华蟾素诱导L1210细胞凋亡及作用机制的研究

目的：探讨华蟾素对离体小鼠急性淋巴细胞白血病 L1210 细胞的作用.方法：将 L1210 细胞分为对照组及药物处理组.MTT 实验检测细胞活性及增殖抑制情况 ;DNA 亲和染料 Hoechst 33258 荧光染色观察细胞核变化 ;Western Blot 检测细胞凋亡通路相关蛋白表达水平的改变.结果：华蟾素在 0.25～1.0 μM 浓度范围内即表现出显著的细胞生长增殖抑制,而且有时间和浓度依赖性 ;Hoechst 33258 染色发现高浓度的华蟾素作用 48 h,L1210 细胞即表现出凋亡形态改变 ;Western Blot 实验表明,细胞凋亡通路相关蛋白 Caspase-3,Caspase-8,Caspase-9表达明显高于对照组.结论：华蟾素有诱导 L1210 细胞凋亡的作用.     

枸杞多糖对人白血病细胞株凋亡的影响

目的 :　探讨枸杞多糖 ( LBP- X)对人白血病 HL- 60细胞凋亡的影响。方法 :　 LBP-X与 HL- 60细胞共培养 ,MTT法检测 LBP- X对 HL- 60细胞增殖的抑制作用 ;以 DPH为荧光探针检测细胞膜流动性的变化 ;用 Hoechst332 5 8染色在荧光显微镜下观察细胞核形态学变化 ;琼脂糖凝胶电泳法和流式细胞仪检测法定性定量检测细胞凋亡。结果 :　LBP- X2 0～ 1 0 0 0 mg/L呈剂量依赖性抑制 HL- 60细胞增殖 ,且可降低 HL- 60细胞膜的流动性 ;LBP- X作用 48h后 ,荧光显微镜下细胞核固缩、凝聚和断裂 ,出现浓染致密的颗粒块状荧光 ;DNA琼脂糖凝胶电泳可见明显的“DNA条带”;流式细胞仪分析图上出现凋亡峰。结论 :　 LBP- X对诱导人白血病 HL- 60细胞凋亡有一定作用。     

家蝇3龄幼虫抗肿瘤肽促K562细胞凋亡作用及其机制研究

目的观察家蝇3龄幼虫抗肿瘤肽对K562细胞核和线粒体膜电位及K562细胞凋亡蛋白半胱天冬氨酸蛋白酶-3(caspase-3)的影响,进而探讨家蝇3龄幼虫抗肿瘤肽是否可以影响线粒体膜电位及促进K562细胞凋亡。方法首先采用Hoechst33258荧光标记法,将家蝇3龄幼虫峰5、峰8组分作用于K562细胞,用荧光显微镜检测K562细胞核荧光强度的变化;其次采用荧光探针罗丹明123标记K562细胞,在激光扫描共聚焦显微镜下观察K562细胞中罗丹明123荧光强度,以细胞内荧光强度表示线粒体膜电位大小;最后用caspase-3检测试剂盒和酶标仪测定K562细胞caspase-3的含量。结果采用Hoechst33258荧光标记法,用荧光显微镜观察到家蝇3龄幼虫峰5、峰8组分作用于K562细胞后部分细胞核的荧光强度增强,说明其可以引起K562细胞发生凋亡;用罗丹明123标记,激光共聚焦显微镜观察发现,家蝇3龄幼虫峰5、峰8组分作用组的K562细胞荧光强度值明显低于对照组(t1=21.30,t2=196.23,P<0.05),说明家蝇3龄幼虫抗肿瘤肽可以使K562细胞线粒体膜电位降低;峰5、峰8组分作用组caspase-3的含量明显高于对照组(t1=146.92,t2=189.56,P<0.05),说明3龄幼虫抗肿瘤肽可以促进K562细胞发生凋亡。结论家蝇3龄幼虫抗肿瘤肽峰5、峰8组分对K562细胞核具有损伤作用,可以引起K562细胞发生凋亡,作用机制是通过降低K562细胞线粒体膜电位,激活caspase-3,扰乱其生理功能,促使其凋亡。     

二十碳五烯酸（EPA）对胃癌细胞增殖与凋亡的影响

目的观察二十碳五烯酸（EPA）对人胃癌细胞系增殖与凋亡的影响,并探讨其作用机制。方法以终浓度为10、20、40μg／ml的EPA作用于SGC-7901和MGC-803人胃癌细胞系24-72h。采用四甲基偶氮唑蓝法检测细胞增殖抑制率,采用流式细胞技术分析细胞周期分布与细胞凋亡,利用荧光探针rhodamine 123测定线粒体膜电位,酶联免疫吸附法测定线粒体和胞浆中细胞色素C水平,荧光光谱法测定凋亡效应酶半胱天冬蛋白酶-3（caspase-3）活性。结果经10-40μg／ml EPA处理的胃癌细胞增殖率显著降低,且呈现时间依赖性。与对照组比较,经40g／ml EPA作用72h后,SGC-7901和MGC-803细胞G0／G1期细胞的比例均增加（P=0.006、P=0.009）。经40g／ml EPA作用24h后,胃癌细胞线粒体膜电位显著低于对照组（P=0.001、P=0.047）;线粒体内细胞色素C的含量明显少于对照组（P=0.001、P=0.000）,而细胞浆中细胞色素C含量显著高于对照组（P=0．001、P=0．000）。在SGC-7901细胞中,caspase-3活性随着EPA（40g／mL）作用时间延长而升高。结论EPA通过诱导细胞周期阻滞和激活线粒体通路,抑制人胃癌细胞增殖,诱导细胞凋亡。     

PBDE-47对人神经母细胞瘤细胞凋亡和线粒体膜电位及细胞色素C蛋白表达的影响

目的探讨PBDE-47对人神经母细胞瘤(SH-SY5Y)细胞线粒体膜电位和细胞色素C(Cyt C)蛋白表达的影响及其与细胞凋亡的关系。方法将处于对数生长期的SH-SY5Y细胞分别加入终浓度为0(溶剂对照组)、1、5、10μmol/L的PBDE-47暴露24 h。采用流式细胞术检测细胞凋亡率和线粒体膜电位变化,采用Western blot技术检测胞浆与线粒体细胞色素C蛋白表达的水平。结果与溶剂对照组相比,10μmol/L PBDE-47染毒组SH-SY5Y细胞凋亡率和5、10μmol/L PBDE-47染毒组胞浆内细胞色素C蛋白表达水平均明显升高,10μmol/L PBDE-47染毒组线粒体膜电位和各浓度PBDE-47染毒组线粒体内细胞色素C蛋白表达水平均明显下降,差异均有统计学意义(P<0.05)。且随着PBDE-47染毒浓度的升高,SH-SY5Y细胞凋亡率和胞浆内细胞色素C蛋白表达水平呈升高趋势,线粒体内细胞色素C蛋白表达水平呈下降趋势。结论 PBDE-47可通过引起线粒体膜通透性转运孔开放和细胞色素C的释放介导SH-SY5Y细胞凋亡,从而对SH-SY5Y细胞产生损伤。     

B型流感病毒细胞凋亡作用的体外研究

[目的]研究B型流感病毒导致的体外培养狗肾传代细胞凋亡作用,初步探讨流感病毒细胞毒性发生机制。[方法]采用Hoechst 33258荧光染色观察形态学变化,DNA片段的琼脂糖凝胶电泳检测细胞凋亡,流式细胞术ANNEXINV-FITC加碘化丙啶(PI)双染定量检测细胞凋亡率和坏死率。[结果]加入不同滴度的B型流感病毒培养6小时后开始出现细胞核荧光强度增强,染色质沿核周分布现象。DNA电泳呈典型的"梯形"条带。流式细胞术检测发现,不同滴度流感病毒作用后,细胞凋亡率明显高于空白对照组,差异有显著性(P﹤0.05)。流感病毒滴度越高,凋亡率也越高,但当流感病毒滴度为1︰128时,细胞坏死率增加。滴度为1︰64的流感病毒诱导的细胞凋亡,随着作用时间的延长,凋亡率明显增高,作用18h达高峰,而后细胞凋亡率下降。[结论]体外培养条件下B型流感病毒能诱导MDCK细胞凋亡,并存在量-效与时-效关系。     

2,5-己二酮诱导的PC12细胞损伤和凋亡

目的建立2,5己二酮诱导的PC12细胞凋亡模型。方法以PC12细胞为神经元的细胞模型,将2,5己二酮(30、40、50mmol/L)加入培养的PC12细胞中,四甲基偶氮唑盐检测细胞活性,Hochest33258观察细胞核变化,琼脂糖凝胶电泳检测DNA断裂、流式细胞仪检测细胞凋亡率,比较各组间的差异。结果随2,5己二酮浓度增加,细胞存活率下降(P<0.01);荧光显微镜观察有特征性的凋亡细胞核;琼脂糖凝胶电泳有明显的DNAladder;流式细胞仪检测凋亡率随2,5己二酮浓度的增大而增加(P<0.05)。结论一定浓度的2,5己二酮有导致PC12细胞损伤和凋亡的作用。     

苦参碱诱导NB4细胞凋亡作用及机制

目的探讨苦参碱(Mat)诱导急性早幼粒白血病细胞株NB4细胞凋亡作用及机制。方法不同剂量M at(0.25、0.50、1.00 mg/m L)作用于NB4细胞(24、48、72 h),噻唑蓝法(M TT)检测细胞增殖抑制率;Hoechst33258染色观测细胞形态学变化;流式细胞术(FCM)检测细胞周期,Annexin V-FITC/PI双标记法测定细胞凋亡率;分光光度法检测NB4细胞内caspase-3和caspase-8活性。结果与对照组比较,Mat可明显抑制NB4细胞增殖,诱导细胞凋亡,呈时间和剂量效应(P0.05);细胞形态学观察可见明显的细胞凋亡特征;与对照组比较,0.50、1.00 mg/m L M at组G0/G1期细胞比例(分别为57.91%、83.00%)明显增加,S期比例(分别为40.95%、16.10%)减少;与对照组比较,0.50、1.00 mg/m L Mat组NB4细胞内caspase-3酶活性[分别为(77.41±4.01)、(111.78±4.05)]、1.00 mg/m L M at组NB4细胞内caspase-8酶活性(44.98±7.63)明显增强(P0.05)。结论 M at可抑制NB4细胞增殖并诱导细胞凋亡,其机制可能与G_0/G_1期细胞阻滞、抑制DNA合成并激活细胞内caspase-3、caspase-8凋亡途径有关。     

线粒体分裂融合基因在氧化应激诱导宫颈癌Hela细胞损伤中的作用

目的:利用维生素K3(VitaminK3,Vk3)复制子宫颈癌Hela细胞损伤模型,观察线粒体融合分裂基因的变化,探讨线粒体融合分裂基因在Vk3诱导Hela细胞凋亡过程中的作用。方法:MTT法检测各组Hela细胞存活率,Hoechst33258染色观察细胞凋亡,RT-PCR方法检测各组Hela细胞中线粒体融合基因Mfn1、Mfn2和Opa1及线粒体分裂基因Drp1、Fist1和MTP18mRNA表达。结果:Vk3能够降低Hela细胞存活率,并且Hoechst33258染色结果显示细胞呈现凋亡形态变化,凋亡率增加,Mfn1、Opa1、和MTP18mRNA表达量明显下降(P<0.05);而与Vk3单独作用组比较,Vk3+NAC联合作用组Hela细胞存活率增加(P<0.05),凋亡率降低,Mfn1、Opa1、和MTP18mRNA表达量明显增加(P<0.05)。结论:线粒体融合和分裂基因表达的降低都有可能参与了氧化应激诱导的细胞损伤作用。     

内皮抑素对异位子宫内膜细胞的作用及机制

目的探讨内皮抑素诱导人异位子宫内膜细胞凋亡的效果及作用机制。方法选取2014年2-12月湖北省人民医院妇产科收治的子宫内膜异位症患者共18例,收集异位子宫内膜标本进行原代分离和培养,分别用10、50和100μmol/L的内皮抑素处理作为实验组,等体积磷酸盐缓冲液(PBS)处理作为对照组。采用流氏细胞术检测细胞凋亡率及线粒体跨膜电位的变化;采用蛋白印记技术检测细胞色素C及含半胱氨酸的天冬氨酸蛋白水解酶-3(Caspase-3)、Caspase-8、Caspase-9的表达水平。结果内皮抑素体外能够诱导人腺上皮细胞和间质细胞发生凋亡,且呈明显的药物浓度依赖性,100μmol/L的内皮抑素作用48 h后,凋亡率分别为24.89%及29.23%。内皮抑素诱导胞质中细胞色素C、Caspase-9、Caspase-3、Caspase-8表达上调,线粒体跨膜电位崩溃。结论内皮抑素能诱导异位子宫内膜细胞发生凋亡,其机制可能与上调细胞色素C基因的表达,激活前体Caspase-9(Pro Caspase-9)、Pro Caspase-3、Pro Caspase-8,最终经线粒体信号转导途径诱导细胞凋亡有关。     

Genistein induces apoptosis in T lymphoma cells via mitochondrial damage

The soy isoflavone genistein has been identified as having antiproliferative and apoptotic effects on various malignant cell types derived from solid tumors. Because little information regarding the effect of genistein on hematopoietic malignancies is available, we undertook this study of T-cell lymphomas. We tested the effect of genistein on murine T-cell lines derived from thymic lymphomas induced by an oncogenic murine leukemia virus. When T lymphoma cells were treated with genistein concentrations of 15 microM and greater, it was observed that the percentage of viable cells was significantly reduced in a dose- and time-dependent manner. The observed cell killing was found to be the result of apoptosis as detected by flow cytometric analysis of cells stained with annexin V and propidium iodide and assays for caspase-3 activation and DNA fragmentation. Cell staining with the mitochondrial specific dye JC-1 and detection of caspase-9 activation revealed that genistein produced mitochondrial depolarization as an early step in the induction of apoptosis. Bongkrekic acid inhibition of mitochondrial depolarization identified the mitochondria permeability transition pore (PTP) as a potential target of genistein activity. These results indicate that the induction of apoptosis by pharmacological concentrations of genistein in T lymphoma cells occurs via mitochondrial damage with the involvement of the PTP.     

β-紫罗兰酮诱导SGC-7901细胞凋亡作用

目的探讨β-紫罗兰酮对胃腺癌SGC-7901细胞的凋亡作用及其可能机制。方法采用细胞生长曲线、核分裂、Hoechst33258荧光染色、透射电镜和WesternBlot方法,观察了β-紫罗兰酮对SGC-7901细胞的生长抑制作用和细胞凋亡诱导情况。结果β-紫罗兰酮对SGC-7901细胞生长和有丝分裂有明显地抑制作用,EC50值为(174.93±12.79)μmol/L;并且可诱导SGC-7901细胞产生凋亡,激活Caspase-3片断和降低ERK1/2蛋白的表达,呈剂量-反应关系。结论β-紫罗兰酮可诱导SGC-7901细胞产生凋亡,不仅作用凋亡途经,而且还影响MAPK途经。     

Induction of apoptosis in human promyelocytic leukemia HL60 cells by an extract from Erythrina suberosa stem bark

In this study, the apoptosis-inducing effect of an alcoholic extract from Erythrina suberosa stem bark (ESB) was investigated using human promyelocytic leukemia HL60 cells. Cell viability was estimated by MTT assay. We found that the ESB inhibited cell proliferation in a dose- and time-dependent manner. A series of well-documented morphological changes, such as cell shrinkage, condensation of nuclear chromatin, and nuclear fragmentation, were observed by fluorescence microscopy. The gold standard scanning electron micrographs showed apoptotic bodies and formation of blebs. Cell cycle analysis showed a significant increase in Sub G(0) population of cells above 50 μg/ml. ESB treatment resulted in a dose-dependent increase in annexin V positive cells. Increase in intracellular ROS production up to sixfold was detected in ESB-treated HL60 cells by DCFH-DA assay. Dissipation of mitochondrial membrane potential of intact cells accompanied by increase in cytosolic cytochrome c was observed, which was followed by activation of caspase-9 and -3 but not caspase-8. DNA fragmentation analysis revealed typical ladders as early as 18 h indicative of caspase-3 role in the apoptotic pathway. The overall results suggest that ESB induces mitochondria-mediated intrinsic apoptotic pathway in HL60 cells and might have therapeutic value against human leukemia.     

Terpenoids Isolated From the Shoot of Plectranthus hadiensis Induces Apoptosis in Human Colon Cancer Cells Via the Mitochondria-Dependent Pathway

The plant Plectranthus hadiensis is a rich source of many bioactive phytochemicals, especially terpenoids. The terpenoid fraction was isolated and phytochemical characterization was done using GC-MS. The aim of the present study was to find out the antiproliferative activity and the mechanism of cell death induction by the terpenoid fraction on human colon cancer cells (HCT-15). MTT assay was performed with different concentrations of the fraction (10, 20, and 50 µg/mL) to obtain IC50 value for 24 h to induce cell death. The induction of apoptosis were studied by Hoechst staining, acridine orange/ethidium bromide staining, Comet assay, DNA fragmentation, and caspase-3 activity assays. The mechanism of apoptosis induction was studied by expression analysis of antiapoptotic Bcl-2 and proapoptotic Bax using RT-PCR and also by Western blot analysis of proteins involved in the apoptotic pathway. The terpenoid fraction induced significant morphological changes and DNA fragmentation in the cells. Positive Hoechst staining and acridine orange/ethidium bromide staining indicated apoptosis induction by the fraction. DNA fragmentation, which is a characteristic feature of apoptosis, was also observed. Upregulation of caspase-3 activity and proapoptotic Bax, and the downregulation of antiapoptotic Bcl-2 and COX-2 confirmed that the apoptosis induction was via the mitochondria-dependent pathway.     

Apelin抑制小鼠成骨细胞MC3T3-E1凋亡

目的观察Apelin对小鼠成骨细胞MC3T3-E1凋亡的作用。方法 ELISA和吖啶橙/溴乙啶（AO/EB）染色法检测细胞凋亡,Western blot检测细胞Bcl-2和Bax表达以及cytochrome c的分泌,通过荧光素（AFC、AMC）标记的底物来检测活性。结果 Apelin抑制无血清饥饿诱导的MC3T3-E1细胞凋亡;Apelin抑制无血清饥饿诱导的cyto-chrome c的分泌和caspase-3、caspase-8c、aspase-9的活化;Apelin抑制地塞米松或肿瘤坏死因子-α（TNF-α）诱导的MC3T3-E1细胞凋亡。结论 Apelin可抑制成骨细胞凋亡。     

Induction of Apoptosis in Human Promyelocytic Leukemia HL60 Cells by an Extract From Erythrina suberosa Stem Bark

In this study, the apoptosis-inducing effect of an alcoholic extract from Erythrina suberosa stem bark (ESB) was investigated using human promyelocytic leukemia HL60 cells. Cell viability was estimated by MTT assay. We found that the ESB inhibited cell proliferation in a dose- and time-dependent manner. A series of well-documented morphological changes, such as cell shrinkage, condensation of nuclear chromatin, and nuclear fragmentation, were observed by fluorescence microscopy. The gold standard scanning electron micrographs showed apoptotic bodies and formation of blebs. Cell cycle analysis showed a significant increase in Sub G₀ population of cells above 50 μg/ml. ESB treatment resulted in a dose-dependent increase in annexin V positive cells. Increase in intracellular ROS production up to sixfold was detected in ESB-treated HL60 cells by DCFH-DA assay. Dissipation of mitochondrial membrane potential of intact cells accompanied by increase in cytosolic cytochrome c was observed, which was followed by activation of caspase-9 and -3 but not caspase-8. DNA fragmentation analysis revealed typical ladders as early as 18 h indicative of caspase-3 role in the apoptotic pathway. The overall results suggest that ESB induces mitochondria-mediated intrinsic apoptotic pathway in HL60 cells and might have therapeutic value against human leukemia.     

Potential anticancer activity of myricetin in human T24 bladder cancer cells both in vitro and in vivo

Myricetin, a naturally occurring phytochemical, has potent anticancer-promoting activity and contributes to the chemopreventive potential of several foods. In this preliminary study, we evaluate the chemopreventive potential of myricetin against bladder cancer and its mechanism of action. The results of a MTT assay showed that myricetin was able to inhibit the viability and proliferation of T24 cells in a dose- and time-dependent manner. It also promoted cell cycle arrest at G2/M in a dose-dependent manner and induced apoptosis detected by flow cytometry and DNA fragmentation analysis. Treatment with myricetin led to G2/M cell cycle arrest in T24 cells by downregulation of Cyclin B1 and cyclin-dependent kinase cdc2. Myricetin-induced apoptosis correlates with the modulation of Bcl-2 family proteins and activation of the caspase-3. Myricetin also inhibited the phosphorylation of Akt, whereas the phosphorylation of p38 MAPK was enhanced. Myricetin had a significantly reduced T24 cell migration that was accompanied by a decreasing MMP-9 expression in vitro. Furthermore, myricetin treatment significantly inhibited the tumor growth on T24 bladder cancer xenografts model. These findings suggest that myricetin has potential anticancer activity and could be an important chemoprevention agent for bladder cancer.     

Potential Anticancer Activity of Myricetin in Human T24 Bladder Cancer Cells Both In Vitro and In Vivo

Myricetin, a naturally occurring phytochemical, has potent anticancer-promoting activity and contributes to the chemopreventive potential of several foods. In this preliminary study, we evaluate the chemopreventive potential of myricetin against bladder cancer and its mechanism of action. The results of a MTT assay showed that myricetin was able to inhibit the viability and proliferation of T24 cells in a dose- and time-dependent manner. It also promoted cell cycle arrest at G2/M in a dose-dependent manner and induced apoptosis detected by flow cytometry and DNA fragmentation analysis. Treatment with myricetin led to G2/M cell cycle arrest in T24 cells by downregulation of Cyclin B1 and cyclin-dependent kinase cdc2. Myricetin-induced apoptosis correlates with the modulation of Bcl-2 family proteins and activation of the caspase-3. Myricetin also inhibited the phosphorylation of Akt, whereas the phosphorylation of p38 MAPK was enhanced. Myricetin had a significantly reduced T24 cell migration that was accompanied by a decreasing MMP-9 expression in vitro. Furthermore, myricetin treatment significantly inhibited the tumor growth on T24 bladder cancer xenografts model. These findings suggest that myricetin has potential anticancer activity and could be an important chemoprevention agent for bladder cancer.     

Radiation-induced apoptosis is independent of caspase-8 but dependent on cytochrome c and the caspase-9 cascade in human leukemia HL60 cells

We investigated the role of the caspase activation cascade in apoptosis induced by ionizing radiation or hydrogen peroxide (H(2)O(2)) in human leukemia HL60 cells. Electron paramagnetic resonance (EPR) spectra revealed that hydroxyl and hydrogen radicals were generated in the culture medium after exposure to radiation or H(2)O(2). Initial accumulation of DNA fragments at 2 h after exposure was delayed in irradiated cells compared with H(2)O(2)-treated cells, although formation of abasic sites immediately after exposure was significantly higher in irradiated cells and similar quantities of hydroxyl radicals were produced under both conditions. Activity assay of caspases revealed that caspase-3, -8 and -9 were activated 2 h after exposure to H(2)O(2), whereas in irradiated cells caspase-3 and -9 activation occurred 4 h after exposure but increased caspase-8 activation was not observed. Release of cytochrome c into cytosol was seen at 2 h after radiation and H(2)O(2) treatment. Radiation did not affect proapoptotic proteins (Bax and Bid), whereas H (2)O(2) increased accumulation of Bax in the mitochondrial membrane 2 h to 6 h after treatment, independently of the truncation of Bid by activated caspase-8. Moreover, treatment with the caspase-8 inhibitor Z-IETD-FMK increased cell survival and prevented accumulation of DNA fragments in H(2)O(2)-treated cells, but not in irradiated cells. These results suggest that, unlike the caspase cascade of H(2)O(2)-induced apoptosis, cytochrome c and caspase-9 are important for the intrinsic pathway of radiation-induced apoptosis, independent of caspase-8.