硫酸化茯苓多糖对人乳腺癌细胞株MCF-7凋亡的影响

为了探讨硫酸化茯苓多糖(SP)对人乳腺癌细胞株MCF-7凋亡的影响,采用MTT法检测不同浓度、作用时间SP对乳腺癌MCF-7细胞的抑制作用,倒置显微镜观察MCF-7细胞的形态学变化,RT-PCR检测SP处理MCF-7细胞凋亡相关基因(Bcl-2,Bax)的表达;Western blotting技术检测SP对乳腺癌细胞凋亡蛋白Bcl-2、Bax表达变化。结果表明,SP对MCF-7细胞增殖有抑制作用,且在一定范围内呈剂量效应;细胞贴壁能力减弱,细胞间隙增大,胞膜褶皱;Bcl-2基因表达水平和蛋白表达水平明显降低(p0.05),Bax基因表达水平和蛋白表达水平明显升高(p0.05)。基于以上研究,SP通过促凋亡基因Bax的表达,抑制抗凋亡基因Bcl-2的表达来下调Bcl-2/Bax比值,激活凋亡途径,诱导MCF-7细胞的凋亡。     

Ad-ING4-IL-24 双基因共表达对人肺腺癌化疗的增敏效应

研究ING4 (肿瘤生长抑制因子4) 和IL-24 (人白细胞介素24) 双基因共表达腺病毒载体 (Ad-ING4-IL-24) 对肺腺癌的化疗增敏效应及分子机制。将Ad-ING4-IL-24感染A549肺癌细胞及联合顺铂 (DDP) 化疗药物作用,RT-PCR和Western blotting检测ING4和IL-24基因在A549肺癌细胞中的转录和表达,MTT法、流式细胞仪 (FCM) 和 Hoechst 染色法检测Ad-ING4-IL-24联合DDP (联合组) 对A549肺癌细胞的生长抑制和凋亡效应。采用A549细胞株建立人肺腺癌裸鼠模型,然后瘤体局部注射干预用药,动态测量肿瘤体积,并计算瘤重抑瘤率,免疫组化检测ING4、IL-24、bax、bcl-2、VEGF等基因的表达。结果表明,Ad-ING4-IL-24感染A549肺癌细胞后可获得成功转录和表达,体外联合组能明显抑制A549肺癌细胞生长和诱导细胞凋亡,呈现出典型细胞凋亡形态学变化。体内联合组同样能显著抑制肿瘤生长,瘤重抑瘤率达52.81％,免疫组化结果显示联合组能上调bax基因表达,同时下调bcl-2、VEGF等基因的表达。以上结果表明Ad-ING4-IL-24具有化疗增敏的作用,该作用机制可能与促进肿瘤细胞凋亡和抑制血管形成有关。     

甲基莲心碱抗乳腺癌 MCF-7细胞的研究

目的:观察甲基莲心碱对乳腺癌细胞系MCF-7增殖和凋亡的影响,并探讨其诱导乳腺癌细胞系MCF-7凋亡的可能作用机制。方法:采用体外培养人乳腺癌细胞系MCF-7,CCK-8实验检测不同浓度甲基莲心碱对MCF-7细胞增殖抑制作用;乳酸脱氢酶(LDH)试剂盒(微板法)检测细胞上清液LDH含量;流式细胞术分析甲基莲心碱对MCF-7细胞周期及凋亡的影响;实时定量PCR(RT-PCR)检测线粒体凋亡相关基因Bax和Bcl-2的表达水平。结果:CCK-8、LDH结果显示甲基莲心碱以时间、浓度依耐性的方式抑制乳腺癌MCF-7细胞的增殖及促进细胞毒性的增加;流式细胞术结果表明不同甲基莲心碱作用下MCF-7的平均凋亡率分别为(15.44±0.52)、(18.81±2.24)、(24.26±2.84)、(36.90±3.15)、(59.27±5.86),且使其周期阻滞于G0/G1期;RT-PCR检测结果证明甲基莲心碱可上调乳腺癌细胞中促凋亡基因Bax的表达,而下调抑制凋亡基因Bcl-2。结论:甲基莲心碱以时间和浓度依赖的方式抑制乳腺癌细胞增殖、细胞毒性增加,导致细胞周期于G0/G1阻滞并促进癌细胞凋亡。甲基莲心碱抗乳腺癌的可能作用机制是激活线粒体凋亡途径。     

靶向VEGF小干扰RNA协同5-FU诱导人乳腺癌细胞MCF-7凋亡机制的研究

探讨慢病毒介导的靶向VEGF小干扰RNA联合应用化疗药物5 FU诱导人乳腺癌细胞MCF-7凋亡的机制。以携带VEGF siRNA的慢病毒载体感染MCF-7细胞,应用RT-PCR和Western blot分别检测各组VEGF mRNA、VEGF蛋白及凋亡相关蛋白的表达,流式细胞术检测细胞凋亡。结果表明,慢病毒VEGF siRNA干扰组细胞VEGF mRNA和蛋白表达水平明显低于对照组,凋亡相关蛋白P53及P21表达上调,而SIRT1、Bcl-2及Survivin表达下调。流式细胞术检测显示慢病毒干扰组及5-FU组细胞凋亡率显著升高,联合治疗组的协同作用更为明显。上述结果表明：慢病毒介导的RNA干扰能明显抑制MCF-7细胞VEGF的表达,通过下调SIRTI蛋白的表达,导致P53蛋白表达上调,并调控其下游P21、bcl-2和Survivin的表达,从而诱导MCF-7细胞的凋亡,并且提高了MCF-7对5-FU的敏感性。     

细胞膜脂肪酸组成改变促进乳腺癌细胞凋亡的研究

目的:研究n-6脂肪酸脱氢酶fat-1基因在人乳腺癌细胞内的表达,改变细胞膜脂肪酸组成,对乳腺癌细胞的凋亡作用.方法:构建含有fat-1基因的重组腺病毒载体(Ad.GFP.fat-1),通过包装细胞系(293)产生的腺病毒,感染人乳腺癌细胞MCF-7.提取细胞的总RNA,以fat-1的反义mRNA作探针,用Northern blot检测fat-1基因在MCF-7细胞内的表达.MTT法分析fat-1基因对MCF-7细胞增殖的影响,凋亡染色试剂盒检测细胞的凋亡.气相色谱仪分析对MCF-7细胞的n-6 PUFAs/n-3 PUFAs含量影响.结果:通过基因重组技术,得到预期的重组病毒;fat-1基因在人乳腺癌细胞MCF-7中能有效异源表达,2d后,可检测到fat-1 mRNA的条带.与对照细胞相比,fat-1基因有效地抑制了MCF-7细胞的增殖(23%,p＜0.05),促进了凋亡(增加35%);同时降低了人乳腺癌细胞MCF-7细胞膜n-6 PUFAs/n-3 PUFAs的比率.结论:腺病毒介导的fat-1基因能在人乳腺癌细胞MCF-7内有效异源表达,且抑制了MCF-7细胞的增殖.机理为降低了细胞膜的n-6 PUFAs/n-3 PUFAs的比率.     

ω-6脂肪酸脱氢酶基因在乳腺癌细胞内的表达和作用

为探讨ω- 6脂肪酸脱氢酶基因fat -1在人类乳腺癌细胞MCF- 7中表达和对其生长的作用,将fat -1基因插入到腺病毒载体中,构建腺病毒重组载体(Ad·GFP·fat1) .通过包装细胞系(2 93)产生重组腺病毒,感染MCF 7细胞.用核糖核酸酶保护性分析技术,检测fat -1基因在MCF- 7细胞内的表达,细胞增殖试剂盒(MTT)和凋亡染色试剂盒染色分析fat 1基因对MCF- 7细胞增殖和凋亡的影响,用酶联免疫分析花生酸类(eicosanoids)前列腺素E2 (prostaglandinE2 )的含量.结果显示,腺病毒介导的fat- 1基因能在MCF- 7细胞内有效异源表达,抑制MCF -7细胞的增殖且导致凋亡,前列腺素的含量也明显地减少.结果说明,fat- 1基因在乳腺癌的基因治疗中具有良好利用价值.     

改变细胞膜的脂肪酸组成可促进乳腺癌细胞凋亡

目的: 研究n-6脂肪酸脱氢酶 fat-1基因在人乳腺癌细胞内的表达,改变细胞膜脂肪酸组成,对乳腺癌细胞的凋亡作用。方法: 构建含有fat-1 基因的重组腺病毒载体 (Ad.GFP.fat-1),通过包装细胞系（293）产生的腺病毒,感染人乳腺癌细胞MCF-7。提取细胞的总RNA,以fat-1的反义mRNA 作探针,用Northern Blot检测fat-1 基因在MCF-7细胞内的表达。MTT法分析fat-1 基因对MCF-7细胞增殖的影响,凋亡染色试剂盒检测细胞的凋亡。气相色谱仪分析对MCF-7细胞的n-6 PUFAs/n-3 PUFAs含量影响。结果: 通过基因重组技术,得到预期的重组病毒;fat-1 基因在人乳腺癌细胞MCF-7 中能有效异源表达,2天后,可检测到fat-1 mRNA的条带。与对照细胞相比,fat-1基因有效地抑制了MCF-7细胞的增殖（23%,p<0.05）,促进了凋亡（增加35%）;同时降低了人乳腺癌细胞MCF-7细胞膜n-6 PUFAs/n-3 PUFAs的比率。结论: 腺病毒介导的fat-1 基因能在人乳腺癌细胞MCF-7内有效异源表达,且抑制了MCF-7细胞的增殖。机理为降低了细胞膜的n-6 PUFAs/n-3 PUFAs的比率。     

槟榔碱对人乳腺癌MCF-7细胞增殖和凋亡的影响

目的：观察槟榔碱对人乳腺癌细胞（MCF-7）增殖和凋亡的影响,并探讨其机制。方法：采用四甲基偶氮唑盐（MTT）法检测不同浓度（0、10、30、50、100、300、500μmol/L）槟榔碱对MCF-7细胞增殖的影响,Hoechst 33342染色和流式细胞术检测细胞凋亡,Western blot法检测Bax,Bcl-2和P53蛋白表达。结果：低浓度（0、10、30、50μmol/L）槟榔碱不影响细胞的增殖和凋亡;而高浓度（100、300、500 μmol/L）槟榔碱呈浓度依赖性抑制MCF-7细胞增殖、诱导MCF-7细胞凋亡、提高P53和Bax蛋白表达、降低Bcl-2蛋白表达。结论：高浓度槟榔碱抑制MCF-7细胞增殖、诱导凋亡,其机制可能与提高P53和Bax蛋白表达,降低Bcl-2蛋白表达有关。     

沉默FBI-1基因对三阴性乳腺癌细胞MDA-MB-231增殖和凋亡的影响

本研究旨在观察短发夹状RNA (short hairpin RNA, shRNA)介导的人类免疫缺陷病毒短转录诱导物连接因子1 (FBI-1)基因沉默对三阴性乳腺癌细胞MDA-MB-231增殖和凋亡的影响。用qRT-PCR和Western blot分别检测FBI-1、Bcl-2、Bax、cleaved-Caspase3和Survivin的mRNA和/或蛋白的表达水平;采用shRNA干扰技术沉默MDA-MB-231细胞中FBI-1基因的表达;用CCK-8法以及克隆形成实验检测细胞增殖;用流式细胞术检测细胞凋亡;用裸鼠皮下成瘤实验检测细胞的成瘤能力。结果显示,MDA-MB-231细胞的FBI-1 mRNA和蛋白的表达水平明显高于正常乳腺上皮细胞MCF-10A;经shRNA靶向沉默FBI-1基因表达后,细胞增殖能力明显降低,细胞凋亡率显著增高,伴随Bcl-2和Survivin蛋白表达明显下调、Bax蛋白表达显著上调和Caspase 3活化,MDA-MB-231细胞的裸鼠皮下成瘤能力受抑制。以上结果提示,靶向沉默FBI-1基因表达可以抑制MDA-MB-231细胞增殖,诱导细胞凋亡并抑制细胞的裸鼠皮下成瘤能力。     

左旋咪唑抑制乳腺癌细胞MCF-7生长的作用机制

左旋咪唑可作为乳腺癌治疗的辅助药物,然其抑制乳腺癌细胞MCF-7生长的作用机制仍未清楚。为探究左旋咪唑抑制乳腺癌细胞生长的分子机制,该研究通过CCK-8法检测左旋咪唑对MCF-7细胞活力的影响,细胞划痕检测细胞迁移变化,显微镜下观察细胞形态学变化,吖啶橙/溴化乙啶双荧光染色法(AO-EB)检测细胞凋亡,免疫印迹法(Western blot,WB)检测PI3K/Akt、Bcl-2/Bax、Caspase-9/3相对表达变化。结果显示,与对照组相比,加药组细胞增殖受到显著抑制,其效应与药物浓度和作用时间均呈正相关;与对照组相比,加药组细胞形态发生皱缩,趋于圆形,胞内出现大量空泡;细胞划痕结果显示,加药组细胞迁移能力受到显著抑制;AO-EB结果表明,加药组细胞凋亡小体增加,细胞凋亡率显著上升;免疫印迹法结果表明,与对照组相比,加药组PI3K/Akt、Bcl-2相对表达量显著下降(P0.01),Bax、Caspase-9、Caspase-3蛋白表达显著上升(P0.001)。结果表明,左旋咪唑可通过抑制PI3K/Akt、Bcl-2/Bax信号途径来抑制乳腺癌细胞MCF-7的增殖、迁移,从而促进细胞凋亡,抑制细胞的生长。     

腺病毒介导的人抑瘤素M基因对A375人黑色素瘤细胞的抑制作用

目的:构建抑瘤素M(OSM)重组腺病毒载体,研究其对人黑色素瘤细胞A375的抑制作用。方法:以PEGZ-OSM重组质粒为模板,通过PCR技术扩增出OSM片段,采用腺病毒载体的基因重组和体外包装技术获得表达与人OSM氨基酸序列相同的重组腺病毒子Ad-OSM,感染A375细胞,用荧光显微镜、RT-PCR、Western blot法检测OSM在A375细胞中的转录和表达;荧光显微镜观察A375细胞的形态学改变;MTT法和流式细胞术(FCM)检测Ad-OSM对A375细胞的生长抑制和细胞周期的抑制效应;半定量RT-PCR法检测OSM基因表达对A375细胞中的Bax、Bcl-2基因表达的影响。结果:基因测序和PCR分析结果显示,成功构建了Ad-OSM腺病毒表达载体;RT-PCR和Western blot法检测到OSM基因在A375细胞中的转录和表达;OSM基因的表达对A375细胞增殖有明显抑制作用,并可诱导细胞凋亡,OSM基因可通过上调细胞中Bax和下调Bcl-2基因表达诱导细胞凋亡。结论:成功构建了Ad-OSM腺病毒表达载体,感染OSM基因可明显抑制A375人黑色素瘤细胞的生长,诱导其凋亡,该现象可能是通过改变Bax、Bcl-2基因表达水平来发挥抗肿瘤作用。     

Transient suppression of X-ray-induced apoptosis by exposure to power frequency magnetic fields in MCF-7 cells

Epidemiological studies suggest that exposure to power frequency magnetic fields may be a risk factor for breast cancer in humans. To study the relationship between exposure to 60-Hz magnetic fields (MFs) and breast cancer, cell cycle distribution, apoptosis, and the expression of related proteins (p21, Bax, and Bcl-2) were determined in MCF-7 cells following exposure to magnetic fields (60 Hz, 5 mT) alone or in combination with X rays. It was found that exposure of MCF-7 cells to 60-Hz MFs for 4, 8, and 24 h had no effect on cell cycle distribution. Furthermore, 60-Hz MFs failed to affect cell growth arrest and p21 expression induced by X rays (4 Gy). Similarly, 60-Hz MFs did not induce apoptosis or the expression of Bax and Bcl-2, two proteins related to apoptosis. However, exposure of cells to 60-Hz MFs for 24 h after irradiation by X rays (12 Gy) significantly decreased apoptosis and Bax expression but increased Bcl-2 expression. The effects of exposure to 60-Hz MFs on X-ray-induced apoptosis and Bax and Bcl-2 expressions were not observed at 72 h. These data suggest that exposure to 60-Hz MFs has no effects on the growth of MCF-7 cells, but it might transiently suppress X-ray-induced apoptosis through increasing the Bcl-2/Bax ratio.     

腺病毒介导的人ING4基因诱导C6鼠胶质瘤细胞凋亡

目的：研究肿瘤抑制基因人ING4 (inhibitor of growth family, member 4)对C6鼠胶质瘤细胞的促凋亡作用。方法：将携有绿色荧光蛋白（GFP）腺病毒空载体Ad及重组腺病毒Ad-hING4-His（由本科室构建）分别感染C6细胞,RT-PCR法检测hING4的转录,Western-blotting法检测目的蛋白的表达。并观测hING4基因表达对C6胶质瘤细胞的作用,用MTT法绘制生长曲线,计算抑瘤率。再取重组腺病毒Ad-hING4-His及空腺病毒Ad作用后的C6细胞分别行激光共聚焦显微镜观察凋亡小体、透射电镜观察亚细胞结构的变化,抽提基因组DNA行琼脂糖凝胶电泳及流式细胞仪检测。结果： Ad-hING4-His感染C6细胞后,RT-PCR及Western-blotting结果提示有目的基因的转录和表达。hING4基因表达可以显着抑制C6细胞生长。激光共聚焦观察可见明显核断裂、透射电镜可见实验组细胞呈凋亡表现、基因组DNA电泳呈现梯形条带,流式细胞仪检测有明显AP峰,凋亡率达18.1％。结论：hING4可以通过促进细胞凋亡作用而显着抑制C6细胞的增殖和生长。     

Differential regulation of protein expression, growth and apoptosis by natural and synthetic retinoids

All-trans retinoic acid (ATRA) can down regulate the anti-apoptotic protein Bcl-2 and the cell cycle proteins cyclin D1 and cdk2 in estrogen receptor-positive breast cancer cells. We show here that retinoids can also reduce expression of the inhibitor of apoptosis protein, survivin. Here we have compared the regulation of these proteins in MCF-7 and ZR-75 breast cancer cells by natural and synthetic retinoids selective for the RA receptors (RARs) alpha, beta, and gamma then correlated these with growth inhibition, induction of apoptosis and chemosensitization to Taxol. In both cell lines ATRA and 9-cis RA induced the most profound decreases in cyclin D1 and cdk2 expression and also mediated the largest growth inhibition. The RARalpha agonist, Ro 40-6055 also strongly downregulated these proteins although did not produce an equivalent decrease in S-phase cells. Only ATRA induced RARbeta expression. ATRA, 9-cis RA and 4-HPR initiated the highest level of apoptosis as determined by mitochondrial Bax translocation, while only ATRA and 9-cis RA strongly reduced Bcl-2 and survivin protein expression. Enumeration of dead cells over 96 h correlated well with downregulation of both survivin and Bcl-2. Simultaneous retinoid-mediated reduction of both these proteins also predicted optimal Taxol sensitization. 4-HPR was much weaker than the natural retinoids with respect to Taxol sensitization, consistent with the proposed requirement for reduced Bcl-2 in this synergy. Neither the extent of cell cycle protein regulation nor AP-1 inhibition fully predicted the antiproliferative effect of the synthetic retinoids suggesting that growth inhibition requires regulation of a spectrum of RAR-regulated gene products in addition even to pivotal cell cycle proteins.     

Acacetin-induced apoptosis of human breast cancer MCF-7 cells involves caspase cascade, mitochondria-mediated death signaling and SAPK/JNK1/2-c-Jun activation

The mechanism of acacetin-induced apoptosis of human breast cancer MCF-7 cells was investigated. Acacetin caused 50% growth inhibition (IC50) of MCF-7 cells at 26.4% 0.7% M over 24 h in the MTT assay. Apoptosis was characterized by DNA fragmentation and an increase of sub-G1 cells and involved activation of caspase-7 and PARP (poly-ADP-ribose polymerase). Maximum caspase 7 activity was observed with 100 microM acacetin for 24 h. Caspase 8 and 9 activation cascades mediated the activation of caspase 7. Acacetin caused a reduction of Bcl-2 expression leading to an increase of the Bax:Bcl-2 ratio. It also caused a loss of mitochondrial membrane potential that induced release of cytochrome c and apoptosis inducing factor (AIF) into the cytoplasm, enhancing ROS generation and subsequently resulting in apoptosis. Pretreatment of cells with N-acetylcysteine (NAC) reduced ROS generation and cell growth inhibition, and pretreatment with NAC or a caspase 8 inhibitor (Z-IETD-FMK) inhibited the acacetin-induced loss of mitochondrial membrane potential and release of cytochrome c and AIF. Stress-activated protein kinase/c-Jun NH4-terminal kinase 1/2 (SAPK/ JNK1/2) and c-Jun were activated by acacetin but extracellular-regulated kinase 1/2 (Erk1/2) nor p38 mitogen-activated protein kinase (MAPK) were not. Our results show that acacetin-induced apoptosis of MCF-7 cells is mediated by caspase activation cascades, ROS generation, mitochondria-mediated cell death signaling and the SAPK/JNK1/2-c-Jun signaling pathway, activated by acacetin-induced ROS generation.     

斜生褐孔菌多糖诱导人肝癌HepG-2细胞凋亡的研究

研究了斜生褐孔菌多糖对人类肝癌HepG-2细胞凋亡的诱导作用。采用噻唑蓝法(MTT法)观察了斜生褐孔菌多糖对HepG-2细胞生长的影响,用透射电镜观察了细胞形态,用DNA Ladder检测了细胞凋亡,用流式细胞仪检测了细胞凋亡率;同时采用逆转录-聚合酶链反应法(RT-PCR法)研究了不同浓度斜生褐孔菌多糖作用后HepG-2细胞中Bax和Bcl-2基因mRNA转录水平的变化。结果表明斜生褐孔菌多糖能抑制HepG-2细胞增殖,并呈时间剂量依赖关系;电镜观察、DNALadder和流式细胞仪检测均证实了斜生褐孔菌多糖能够诱导HepG-2细胞凋亡;经斜生褐孔菌多糖处理后,HepG-2细胞中Bax基因mRNA转录水平增强,而Bcl-2基因mRNA无明显变化。证明了斜生褐孔菌多糖具有抑制HepG-2细胞生长及诱导HepG-2细胞凋亡的作用,这可能与调节Bcl-2和Bax基因表达水平有关。     

软骨多糖诱导MCF-7乳腺癌细胞凋亡的实验研究

研究软骨多糖诱导MCF-7乳腺癌细胞凋亡及其作用机理。方法:选用MCF-7人类乳腺癌细胞系体外培养,应用MTT法检测细胞生长抑制率,TUNEL法检测细胞凋亡率,HE染色法观察细胞形态学改变,流式细胞仪检测细胞周期的变化,免疫荧光方法检测BCL-2BAD及波形蛋白Vimentin的表达率。结果:软骨多糖对MCF-7细胞体外生长具有明显的抑制作用,且呈时间和浓度依赖性;软骨多糖可诱导MCF-7细胞发生凋亡并伴随有凋亡小体出现等形态学变化;软骨多糖促进BCL-2蛋白的表达水平下降,BAD表达水平上升,及Vimentin的降解。结论:软骨多糖能够在体外诱导MCF-7细胞凋亡,是一种新型的抗乳腺癌活性物质。     

Bcl-2 controls caspase activation following a p53-dependent cyclin D1-induced death signal

MCF-7 and ZR-75 breast cancer cells infected with an adenovirus constitutively expressing high levels of cyclin D1 demonstrated widespread mitochondrial translocation of Bax and cytochrome c release that was approximately doubled after the addition of all-trans retinoic acid (RA) or Bcl-2 antisense oligonucleotide. By comparison, the percentage of cells in Lac Z virus-infected cultures containing translocated Bax and cytoplasmic cytochrome c was markedly less even after RA treatment. Despite this, RA-treated Lac Z and untreated cyclin D1 virus-infected cultures contained similarly low proportions of cells with active caspase or cells that were permeable to propidium iodide. Bax activation was p53-dependent and accompanied by arrest in G(2) phase. Although constitutive Bcl-2 expression prevented Bax activation, it did not alter cyclin D1-induced cell cycle arrest, illustrating the independence of these events. Both RA and antisense Bcl-2 oligonucleotide decreased Bcl-2 protein levels and markedly increased caspase activity and apoptosis in cyclin D1-infected cells. Thus amplified cyclin D1 expression initiates an apoptotic signal inhibited by different levels of cellular Bcl-2 at two points. Whereas high cellular levels of Bcl-2 prevent mitochondrial Bax translocation, lower levels can prevent apoptosis by inhibition of caspase activation.