卡铂联合TRAIL对肺癌A549细胞增殖和凋亡的影响

目的:观察卡铂联合TNF相关凋亡诱导配体(TRAIL)对人肺腺癌细胞A549增殖和凋亡的影响。方法:经20、40、80 μg/mL卡铂和100 ng/μL TRAIL单用或联用处理后,用MTS法检测A549细胞的增殖能力,在光镜下观察细胞形态学变化;并采用流式细胞术检测细胞凋亡情况;RT-PCR和Western blot法检测死亡受体4(DR4)、死亡受体5(DR5)、Survivin和X连锁凋亡抑制蛋白基因(XIAP)mRNA与蛋白表达的变化。结果:卡铂和TRAIL单用或联用均可浓度依赖性抑制A549细胞的增殖,诱导其凋亡,两药联用比单用卡铂时抑制率和凋亡率更高(P<0.05)。单用卡铂或TRAIL可使A549细胞数减少,漂浮细胞增多,出现明显的凋亡形态变化,且明显降低Survivin和XIAP的mRNA和蛋白表达水平(P均<0.05);但对A549细胞DR4和DR5 mRNA表达均无明显影响,而单用卡铂或TRAIL却能升高A549细胞DR5蛋白的表达(P<0.05)。与单用组相比,TRAIL与卡铂联用A549细胞凋亡形态变化更明显,可明显降低A549细胞Survivin和XIAP mRNA和蛋白的表达水平及升高DR5蛋白表达水平(P<0.05)。结论:卡铂与TRAIL联用可协同抑制肺癌细胞A549细胞增殖,促进其凋亡,且与卡铂能够增加A549细胞DR5蛋白的表达和降低Survivin及XIAP的表达相关。     

JAK2/STAT3信号通路对A549细胞株中HIF-1α、VEGF蛋白表达的影响

目的:研究人肺腺癌细胞A549细胞中JAK2/STAT3信号通路对HIF-1α、VEGF蛋白表达的影响.方法:AG490处理在氧含量正常及缺氧条件下(CoCl2 200μmol/L)48h后Western blot法测定A549细胞中HIF-1α、VEGF蛋白表达变化(分组为对照组、AG490 50μmol/L、AG490 100μmol/L、CoCl2、CoCl2+AG49050μmol/L和CoCl2+AG490 100μmol/L组).IL-6 (3.85nmol/L)处理A549细胞24h后,检测细胞中STAT3、p-STAT3、HIF-1α、VEGF蛋白表达变化(分组为对照组,IL-6组).结果:JAK2特异性抑制剂AG490作用48h后,相对于对照组AG490能够下调HIF-1α、VEGF的蛋白表达,且呈浓度依赖性;随浓度的升高,抑制作用更明显.缺氧条件下(CoCl2200μmol/L)能够上调HIF-1α、VEGF的蛋白表达.AG490也能够下调CoCl2诱导的HIF-1α、VEGF的蛋白表达,且呈浓度依赖性,随浓度的升高,抑制作用更显著.IL-6能够上调HIF-1α、VEGF的蛋白表达.结论:在人肺腺癌细胞A549细胞中,缺氧可上调HIF-1α和VEGF蛋白表达.JAK2/STAT3信号通路对HIF-1α和VEGF蛋白的表达具有调节作用.     

缺氧诱导对人结肠癌SW480细胞增殖和缺氧诱导因子-1α、血红素氧合酶-1基因表达的影响

目的构建缺氧诱导模型和观察缺氧对人结肠癌SW480细胞系生长和缺氧诱导因子-1α(hypoxiainduciblefactor-1alpha,HIF-1α),血红素氧合酶-1(hemeoxygenase-1,HO-1)表达的影响。方法利用氯化钴(cobaltchloride,CoCl2)构建缺氧诱导模型,以MTT试验观察不同程度缺氧条件对SW480细胞生长曲线的影响;利用RT-PCR方法测定氯化钴缺氧诱导与SW480细胞HIF-1α和HO-1基因mRNA表达的时效和量效关系。结果适度缺氧(CoCl2浓度<200μmol/L)可刺激肿瘤细胞增殖,过度缺氧(CoCl2浓度>250μmol/L)则抑制其生长;HIF-1α、HO-1基因mRNA表达与氯化钴缺氧呈明显的量效关系和时效关系;HIF-1α与HO-1基因mRNA的表达在缺氧诱导量效关系(相关系数r=0.786,P<0.05)和时效关系(相关系数r=0.863,P<0.05)中均存在显著的正相关性。结论缺氧可以刺激SW480细胞增殖;HIF-1α和HO-1基因的表达与肿瘤缺氧程度密切相关;HO-1的适量表达对缺氧细胞具有细胞保护作用,其过度表达可能导致细胞损伤和凋亡。     

单唾液酸神经节苷脂3对人肺腺癌细胞增殖凋亡及血管内皮生长因子表达的影响

目的 研究外源性单唾液酸神经节苷脂3(GM3)对人肺腺癌细胞增殖、凋亡和血管内皮生长因子(VEGF)表达的影响,探讨GM3的抗肿瘤作用.方法 以不同浓度外源性GM3干预人肺腺癌A549细胞48 h,采用四甲基偶氮唑蓝(MTT)法检测细胞增殖变化,Annexin V-FITC/PI双染流式细胞学技术检测细胞凋亡变化,逆转录聚合酶链反应(RT-PCR)检测细胞VEGF mRNA的表达水平,激光共聚焦显微镜观察细胞VEGF蛋白的表达变化.结果 2.5、10、40、160和640 μmol/L GM3干预A549细胞48 h,增殖抑制率分别为4.1%、8.9%、29.9%、34.2%和52.6%,GM3作用于A549细胞48 h的半数抑制浓度(IC50)为412 μmol/L.与对照组相比,当GM3＞10 μmol/L时,对细胞增殖抑制作用明显(P＜0.05),且具有浓度依赖性.10、40和160μmol/L GM3干预A549细胞48 h,A549细胞的凋亡率分别为(1.3±0.6)%、(4.8±0.4)%、(14.2±1.0)%.与对照组相比,当GM3＞40 μmol/L时,其促进细胞凋亡作用明显(P＜0.05).与对照组相比,经浓度＞40 μmol/L的GM3干预后,A549细胞的VEGF mRNA表达水平显著下降(P＜0.05).随着GM3浓度增高,A549细胞的VEGF荧光强度明显减弱.结论 GM3能呈浓度依赖性抑制人肺腺癌细胞株A549增殖,促进A549细胞凋亡,下调A549细胞VEGF mRNA和蛋白水平的表达,提示GM3可能通过调节肿瘤细胞凋亡和肿瘤血管生成而发挥双重抗肿瘤作用.

Abstract：

Objective To determine the effect of exogenous GM3 on proliferation, apoptosis and VEGF expression in human lung adenocarcinoma cell line A549 cells.Methods A549 cells were treated with GM3 at different concentrations for 48 hours.MTT assay was used to detect the cell proliferation and flow cytometry was applied to analyze cell apoptosis.RT-PCR was used to detect the expression level of VEGF mRNA and confocal laser scanning microscopy was applied to observe the localization and fluorescence intensity of VEGF.Results Comparing with the control, being treated with higher than 10μmol/L GM3 significantly inhibited A549 cell proliferation ( P ＜ 0.05 ), and the suppressive effect could be enhanced following increasing doses.The IC50 was 412 μ mol/L.Comparing with the control, being treated with higher than 40 μmol/L GM3 significantly promoted the apoptotic rate of A549 cells ( P ＜ 0.05 ).Comparing with the control, being treated with higher than 40 μ mol/L GM3 significantly decreased the VEGF mRNA level of A549 cells (P＜0.05), and the fluorescence intensity of VEGF distinctly weakened.Conclusions Exogenous ganglioside GM3 can inhibit the proliferation, promote apoptosis, and down-regulate the VEGF expression level in A549 cells.This may be considered as two mechanisms of GM3 for its anti-tumor effect by modulating cell apoptosis and angiogenesis.     

JAK2/STAT3信号通路对A549细胞株中HIF-1ɑ、VEGF蛋白表达的影响

目的：研究人肺腺癌细胞A549细胞中JAK2/STAT3信号通路对HIF-1ɑ、VEGF蛋白表达的影响。方法：AG490处理在氧含量正常及缺氧条件下（CoCl2200μmol/L）48h后Westernblot法测定A549细胞中HIF-1ɑ、VEGF蛋白表达变化（分组为对照组、AG49050μmol/L、AG490100μmol/L、CoCl2、CoCl2＋AG49050μmol/L和CoCl2＋AG490100μmol/L组）。IL-6（3.85nmol/L）处理A549细胞24h后,检测细胞中STAT3、p-STAT3、HIF-1ɑ、VEGF蛋白表达变化（分组为对照组,IL-6组）。结果：JAK2特异性抑制剂AG490作用48h后,相对于对照组AG490能够下调HIF-1ɑ、VEGF的蛋白表达,且呈浓度依赖性;随浓度的升高,抑制作用更明显。缺氧条件下（CoCl2200μmol/L）能够上调HIF-1ɑ、VEGF的蛋白表达。AG490也能够下调CoCl2诱导的HIF-1ɑ、VEGF的蛋白表达,且呈浓度依赖性,随浓度的升高,抑制作用更显著。IL-6能够上调HIF-1ɑ、VEGF的蛋白表达。结论：在人肺腺癌细胞A549细胞中,缺氧可上调HIF-1α和VEGF蛋白表达。JAK2/STAT3信号通路对HIF-1α和VEGF蛋白的表达具有调节作用。     

三氧化二砷诱导A549细胞凋亡及对survivin、cyclinD1表达的影响

目的:了解三氧化二砷对肺腺癌A549细胞株细胞增殖、细胞凋亡以及survivin、cylin D1基因表达的影响. 方法:分别以不同浓度的三氧化二砷作用于A549细胞,作用不同时间对细胞增殖活性、细胞凋亡及细胞survivin、cylin D1基因表达的变化进行检测. 结果:稍高浓度(＞2μmol/L)的三氧化二砷可抑制A549细胞增殖,并有剂量和时间依赖性.逆转录聚合酶链式反应(RT-PCR)检测survivin、cylin D1基因转录水平和细胞爬片免疫组化检测survivin、cyclin D1基因蛋白表达的相对强度,发现三氧化二砷作用组survivin 和cyclin D1基因的表达与对照组相比明显减低(P＜0.01). 结论:三氧化二砷可有效抑制A549细胞增殖,诱导A549细胞凋亡,与作用剂量和作用时间呈正相关,这一过程可能与三氧化二砷抑制survivin、cyclin D1表达有关.     

尼美舒利对胆管癌细胞增殖及PCNA、Survivin蛋白表达的影响

目的:观察选择性环氧合酶-2(cyclooxygenase-2,COX-2)抑制剂尼美舒利对人胆管癌细胞系QBC939生长的抑制作用及Survivin基因表达的变化.方法:应用MTT比色法、细胞群体倍增时间观察尼美舒利对人胆管癌细胞QBC939增殖的影响,流式细胞仪检测细胞凋亡,免疫组织化学法观察尼美舒利对QBC939细胞PCNA,Survivin蛋白表达的影响.结果:尼美舒利呈时间、剂量依赖性抑制胆管癌增殖,高浓度(200μmol/L)尼美舒利不仅抑制胆管癌细胞增殖,而且诱导其凋亡.流式细胞仪研究显示,随药物浓度增加,细胞凋亡率显著增加.免疫组化结果显示尼美舒利处理后的QBC939细胞PCNA,Survivin蛋白的表达明显减弱.结论:尼美舒利能抑制QBC939细胞增殖,诱导其凋亡,其机制可能与PCNA,Survivin蛋白的表达下调有关.     

吗啡对人肺腺癌A549细胞增殖的作用研究

目的 观察不同浓度吗啡对A549细胞增殖及凋亡的影响。方法 采用不同浓度的吗啡（0.3、3、30 μg/ml）处理A549细胞48 h后采用CCK-8法检测其增殖情况,Annexin-V FITC/PI双染法检测30 μg/ml吗啡作用48 h后的细胞凋亡率,Western blotting检测30 μg/ml吗啡作用48 h后的X连锁凋亡抑制蛋白（XIAP）、Survivin、Bcl-2、caspase-3和Bax蛋白表达水平。 结果 吗啡可以剂量依赖性促进A549细胞增殖,各浓度间的差异均有统计学意义（P＜0.05）。与对照组相比,30 μg/ml吗啡降低了A549细胞的凋亡率（P＜0.05）,且Survivin和XIAP蛋白水平升高,而caspase-3蛋白水平降低（P＜0.05）。 结论 吗啡可促进人肺腺癌A549细胞增殖并抑制其凋亡,可能与吗啡上调XIAP、Survivin蛋白的表达和抑制caspase-3蛋白活化有关。     

Survivin反义寡核苷酸抑制EC9706细胞增殖并诱导细胞凋亡

目的 观察Sunrivin反义寡核苷酸(ASODN)对人食管癌细胞系EC9706细胞增殖和凋亡的影响.方法 人工合成Survivin基因反义和正义ODN,并进行硫代磷酸化修饰,通过脂质体途径分别转染 EC9706 细胞;应用 RT-PCR 和 Western Blot 检测 SurvivinmRNA和蛋白表达;应用MTT法检测 Survivin ASODN 对 EC9706 细胞增殖的影响;流式细胞仪检测细胞周期变化及细胞凋亡比率.结果 体外培养的 EC9706 细胞可表达较强的 Survivin mRNA 和蛋白;Survivin ASODN 可呈浓度依赖性地抑制 Survivin mRNA 和蛋白表达,50μmoL/L ASODN 几乎可以完全抑制.MTT 研究结果表明,Survivin ASODN 可呈浓度依赖性地抑制 EC9706 细胞增殖,50μmol/L ASODN 对细胞生长的抑制率可达78.5%,诱导细胞凋亡,使细胞阻滞于G2/M 期.Survivin SODN 对 Survivin mRNA 和蛋白以及 EC9706 细胞的增殖和细胞周期无明显的抑制作用.结论 脂质体介导转染 Survivin 反义寡核苷酸可抑制细胞增殖、诱导细胞G2/M 期阻滞而促进细胞凋亡.     

Vitexicarpin诱导肺癌A549细胞凋亡和对细胞周期作用的研究

目的:研究蔓荆子黄素(Vitexicarpin)对人肺癌细胞株A549细胞增殖和凋亡的影响及其机制.方法:设计长春新碱(Vineristine)阳性对照组和空白阴性对照组及不同浓度的Vitexiearpin溶液作用于A549细胞,应用细胞增殖抑制试验和流式细胞仪检测技术,观察Vitexicarpin对A549细胞增殖的影响,以及其诱导A549细胞在不同时段的凋亡情况和对细胞周期的影响.结果:Vitexicarpin浓度为10μmol/L时对A549细胞的增殖抑制率超过50%,IC50为8.21μmol/L.另外,Vitexicarpin能有效诱导A549细胞凋亡.从Vitexicarpin作用4h开始,细胞被阻滞于G2/M期.随时间增加,G1/S期细胞逐渐减少,G2/M期细胞逐渐增多,且凋亡细胞逐渐增多.结论:Vitexicarpin能抑制A549细胞增殖,诱导A549细胞发生凋亡,将细胞阻滞于G2/M期是其可能作用机制.     

抑制Survivin基因对人卵巢癌SKOV3细胞增殖和凋亡的影响

目的:探讨应用RNAi技术沉默Survivin基因对人卵巢癌SKOV3细胞的影响。方法:构建Survivin基因shRNA真核表达载体,转染人卵巢癌SKOV3细胞。RT-PCR及Western blot检测Survivin基因的表达,MTT实验、流式细胞仪检测细胞增殖、凋亡的变化。结果:siRNA实验组细胞Survivin蛋白及mRNA表达水平明显下降,细胞增殖能力显著降低,细胞凋亡率显著升高。结论:应用RNAi技术沉默Survivin基因可以降低卵巢癌SKOV3细胞Survivin基因的表达,进而抑制肿瘤细胞的生长、增殖并诱导细胞凋亡。因此,Survivin基因可能成为抗肿瘤治疗的潜在靶点。     

Hypoxia down-regulates DNA double strand break repair gene expression in prostate cancer cells.

BACKGROUND AND PURPOSE: Intratumoral hypoxia has been correlated with poor clinical outcome in prostate cancer. Prostate cancer cells can be genetically unstable and have altered DNA repair. We, therefore, hypothesized that the expression of DNA double-strand break (DNA-dsb) repair genes in normal and malignant prostate cultures can be altered under hypoxic conditions. METHODS AND MATERIALS: The expression of homologous recombination (HR) and non-homologous recombination (NHEJ) genes following gas hypoxia (0.2%) or exposure to HIF1alpha-inducing agent, CoCl2 (100 microM), was determined for normal diploid fibroblasts (GM05757) and the pre-malignant and malignant prostate cell lines, BPH-1, 22RV-1, DU145 and PC3. RNA and protein levels were determined using RT-PCR and Western blotting. Additionally, p53 genotype and function, the level of hypoxia-induced apoptosis, and cell cycle distribution, were determined to correlate to changes in DNA-dsb gene expression. RESULTS: Induction of hypoxia was confirmed using HIF1alpha and VEGF expression in gas- and CoCl2-treated cultures. Hypoxia (48-72 h of 0.2% O2) decreased RNA expression of a number of HR-related genes (e.g. Rad51, Rad52, Rad54, BRCA1, BRCA2) in both normal and malignant cultures. Similar decreases in RNA pertaining to the NHEJ-related genes (e.g. Ku70, DNA-PKcs, DNA Ligase IV, Xrcc4) were observed. In selected cases, hypoxia-mediated decreases in RNA expression led to decreased DNA-dsb protein expression. CoCl2-treated cultures did not show decreased DNA-dsb protein expression. The ability of hypoxia to down-regulate Rad51 and other HR-associated genes under hypoxia was not correlated to c-Abl or c-Myc gene expression, p53 genotype or function, propensity for hypoxia-mediated apoptosis, or specific changes in cell cycle distribution. CONCLUSIONS: Hypoxia can down-regulate expression of DNA-dsb repair genes in both normal and cancer cells. If associated with a functional decrease in DNA-dsb repair, this observation could provide a potential basis for the observed genetic instability within tumor cells exposed to hypoxia.     

氯化钴诱导化学性低氧对经HIF-1α基因转染后的大鼠骨髓间充质干细胞生物学特性的影响

目的:探讨氯化钴（CoCl2）诱导的化学性低氧对经低氧诱导因子-1α（hypoxia-inducible factor-1α,HIF-1α） 基因转染后的大鼠骨髓间充质干细胞（bone marrow mesenchymal stem cells,BMSCs）生物学特性的影响。方法:第3代BMSCs以及转染有HIF-1α全长基因的第 3 代BMSCs,根据低氧诱导方式不同分为4组。BMSCs组、BMSCs-HIF-1α组:BMSCs或经HIF-1α基因转染后的BMSCs在H-35低氧工作站诱导的物理性低氧环境下培养;BMSCs-CoCl2组、BMSCs-HIF-1α-CoCl2组:BMSCs或经HIF-1α基因转染后的BMSCs在CoCl2诱导的化学性低氧环境下培养。低氧培养8天后,Western blot 测定各组内HIF-1α蛋白表达;流式细胞仪检测细胞周期内G1/G2/S期细胞比例,统计增殖指数（PI）和凋亡率。MTT法绘制细胞生长曲线,比较细胞数目。结果:与BMSCs-HIF-1α组和BMSCs-CoCl2组比较,BMSCs-HIF-1α-CoCl2组内HIF-1α蛋白表达上调（P<0.05）,细胞周期内处于G2期和S期的细胞数目增多（P<0.05）,PI升高（P<0.05）,细胞凋亡率下降（P<0.05）。BMSCs-HIF-1α-CoCl2组细胞在培养的3~8天内,细胞数目均多于其他三组细胞（P<0.05）。结论:CoCl2诱导的化学性低氧上调了经基因转染后BMSCs内HIF-1α的蛋白表达,提高了细胞增殖能力,降低了细胞凋亡率。     

三氧化二砷诱导Raji细胞凋亡与Survivin基因关系的研究

Objective:To investigate the apoptosis induction by arsenic trioxide (As2O3) in Raji cells and its correlation with cell cycle arrest and expression of the Survivin gene.Methods:After Raji cells were treated with As2O3 in different concentrations (1,2,4 and 8 μM),for 24,48 and 72 h,respectively,and cell proliferation was tested by MTT assay.Apoptosis was observed with electron microscope end DNA electrophoresis.The distribution of cell cycles and cell apoptosis were detected by flow cytometry.Expression of the Survivin gene was determined by real-time quantitative RT-PCR.Results:As2O3 (1-8 μM) inhibited Raji cells growth effectively in a dose- and time-dependent manner.As2O3 at 2-8μM could induce cell apoptosis and cell cycle arrest.However,As2O3 (1 μM) inhibited Raji proliferation only by cell cycle arrest,without any symptoms of cell apoptosis.At the same time,Survivin gene expression was down-regulated after the treatment.Conclusion:As2O3 could induce substantial proliferation inhibition,cell cycle arrest and apoptosis in Raji cell.Cell cycle arrest might be a reason why apoptosis occurs.As2O3 can markedly down-regulate expression of the Survivin gene in a dose- and timedependent manner.The down-regulated Survivin gene might be leading to cell apoptosis by As2O3.     

Effect of Mimic Hypoxia on the Proliferation and Expression of miR-27a,miR-9, miR-370 and their Target Genes in MOLT-4 and KG1a Cell Lines

Objective: The aim of this study was to investigate the effect of mimic hypoxia on proliferation, the expression of significant miRNAs, and genes involved in drug resistance in MOLT-4 and KG1 cell lines. Materials and Methods: The KG1 and MOLT-4 cell lines were cultured in RPMI 1640 medium supplemented with 20% FBS and 10% FBS respectively. The MTT test was used for determining  the optimum dose of CoCl2 for KG1 and MOLT-4 cell lines. Western blotting was used for the detection of HIF-1a protein and the confirmation of mimic hypoxia induced by CoCl2. For evaluating the effect of mimic hypoxia on proliferation of MOLT-4 and KG1 cell lines, cell counting was done using trypan blue at 24, 48, and 72 hours. Furthermore, the results obtained from cell counting were confirmed with the MTT test. Total RNA was extracted  using the RNX Plus solution kit according to the manufacturer’s protocol. The expression of genes and miRNAs was evaluated with real time PCR. Results: According to this study, mimic hypoxia induced by CoCl2 contributes to the overexpression of drug resistance related genes including MDR1, MRP1, FOXM1, BCL-xl genes, and the suppression of PUMA gene compared to the control group. The results also showed that mimic hypoxia condition leads to the up-regulation of miR-9 and down-regulation of miR-27a and miR-370. Additionally, our outcomes demonstrated that mimic hypoxia has an inhibitory effect on the proliferation of MOLT-4 and KG1 cell lines. Conclusion: Treatment with CoCl2 has an inhibitory effect on the proliferation of MOLT-4 and KG1 cell lines independent from real hypoxia. Additionally, mimic hypoxia has a substantial effect on the expression of  genes and miRNAs involved in drug resistance. Finally, we are still far away to discover the exact functional mechanisms of hypoxia on drug resistance but these evaluations can provide new perspectives into this field for the upcoming studies.     

CoCl2诱导缺氧对Eca109细胞细胞周期及放射敏感性的影响

Objective:To investigate the change of the cell cycle,apoptosis and radiosensitivity effect by CoCl2 induced hypoxia in esophageal cancer line Eca109 cells in vitro.Methods:The hypoxia culture model induced by 150 microM CoCl2 was established.The cell cycle and apoptosis were measured with flow cytometry (FCM).The radiosensitivity was analysized with clonogenic assay after irradiation alone or combined with hypoxia in Eca109 cells in vitro.Results:Eca109 cells were treated with 150 microM CoCl2 for 24 h,cell cycle arrest in G0/G1 phase increase and decreasing arrest in S phase with longer of hypoxiac time (0-24 h),the other rate of cell cycle and apoptosis did not change obviously.The G2/M phase block was arrested obviously in radiation alone comparing with the hypoxia plus irradiated group,apoptosis did not occur in Eca109 cell line following irradiation.The DO value and cell surviving fraction of Eca109 cell was 2.48 Gy,2.44 Gy and 97.33%,96.33% in hypoxia and control group,respectively;the Dq value of Eca109 cell was 2.89 Gy,0.52 Gy,the cell surviving fraction after radiation with 4 Gy was 48.3%,21.7% in hypoxia and control group,respectively.The hypoxia decreased the radiosensitivity in esophageal cancer Eca109 cells with clonogenic assay.Conclusion:Hypoxia induced by CoCl2 influences radiosensitivity of Eca109 cell through regulating cellular proliferation rates.     

二氯化钴对腺病毒基因表达的影响

目的:研究二氯化钴(CoCl2)对缺氧反应元件调控E1AE1B表达的条件复制型腺病毒载体(Ad-5HRE-E1AE1B-RFP)和缺乏缺氧反应元件的复制缺陷型腺病毒载体(Ad-EGFP)在肿瘤细胞内表达和复制的影响. 方法:肿瘤细胞经不同浓度的CoCl2 处理后,Western 印迹法检测缺氧诱导因子(hypoxia inducible factor-1α,HIF-1α)的表达;倒置荧光显微镜、FCM法及空斑形成实验等观察经CoCl2处理后,感染条件复制型腺病毒和(或)复制缺陷型腺病毒的肿瘤细胞中外源基因的表达水平和病毒复制情况;小动物成像仪观察缺氧调控的条件复制型腺病毒在肿瘤内提高复制缺陷型腺病毒表达的效率. 结果:适当浓度的CoCl2 (0.4和0.08 μg/mL) 能使胃癌细胞株(SGC7901)中HIF-1α蛋白稳定表达并积聚,可较好地模拟缺氧状态;在0.4 μg/mL CoCl2作用下,Ad-5HRE-E1AE1B-RFP对肿瘤细胞的感染效率明显提高,表现为外源基因RFP阳性表达的百分率和荧光强度均明显提高,但空斑形成实验显示Ad-5HRE-E1AE1B-RFP并没有复制.0.4 μg/mL CoCl2也能提高其他非缺氧调控的复制缺陷型腺病毒如Ad-EGFP、Ad-Luc的感染效率和表达水平; Ad-5HRE-E1AE1B-RFP与复制缺陷型腺病毒Ad-Luc联合注射裸鼠肿瘤能显著提高Ad-Luc的表达.结论:CoCl2 能明显提高腺病毒的基因表达水平,其作用机制不仅与CoCl2诱导缺氧有关,也可能与影响基因转录有关.     

Effect of Celecoxib on Apoptosis of Endometrial Carcinoma Cell

Objective:To investigate the effect of Celecoxib on proliferation and apoptosis of the endometrial carcinoma cell HEC-1B and the effect on the expression of Fas and Survivin mRNA.Methods:The inhibition on the growth of human endometrial carcinoma cell HEC-1B was investigated by cell culture and MTT experiment when treated with different concentrations of Celecoxib.The cell apoptosis was detected by flow cytometry and DNA Ladder Electrophoresis.The change of the expression of Fas and Survivin mRNA after the treatment of Celecoxib was detected With RT-PCR.Results:Celecoxib could effectively inhibit the growth of HEC-1B cells and induce apoptosis.Survivin mRNA expression was decreased and Fas mRNA expression was increased after treating with Celecoxib.Conclusion:Celecoxib could inhibit HEC-1B cell proliferation and induce its apoptosis.     

缺氧对人乳腺癌MCF-7细胞miRNA-21表达的影响及与细胞增殖和凋亡的关系

[目的]观察缺氧环境下人乳腺癌MCF-7细胞增殖及凋亡的情况,初步探讨缺氧对miRNA-21表达的影响及其与细胞增殖、凋亡的关系。[方法]CoCl2人工模拟缺氧环境,四甲基偶氮唑蓝比色法（MTT）检测缺氧环境下细胞增殖状况,流式细胞仪检测细胞的凋亡率．实时荧光聚合酶链反应（RT—PCR）测定不同缺氧程度下HIF-1α 和miRNA-21表达水平的变化。[结果]与常氧对照组相比较,CoCl2处理组的细胞明显受到抑制,但100、200μmol／LCoCl2处理组的细胞在一定时间内仍增殖,抑制率随缺氧时间和药物浓度增加而上升。流式细胞仪检测结果显示,CoCl2处理组细胞凋亡率高于对照组（P〈0．05）。RT-PCR检测显不,200、400μmol／L的CoCl2处理组培养24h后细胞的HIF-1α／和miRNA-21表达高于对照组（P〈0．05）。[结论]人乳腺癌MCF-7细胞可在一定缺氧程度和时间内生长,缺氧诱导细胞凋亡率增加,缺氧能上调HIF-1α、miRNA-21的表达,使之进一步耐受缺氧,其机制可能是上调的miRNA-21通过多个信号通路调控靶基因进而影响癌细胞的增殖及凋亡。