管癌细胞株凋亡的实验研究

目的：探讨硒蛋氨酸对食管癌细胞系EC 9706凋亡的影响。方法：采用MTT比色法，细胞生长曲线描绘观察硒蛋氨酸对食管癌细胞系EC 9706增殖的影响。采用流式细胞仪观察硒蛋氨酸对EC 9706细胞诱导其凋亡的作用及对细胞周期的影响。琼脂糖凝胶电泳法检测DNA ladder。结果：硒蛋氨酸呈时间、剂量依赖性方式抑制EC 9706细胞增殖，改变细胞周期分布，增加G0／G1期细胞比例，诱导细胞凋亡。结论：硒蛋氨酸可能通过影响细胞周期分布和诱导细胞凋亡，从而抑制EC 9706细胞增殖。硒蛋氨酸可能是预防和治疗食管癌的一种新制剂。     

环氧合酶-2选择性抑制剂NS-398诱导食管癌细胞EC 9706凋亡及其机制的探讨

目的 观察环氧合酶-2(COX-2)选择性抑制剂NS一398对食管癌细胞株EC 9706增殖及凋亡的影响,砌究其对凋亡抑制蛋白Survivin和Caspase-3表达的影响,探讨NS-398诱导Ec 9706细胞凋亡的作用机制.方法 NS-398作用EC 9706细胞后,MTT法测定NS-398对人食管癌EC 9706细胞增殖的抑制率;DNA片段分析法和流式细胞仪检测细胞凋亡;免疫细胞化学检测Survivin和Caspase-3蛋白表达变化.结果 NS-398(10～100μmol/L)对EC 9706细胞生长有抑制作用,随浓度升高、时间延长抑制作用增强,并诱导EC 9706细胞凋亡,呈剂量-时间效应关系;NS-398可降佴Survivin蛋白表达,增加Caspase-3蛋白表达.结论 NS-398可诱导人食管癌细胞株EC 9706凋亡,其机制可能与下调Survivin表达及激活Capase-3表达有关.     

表皮生长因子受体抑制剂吉非替尼对食管癌EC9706细胞增殖、凋亡及细胞周期的影响

目的探讨表皮生长因子受体(EGFR)抑制剂吉非替尼对食管癌EC9706细胞增殖、凋亡和细胞周期的影响。方法将EC9706细胞培养并加入不同浓度的吉非替尼处理不同时间后,采用MTT法检测细胞增殖抑制率;Annexin V-FITC/PI双染法、流式细胞仪检测细胞凋亡率。PI染色、流式细胞仪检测细胞周期。结果吉非替尼对食管癌EC9706细胞增殖具有明显的抑制作用,且表现为剂量和时间依赖性;吉非替尼组细胞凋亡率明显高于正常对照组,且随着吉非替尼剂量的增加,凋亡率逐渐增加(P0.05);与对照组相比,10μg/ml吉非替尼处理24 h后的EC9706细胞,处于G0/G1期细胞比例明显增加,处于S期细胞比例明显减少(P0.05)。结论吉非替尼对食管癌EC9706细胞具有明显的增殖抑制作用,其机制与诱导凋亡及阻滞细胞周期有关。     

孕烷X受体抗食管癌EC9706细胞凋亡的作用机制

目的 研究孕烷X受体(PXR)抗食管癌EC9706细胞凋亡的作用机制.方法 使用利福平活化食管鳞癌EC9706细胞中的PXR,阿霉素(ADM)诱导高表达PXR的EC9706细胞凋亡,采用流式细胞仪观察细胞的增殖周期,MTT法观察细胞凋亡率,Western印迹和免疫组化法检测Caspase-3,Bcl-2,Bax蛋白表达情况.结果 ADM处理可以明显抑制细胞生长;使细胞呈明显凋亡改变;利福平诱导PXR高表达的EC9706细胞凋亡减少,抑制Caspase-3的蛋白水平,上调蛋白Bcl-2的表达,表明PXR在抗食管鳞癌细胞凋亡中发挥重要的作用.结论 PXR可能是通过降低Caspase-3和升高Bcl-2蛋白的表达抑制食管癌细胞EC9706的凋亡.     

Pinl抑制剂Juglone对食管癌EC1细胞增殖的抑制作用

目的:测定Pin1抑制剂(Juglone)对食管癌细胞EC1生长增殖的影响,探讨Juglone的抗肿瘤作用.方法:体外培养人食管癌细胞系EC1,用MTT试验观察细胞生长增殖状况,流式细胞仪检测细胞周期以及细胞凋亡.结果:MTT试验表明,Juglone对EC1细胞生长有明显的抑制作用,且抑制作用随作用浓度和作用时间增加而增强.流式细胞仪检测表明,加入Juglone培养48 h后,EC1细胞出现G2期阻滞.Juglone药物(10、20、30 μmol/L)培养48 h后,EC1细胞的凋亡率明显增加,与对照组相比有统计学意义(9.06%,32.88%,53.18% vs 8.77%.均P<0.05).结论:Pin1抑制剂Juglone可以通过抑制Pin1表达从而抑制食管癌细胞的增殖,Pin1抑制剂有望成为新型的抗肿瘤治疗靶点.     

顺铂诱导食管癌EC9706耐药细胞中Bcl-2的表达

目的探讨凋亡抑制蛋白Bcl-2在顺铂(DDP)诱导食管癌EC9706耐药细胞中的表达。方法建立EC9706/DDP细胞,流式细胞仪检测细胞凋亡率,四甲基偶氮唑蓝(MTT)法检测细胞存活率,Western印迹检测DDP处理后EC9706细胞及EC9706/DDP细胞Bcl-2蛋白表达,RTPCR、荧光定量PCR检测Bcl-2 mRNA水平。结果 EC9706/DDP细胞对DDP诱导的凋亡不敏感,Western印迹检测到EC9706/DDP细胞中Bcl-2表达上调,与EC9706细胞相比具有统计学意义(P0.05)。RT-PCR、荧光定量PCR检测到EC9706/DDP细胞中Bcl-2 mRNA水平上调,与EC9706细胞相比具有统计学意义(P0.05)。结论 Bcl-2表达上调可能为食管癌EC9706细胞对DDP耐药的重要机制。     

曲古霉素A对食管癌细胞系EC9706细胞凋亡的作用及机制

目的探讨曲古霉素A（TSA）对食管癌细胞系EC9706细胞凋亡的影响及机制。方法用AnnexinV-FITC和PI进行双染色,流式细胞仪检测细胞凋亡率,Western blot检测TSA对食管癌细胞凋亡相关基因表达的影响。结果 1.0μmol/L的TSA诱导EC9706细胞凋亡率增加（P〈0.05）,且呈浓度依赖性;0.5μmol/L的TSA作用48 h后细胞凋亡率增加（P〈0.05）,呈时间依赖性。TSA处理的EC9706细胞Bax蛋白表达增加,Bcl-2蛋白表达减少;TSA诱导EC9706细胞caspase-8及caspase-9裂解活化,且随作用时间延长逐步升高。结论一定量的TSA可以诱导EC9706细胞凋亡,凋亡原因与Bax表达增强、Bcl-2减少以及凋亡细胞中caspase-8及caspase-9介导的caspase-3活化有关。     

人参皂甙Rg3对人食管癌EC-9706细胞及内源性VEGF的影响

培养人食管癌细胞株EC0706,不同浓度中药20（R）-人参皂甙Rg3和不同时间点进行干预,采用MTT法、流式细胞术、免疫细胞化学等技术了解不同浓度Rg3对此细胞增殖、凋亡、细胞周期及内源性VEGF的影响。MTT法示不同浓度Rg3对人食管癌细胞株EC0706的增殖均有明显抑制作用;流式细胞术示Rg3能诱导细胞凋亡及调节细胞周期;免疫细胞化学示Rg3能抑制细胞内源性VEGF的表达。认为适当浓度的RS3作用一定时间后,可促进人食管癌细胞株EC0706的凋亡,并可抑制肿瘤细胞内源性分泌的VEGF的表达,Rg3抑制肿瘤血管生成的机制之一是通过影响细胞分泌上述因子而起作用的。     

玉竹提取物B对人食管癌细胞Eca-109增殖与凋亡的影响

目的观察玉竹提取物B（EB-PAOA）对人食管癌细胞Eca-109增殖与凋亡的影响。方法将体外培养的Eca-109细胞与不同浓度的EB-PAOA共育,采用MTT法检测Eca-109细胞增殖抑制率,采用流式细胞仪检测Eca-109细胞凋亡率。结果随着EB-PAOA浓度增大、作用时间延长,Eca-109细胞的增殖抑制率逐渐升高（P均〈0.05）,呈时间、剂量依赖性;随着EB-PAOA浓度增加,Eca-109细胞凋亡率逐渐增加,呈一定浓度依赖性（P均〈0.05）。结论EB-PAOA能够抑制人食管癌细胞Eca-109的增殖,并诱导其凋亡。     

伊班膦酸钠对食管鳞癌EC9706细胞增殖及VEGF表达的作用

目的了解双膦酸盐(BPs)对人食管癌EC9706细胞株增殖的影响,探讨伊班膦酸钠对VEGF表达的作用。方法倒置显微镜观察细胞形态变化,进行细胞形态学观察;采用MTT法检测伊班膦酸钠对EC9706细胞增殖的抑制作用;采用免疫细胞化学检测伊班膦酸钠对EC9706细胞VEGF表达的影响。结果随着伊班膦酸钠药物浓度的增加和作用时间的延长,实验组细胞密度下降,细胞碎片增多,核染色质和胞浆皱缩,胞膜和核膜增厚,折光性差,细胞贴壁能力下降。实验组不同浓度的伊班膦酸钠对食管癌EC9706细胞均有抑制作用,且与药物浓度和作用时间呈正相关,与对照组相比,差异均有统计学意义(P0.05)。对照组及10、20、40μg/ml浓度VEGF蛋白的表达率分别为43.81±3.61、30.92±3.30、21.57±3.17、9.05±1.38,各实验组与对照组比较、各实验组组间比较,差异均有统计学意义(P0.05)。结论伊班膦酸钠对食管癌EC9706细胞有增殖抑制作用,并且具有时间及剂量依赖性。伊班膦酸钠可以从蛋白水平抑制食管癌EC9706细胞VEGF的表达。     

miR-451对食管癌EC9706细胞增殖、凋亡及侵袭能力的影响

目的:探讨miR-451对食管癌EC9706细胞增殖、凋亡及侵袭能力的影响.方法:化学合成miR-451mimics,脂质体包裹转染EC9706细胞为miR-451组,同时设立无关序列(Scramble-miR)对照组、脂质体对照组和空白对照组.转染后48h,荧光定量RT-PCR检测miR-451表达量的变化,Westernblot检测Bcl-2、AKT和磷酸化AKT蛋白表达水平,流式细胞仪检测细胞凋亡情况,Transwell侵袭实验检测细胞侵袭能力的改变;MTT法检测转染后l、2、3、4、5、6d各组细胞增殖率.结果:miR-451组的miR-451表达水平显著上调(P<0.01,F=69.26),为空白对照组的15.84倍;miR-451组细胞Bcl-2、AKT和磷酸化AKT蛋白表达均显著下调(P<0.05,F=5.83);miR-451组细胞凋亡率为12.07%±1.12%,与3个对照组比较显著升高(P<0.01,F=26.72);miR-451组平均侵袭细胞数为47.4±7.4,与3个对照组比较显著降低(P<0.01,F=34.55).miR-451组细胞的生长在转染后2d出现显著抑制(P<0.05,F=5.95),并且随时间的延长而日益显著.结论:上调miR-451表达可抑制食管癌EC9706细胞增殖和侵袭,促进细胞凋亡.     

Inhibition of adenovirus-mediated p27kip1 gene on growth of esophageal carcinoma cell strain

AIM: To investigate the inhibition of p27kip1 gene on the growth of esophageal carcinoma cell strain (EC9706). METHODS: Recombinant adenovirus Ad-p27kip1 was constructed and transfected into esophageal carcinoma cell EC-9706, and its effect on p27kip1 expression, the growth of esophageal carcinoma cell, DNA replication, protein synthesis, cell multiplication and apoptosis were explored by means of cell growth count, 3H-TdR, 3H-Leucine incorporation, flow cytometry, DNA fragment analysis and TUNEL. RESULTS: Recombinant adenovirus Ad-p27kip1 was successfully constructed with a virus titer of 1.24 X 10(12) pfu/ml. p27kip protein expression increased markedly after EC-9706 transfection, while incorporation quantity of 3H-TdR and 3H-Leucine decreased significantly. The growth of esophageal carcinoma cell was inhibited obviously. Testing of flow cytometry displayed a typical apoptosis peak, and DNA gel electrophoresis showed a typical apoptosis ladder. TUNEL showed the apoptosis rate of Ad-p27kip1 group and control group to be 37.3% and 1.26% (P<0.001) respectively. CONCLUSION: Ad-p27kip1 can inhibit the growth and multiplication of esophageal carcinoma cells and induce apoptosis. Therefore, enhanced p27kip1 expression may be a new way to treat esophageal carcinoma.     

Inhibitory effect of ubiquitin-proteasome pathway on proliferation of esophageal carcinoma cells

AIM: To investigate the inhibitory effect of ubiquitin-proteasome pathway (UPP) on proliferation of esophageal carcinoma cells.METHODS: Esophageal carcinoma cell strain EC9706 was treated with MG-132 to inhibit its UPP specificity. Cell growth suppression was evaluated with 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. DNA synthesis was evaluated by ^3H-thymidine (^3H-TdR) incorporation. Morphologic changes of cells were observed under microscope. Activity of telomerase was examined by telomeric repeat amplification protocol (TRAP) of PCRELISA. Cell cycle and apoptosis were detected by flow cytometry (FCM). DNA fragment analysis was used to confirm the presence of apoptosis. Expression of p27^kip1 was detected by immunocytochemical technique. RESULTS: After exposed to MG-132, the growth and value of ^3H-TdR incorporation of EC9706 cells were obviously inhibited. Cells became round, small and exfoliative under microscope. TRAP PCR-ELISA showed that light absorption of cells gradually decreased after exposed to 5 μmol/L of MG-132 for 24, 48, 72 and 96 h (P&lt;0.01). The percentage of cells at G0/G1 phase was increased and that at S and G2/M was decreased (P&lt;0.01). The rate of apoptotic cells treated with 5 μmol/L of MG-132 for 48 and 96 h was 31.7% and 66.4%, respectively. Agarose electrophoresis showed marked ladders. In addition, the positive signals of p27^kip1 were located in cytoplasm and nuclei in MG-132 group in contrast to cytoplasm staining in control group. CONCLUSION: MG-132 can obviously inhibit proliferation of EC9706 cells and induce apoptosis. The mechanisms include upregulation of p27^kip1 expression, G1 arrest and depression of telomerase activity. The results indicate that inhibiting UPP is a novel strategy for esophageal carcinoma therapy.     

Detachment of esophageal carcinoma cells from extracellular matrix causes relocalization of death receptor 5 and apoptosis

AIM： To investigate the effect of detachment of esophageal cancer cells from extracellular matrix on the localization of death receptor 5 （DR5） and apoptosis. METHODS： Anchorage-dependent EC9706 cells of esophageal squamous cell carcinoma were pretreated or not treated with brefeldin A. Detached cells were harvested by ethylenediaminetetraacetic acid digestion. Expression and localization of DR5 in these cells were determined by immunocytochemical and immunofluorescence assays, as well as flow cytometry analysis. Apoptosis of EC9706 cells was detected by flow cytometry after stained with fluorescein isothiocyanate-labeled annexin V/propidium iodide. Activation of caspase 8 was detected by Western blot analysis. RESULTS： Immunocytochemical assay indicated that DR5 was predominantly perinuclear in adherent cells but was mainly localized in cell membrane in detached cells. In addition, immunofluorescence assay also confirmed the above-mentioned results, and further demonstrated that DR5 was present in the form of coarse granules in detached cells, but in the form of fine granules in adherent cells. Cytometry analysis revealed higher levels of DR5 expression on the surfaces of brefeldin-A-untreated cells than on the surfaces of brefeldin-A-treated cells, but brefeldin A treatment did not affect the total DR5 expression levels. Moreover, nocodazole did not influence the extracelluar DR5 expression levels in EC9706 cells. Apoptosis assay revealed that detached cells were more sensitive to DR5 antibody-induced apoptosis than adherent ceils. Western blotting showed that caspase 8 was activated in temporarily detached cells 4 h earlier than in adherent cells. CONCLUSION： Progress from adhesion to detachment of EC9706 cells causes DR5 relocalization, and promotes cytoplasmic translocation of DR5 to cell surfaces via a Golgi-dependent pathway. Moreover, it might also result in DR5 aggregation to render apoptosis of detached cells.     

己酮可可碱对人食管癌EC9706细胞增殖及NF—κB p65、COX-2、Ki-67蛋白表达的影响

目的探讨己酮可可碱（PTX）对肿瘤细胞的增殖抑制作用及机制。方法体外培养人食管癌EC9706细胞,用不同质量浓度的PTX进行干预,采用MTT法检测细胞增殖抑制率（IR）,免疫组化染色SP法检测核转录因子-κB（NF—κB p65）、环氧化酶-2（COX-2）、Ki-67蛋白表达。结果PTX处理后EC9706细胞IR明显升高,NF-κB P65、COX-2蛋白及Ki-67蛋白表达明显下降（P〈0．05）,且呈时间-浓度依赖性。结论PTX可抑制EC9706细胞增殖,可能机制为下调NF-κB、COX-2及Ki-67蛋白表达;此为PTX治疗食管癌提供了理论依据。     

干扰LINC00707对食管癌细胞生物行为的影响

目的 探讨干扰LINC00707对食管癌细胞生物行为的影响及分子机制.方法 选取51例食管癌患者癌组织及癌旁正常组织,用实时荧光定量-聚合酶链反应(RT-qPCR)检测LINC00707和miR-382-5p的表达水平;将食管癌细胞EC9706随机分为对照(con)组、si-LINC00707组、si-NC组、miR-...     

环氧合酶-2反义RNA在不同时间点对食管癌细胞系EC9706生长的影响

目的:探讨环氧合酶-2(COX-2)反义RNA在不同时间点对人食管癌细胞系EC9706生长的影响。方法:培养293细胞,扩增、纯化COX-2反义RNk的重组腺病毒-Ad-AShcox-2,转染食管癌细胞EC9706,在不同时间点进行活细胞计数及3H-TdR掺入量测定。结果:扩增、纯化获得编码COX-2反义RNA的重组腺(?)毒Ad-AShcox-2,滴度达1.2×1012 PFU/ml;Ad-AShcox-2传染EC9706后在24 h、48 h、72 h、96 h对细胞生长的抑制率分别为8.60％、24.33％、50.21％、75.26％,对3H-TdR掺入量在24 h开始减少,以72～96 h最低,与对照组比较P<0.001。结论:表达COX-2反义RNA重组腺病毒感染人食管癌细胞后,对癌细胞的抑制从24 h开始,48～72 h最明显。