南方大斑蝥体内结合斑蝥素对人肝癌HepG2细胞凋亡的影响

利用水提法从来自罗甸县茂井镇的南方大斑蝥中提取得到结合斑蝥素,作用于人肝癌HepG2细胞后,分别利用Annexin V-FITC/PI双染法检测HepG2细胞的凋亡率,RT-PCR法检测Bax、Bcl-2mRNA相对表达情况,利用Western blot法检测HepG2细胞中Bax、Bcl-2蛋白表达的影响。实验结果表明罗甸县茂井镇产的南方大斑蝥体内结合斑蝥素可诱导人肝癌HepG2细胞凋亡,其机制可能与上调Bax和下调Bcl-2表达有关。     

阿帕替尼对人肝癌细胞增殖及凋亡的作用机制研究

目的:探讨阿帕替尼抑制肝癌细胞增殖促进凋亡的作用机制。方法:选取肝癌细胞系SNU739、HepG2,以CCK-8细胞增殖实验、平板克隆实验测定阿帕替尼对肝癌细胞增殖及克隆形成能力的影响;流式细胞术检测阿帕替尼对肝癌细胞凋亡的影响;蛋白免疫印迹法检测阿帕替尼影响肝癌细胞凋亡相关蛋白Bax、Bcl-2及Caspase3的表达情况。结果:与对照组相比,阿帕替尼可显著抑制肝癌细胞增殖(P0.05)。平板克隆实验提示与对照组相比,10μM和20μM阿帕替尼组肝癌细胞克隆数明显减少(P0.05)。流式细胞术结果提示10μM和20μM阿帕替尼处理组细胞凋亡率明显增加(P0.05)。蛋白免疫印迹法结果显示经阿帕替尼处理的肝癌细胞,促凋亡蛋白Bax及Caspase3的活性片段Cleaved-caspase3表达水平显著上调,抗凋亡蛋白Bcl-2显著下调(P0.01)。结论:阿帕替尼通过调节肝癌细胞凋亡相关蛋白从而抑制肝癌细胞增殖、促进其凋亡。     

半边旗二萜类成分PAG通过ROS诱导肝癌细胞凋亡的作用研究

探讨半边旗二萜类成分Pteisolic acid G(PAG)对人肝癌细胞HepG2增殖和凋亡的影响及作用机制。用不同浓度的PAG处理HepG2细胞后,采用MTT法检测细胞存活率;采用PI单染法检测细胞周期分布;采用Annexin V-FITC/PI双染法检测细胞凋亡率;采用RT-PCR和Western Blotting检测细胞内mRNA和蛋白表达情况;采用DCFH-DA法检测细胞内ROS水平,采用ROS抑制剂乙酰半胱氨酸(NAC)评价PAG细胞增殖抑制作用对ROS的依赖性。结果表明,在24 h、48 h和72 h时,PAG可剂量依赖性地抑制HepG2细胞的增殖(p0.05),IC_(50)分别为64.8μmol/L,38.5μmol/L和24.8μmol/L;用药24 h时PAG可剂量依赖性地使HepG2细胞阻滞在G_2/M期,同时增加HepG2细胞凋亡率(p0.05);PAG可剂量依赖性地降低HepG2细胞内Bcl-2 mRNA和caspase 3、PARP、Bcl-2蛋白的表达(p0.05),增加Bax mRNA和actived-caspase 3、cleaved-PARP、Bax蛋白的表达(p0.05)。当使用1 mmol/L的ROS抑制剂NAC预处理HepG2细胞时,PAG对HepG2细胞增殖抑制作用被显著阻断。上述结果表明,半边旗二萜类成分PAG可提高Bax/Bcl-2的基因和蛋白表达比值,从而诱导肝癌细胞HepG2凋亡,该作用可能是通过升高细胞内ROS水平来实现的。     

维甲酸对肝癌细胞HepG2糖基化磷脂酰肌醇特异性磷酯酶D表达及细胞生物学特性的影响

应用MTT、流式细胞仪、免疫印迹法检测全反式维甲酸(ATRA)单独或联合糖基化磷脂酰肌醇特异性磷酯酶D(GPI-PLD)特异性抑制剂1,10-二氮杂菲对肝癌细胞HepG2生物学特性的改变.ATRA使肝癌细胞HepG2 GPI-PLD基因表达及酶活性上调,并呈现剂量和时间依赖性.ATRA可抑制肝癌细胞HepG2增殖,使肝癌细胞Caspase-3表达水平显著增加,Bcl-2表达水平下调,促进肝癌细胞凋亡(P<0.05).ATRA联合1,10-二氮杂菲诱导组细胞,Bcl-2、细胞增殖活性较ATRA单独诱导组显著增强,Caspase-3、凋亡率显著下降.维甲酸可促进肝癌细胞HepG2 GPI-PLD基因表达上调,高活性的GPI-PLD有助于维甲酸抑制肝癌细胞增殖,促进肝癌细胞凋亡.     

龙牙百合多糖对人肝癌细胞HepG2凋亡的影响

为研究龙牙百合多糖(LP)对人肝癌HepG2细胞抑制和凋亡的影响,采用MTT法考察了3组LP不同浓度对人肝癌HepG2细胞抑制作用,采用Annexin-V/PI双染法和Western blotting法探究了LP1和LP42个组分对人肝癌HepG2细胞凋亡作用机制。LP对人肝癌HepG2细胞抑制呈剂量依赖关系,LP4组1 mg/mL对HepG2细胞的抑制率达46.36%,效果最佳;LP1组8 mg/mL最高抑制率达43.23%;LP2组4 mg/mL最高抑制率达24.35%;LP1和LP4对HepG2细胞的晚期凋亡率显著高于早期。激活Caspase-3、Bax、蛋白表达显著升高(p0.05),Bcl-2蛋白表达水平明显降低(p0.05)。研究表明,LP通过激活死亡受体凋亡途径和线粒体途径,诱导人肝癌HepG2细胞凋亡。     

沉默信息调节因子3促进肝癌细胞凋亡及其机制研究

该文旨在探讨沉默信息调节因子3(sirtuin 3,SIRT3)对肝癌细胞凋亡的影响,并研究SIRT3调节肝癌细胞凋亡的分子机制。运用流式细胞术检测SIRT3过表达对肝癌细胞系(SMMC-7721和SK-Hep-1)凋亡的影响;通过si RNA靶向沉默SIRT3并检测SIRT3沉默对肝癌细胞凋亡的影响;实时荧光定量PCR(Real-time PCR)分析SIRT3对Bcl-2家族成员m RNA水平的影响,筛选受SIRT3调节的Bcl-2家族成员;Western blot进一步检测SIRT3对目标Bcl-2家族成员蛋白水平的影响;流式细胞术分析目标Bcl-2家族成员在SIRT3诱导肝癌细胞凋亡中的作用。结果显示,SIRT3过表达促进肝癌细胞凋亡并引起Bax mRNA和蛋白水平升高;SIRT3沉默抑制肝癌细胞凋亡,同时也抑制Bax蛋白水平表达,Bax沉默显著减少了SIRT3过表达细胞中的凋亡数目。该研究结果提示,SIRT3通过凋亡调节基因Bax诱导肝癌细胞凋亡。     

根皮素对HepG2肝癌细胞增殖和凋亡影响的实验研究

目的：探讨根皮素对人肝癌HepG2细胞增殖和凋亡的影响。方法：MTT法检测不同浓度（30、40、50 ug/mL）根皮素对肝癌 HepG2 细胞增殖的抑制作用,流式细胞技术检测根皮素对HepG2细胞凋亡的影响,ELISA 法检测不同浓度根皮素干预后细胞中 Bcl-2 和Bax表达的变化,Western blot法检测30 ug/mL根皮素在8,16,24 小时后检测p-AKT 蛋白表达情况。结果：30、40 和50 ug/mL 的根皮素对肝癌HepG2 细胞的增殖均有抑制作用（P＜0.01）,同时,30、40 和50 ug/mL 的根皮素在24 小时后可诱导 HepG2 细胞发生早期凋亡,凋亡率分别为0.1321± 0.0224, 0.2607± 0.0457, 0.3712± 0.0884（P＜0.01）;另外,30、40 和50 ug/mL的 根皮素作用24 小时后细胞内Bcl-2 表达降低,Bax 表达增高（P＜0.01）;最后,30 ug/mL根皮素可以明显减少p-AKT 表达量,这 种作用呈现时间依赖性。结论：根皮素能够抑制肝癌HepG2 细胞的增殖和促进细胞凋亡。     

香蜂草苷诱导人肝癌细胞株HepG2凋亡及其作用机制

为了研究香蜂草苷对肝癌细胞株HepG2凋亡的影响,并探讨其作用机制,利用MTT法检测香蜂草苷对HepG2细胞增殖的抑制作用,流式细胞术检测细胞凋亡率,试剂盒检测caspase-3和caspase-9活性,Western blot检测Bcl-2、Bax、RKIP、ERK、p-ERK蛋白表达。实验结果表明香蜂草苷可抑制HepG2细胞增殖并诱导其凋亡,其机制可能与提高caspase-3和caspase-9活性,上调Bax和下调Bcl-2表达有关。此外,我们的研究显示香蜂草苷诱导HepG2细胞凋亡可能还与其增加RKIP表达,抑制ERK/MAPK信号通路有关。     

四逆散对人肝癌HepG2细胞增殖、凋亡的影响及其机制*

目的: 探讨含四逆散药液血清对人肝癌HepG2细胞增殖、凋亡的影响及机制。方法: 将人肝癌HepG2细胞分为5组,每组3个复孔。实验组细胞用五氟尿嘧啶(5-FU)或不同浓度的含四逆散药液血清处理48 h后,用倒置显微镜观察含四逆散药液血清处理后人肝癌HepG2细胞形态的变化;MTT法检测含四逆散药液血清对HepG2细胞生长的抑制作用;荧光染色和流式细胞术分别分析含四逆散药液血清对HepG2细胞凋亡的影响。Rho123染色法检测线粒体膜电位变化,Western blot检测细胞凋亡相关蛋白的表达。结果: 与对照组比较,含四逆散药液血清处理人肝癌HepG2细胞后,细胞数量显著减少(P＜0.01),形态发生改变,呈现典型的凋亡细胞形态;G1期细胞数明显增加,而G2 期细胞数量显著减少(P＜0.05);Bax、Caspase-3、-9和Cyt-c的表达显著升高,而Bcl-2的表达显著降低(P＜0.05);随着含四逆散药液血清浓度增大,HepG2细胞线粒体膜电位显著下降(P＜0.05)。结论: 四逆散可以抑制HepG2细胞增殖,并通过线粒体途径诱导细胞凋亡。     

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。     

Dihydromyricetin Reduced Bcl-2 Expression via p53 in Human Hepatoma HepG2 Cells

Dihydromyricetin (DHM) is a major active ingredient of flavonoids compounds. It exhibited anticancer activity and induced apoptosis in human hepatocellular carcinoma HepG2 cells according to our previous data. In this study, we investigated whether p53 is involved in DHM-triggered viability inhibition and apoptosis induction in cancer cells. MTT [3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay was employed to evaluate the viability of HepG2 cells after DHM treatment. Meanwhile, p53 small interfering RNA (siRNA) was adopted to silence p53 expression. Protein level of p53 and Bax/Bcl-2 were evaluated by western blot analysis. Cell counting assay showed that DHM inhibited HepG2 cell growth effectively in a time- and dose-dependent manner. P53 expression was significantly increased after DHM treatment, whereas Bcl-2 was reduced potently. Furthermore, after co-treatment with Pifithrin-α (PFT-α, p53 inhibitor), Bcl-2 expression was reversed. The expression of Bax was no significant change, which was also observed after p53 silence. These findings defined and supported a novel function that DHM could induce human hepatocellular carcinoma HepG2 cells apoptosis by up-regulating Bax/Bcl-2 expression via p53 signal pathway.     

The anti-proliferative effects of norcantharidin on human HepG2 cells in cell culture

Many lines of evidence have shown that Chinese medicine contains many chemical compounds with anticancer effects. Therefore, we tested whether the active ingredients of blister beetles have a therapeutic effect on hepatoma. The aim of this study was to investigate the inhibitive effects of norcantharidin which is extracted from blister beetles on human hepatoma cells HepG2 in vitro and its anticancer mechanism.MTT assay, agarose gel electrophoresis and flow cytometry were used to evaluate HepG2 cells proliferation and apoptosis. The role of caspase activities were assayed using caspase apoptosis detection kit. Western blot analysis was used to evaluate the level of Bcl-2/Bax expression. Our results indicate that norcantharidin inhibited HepG2 cell growth in a time- and dose-dependent manner by MTT assay. HepG2 cells treated with norcantharidin showed typical characteristics of apoptosis including the DNA fragmentation. The activities of caspase-3, -9 were up-regulated after norcantharidin treatment. By western blot analysis, we found the level of Bcl-2 were down-regulated, whereas, the level of Bcl-2 Up-regulated.so we suggest that up-regulation of mitochondrial Bax expression and down-regulation of Bcl-2 expression participated in the apoptosis induced by NCTD. These results suggest that norcantharidin triggers apoptosis in hepato cancer cell lines via the activation of the caspses, mitochondrial pathways, and that this agent may be useful for developing new therapeutic regimens for the treatment of colorectal carcinoma.     

重组荞麦胰蛋白酶抑制剂对人肝癌细胞的凋亡及半胱氨酸天冬酶活性的影响

先前的研究表明,基因重组荞麦胰蛋白酶抑制剂 (rBTI) 具有诱导不同肿瘤细胞凋亡的作用.为了揭示其诱导肿瘤细胞凋亡的可能机理,从基因水平上探讨与凋亡有关的分子事件,本研究用不同浓度的 rBTI 体外作用于人肝癌细胞 HepG2 后,采用 MTT 比色法检测抑制剂对epG2 细胞的抑制率,用 DNA 凝胶电泳和细胞核的形态学观察检测 HepG2 细胞的凋亡.结果表明,rBTI 在体外能够明显抑制 HepG2 细胞的增长,并诱导细胞凋亡.另外,细胞凋亡与Bcl-2/Bax mRNA 水平有关.通过 RT-PCR 检测发现,细胞经过rBTI处理后,抗凋亡基因Bcl-2 mRNA 水平下调,促凋亡基因 Bax mRNA 有所上调,而对照 GAPDH 无变化.对 HepG2细胞中 Fas/Fas 配体及半胱氨酸天冬酶（caspase）的研究证明,细胞经过 rBTI 处理后,对死亡受体 Fas mRNA没有影响; rBTI 可明显激活caspase-3 和 caspase-9 酶活性, 对caspase-8 活性几乎无影响.上述结果表明,rBTI 对HepG2 细胞具有明显的诱导凋亡作用,其诱导细胞凋亡的机制与 caspase-3 依赖性凋亡调节信号通路有关,未涉及 Fas/Fas 配体途径.     

Effects of Bcl-2 and Bcl-XL protein levels on chemoresistance of hepatoblastoma HepG2 cell line.

The ratio between apoptotic promoters and repressors in the Bcl-2 family determines the chemosensitivity of cells to apoptotic stimuli. This study examines the chemoresistance of a transfected human hepatoblastoma HepG2 cell-line during Taxol and Doxorubicin application. Sense bcl-2, and anti-sense bcl-XL gene fragments were separately inserted into HepG2 cells via stable transfection. The expression profile of the Bcl-2 family proteins was determined by Western blot analysis. Chemosensitivity of the transfected cells was measured by Trypan blue exclusion assay and XTT reduction assay during drug application. In the absence of Bax protein, HepG2 cells with elevated Bcl-2 protein levels did not exhibit any significant increase in chemosensitivity towards the drugs. Transfected cells with reduced Bcl-XL levels became more sensitive to the drugs, and a significant difference in IC50 values was observed. The chemosensitivity of HepG2 cells to Taxol and Doxorubicin was not affected by Bcl-2 levels, while reduction of Bcl-XL levels rendered the cells more sensitive to the drugs. This suggests that the Bcl-2 protein alone could not protect HepG2 cells from drug-induced apoptosis, and that the Bcl-XL protein may be a target for gene therapy in hepatoblastoma treatment.     

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。     

Shikonin inhibits the proliferation and induces the apoptosis of human HepG2 cells

This study investigated the potential of shikonin as an anticancer agent against liver cancer and an in vitro human hepatoma cancer model system. The HepG2 cell line was the hepatoma cancer model in the present study. The inhibitory effect of shikonin on the growth of HepG2 cells was measured by MTT assay. To explore the underlying mechanism of cell growth inhibition of shikonin, the cell cycle distribution, DNA fragmentation, mitochondrial membrane potential (ΔΨm) disruption, and expression of Bax and Bcl-2 were measured in HepG2 cells. The activity of shikonin in inducing apoptosis was investigated through the detection of Annexin V signal and CD95 expression by flow cytometry and electron microscopy, respectively. Shikonin inhibited the growth of HepG2 cells in a dose-dependent manner. The IC50 value (inhibiting cell growth by 50%) was 4.30 mg/mL. Shikonin inhibited cell growth in a dose-dependent manner and blocked HepG2 cell cycle progression at the S phase. The changes in mitochondrial morphology, dose-dependently decreased in ΔΨm, were observed in different concentrations of the drug treatment group. Western blot analysis showed that cajanol inhibited Bcl-2 expression and induced Bax expression. Furthermore, we show that shikonin increases Annexin V signal and CD95 (Fas/APO) expression, resulting in apoptotic cell death of HepG2 cells. In addition, lump formation of intranuclear chromatin, pyknosis of cell nucleus, deletion of microvillus, vacuolar degeneration of mitochondria, reduction of rough endoplasmic reticulum, and resolution of free ribosome, etc., associated with apoptosis were discovered by electron microscopy in HepG2 cells after 48 h treatment. Shikonin inhibited HepG2 cells, possibly through the pathway of inducing early apoptosis, and was beneficial for restoring the apoptotic sensitivity of HepG2 cells by CD95, and should therefore be considered as a candidate agent for the prevention or treatment of human hepatoma.     

Altered Sensitivity to Retinoid-Induced Apoptosis Associated with Changes in the Subcellular Distribution of Bcl-2

In the acute promyelocytic leukemia cell line NB4, Bcl-2 downregulation occurred as a late event of retinoid-induced differentiation. In the maturation-resistant NB4-R1 subclone, retinoids failed to downregulate Bcl-2 even in the situation of apoptosis massively induced by pan-agonists and RXR-selective agonists. We observed that NB4 and NB4-R1 cells differed with respect to the intracellular localization of Bcl-2 which showed a perinuclear localization in NB4-R1 cells, while Bax was broadly expressed in the cytoplasm and to only a minor extent in the perinuclear area. Therefore, the distinct intracellular localization of Bcl-2 and Bax was in general nonoverlaping. Bcl-2 remained massively expressed until cell disruption. Bax was not significantly upregulated in cells committed to death. However, Bax localization changed from a diffuse pattern to concentrate in few specific cytoplasmic area at a stage preceding the formation of apoptotic bodies. A human Bcl-2 transgene was transiently overexpressed in NB4-R1 cells which showed increased resistance to apoptosis induced by retinoids. Stably transfected clones of NB4-R1 cells showed an increased expression of Bcl-2 and a marked resistance to apoptosis. Interestingly, the overexpression of Bcl-2 restored a pattern of uniform Bcl-2 labeling in the cytoplasm and, remarkably, the colocalization of Bcl-2 with Bax. This work demonstrates that the ability of retinoid-induced cells to undergo apoptosis depends on the level of expression and the functional interaction between Bcl-2 and Bax.     

妊娠兔胎盘细胞凋亡及凋亡相关基因Bcl-2和Bax表达的动态变化

以新西兰雌兔为动物模型。研究妊娠期间胎盘细胞凋亡及其凋亡调控蛋白Bcl-2和Bax表达的动态变化,基因组DNA凝胶电泳实验检测到妊娠中期和晚期胎盘基因组DNA中出现典型的凋亡特征-DNA梯带,而且DNA断裂值在妊娠早、中、晚期分别为：0.14,0.49和1.43,与妊娠早期相比,妊娠中,晚期胎盘基因组DNA断裂值有显著性增加,TUNEL实验和活化caspase-3的免疫定位实验表明,在妊娠早期胎盘中存在细胞凋亡,而且在各妊娠期中细胞凋亡主要发生于合体滋养层,免疫印迹法分析表明,Bcl-2和Bax随妊娠的进行其表达量明显增加,Bax:Bcl-2比值在妊娠早、中、晚期分别为：0.89,0.91和1.25,呈增加趋势,实验结果说明,在兔正常妊娠中,胎盘合体滋养层细胞发生凋亡,且随妊娠的进行,凋亡细胞数量增多,胎盘细胞凋亡主要与细胞中Bax:Bcl-2的比例相关。     

镉诱导小鼠睾丸细胞凋亡与Caspase-3、Bcl-2和Bax mRNA的表达

一个月大雄性小鼠24只,随机分为6组,用30 μmol/kg CdCl2作用小鼠睾丸不同的时间(3 h、6 h、12 h、18 h、24 h)后,利用DNA电泳、免疫组化和半定量RT-PCR技术,分析生殖细胞凋亡过程中三种关键物质Caspase-3、Bcl-2、Bax蛋白和mRNA的表达量变化.结果显示:1) DNA各组 (除对照组外)均出现不同程度断裂.2)Caspase-3蛋白表达量一直上升,与对照组相比差异极显著;Bax蛋白在12 h前一直上升,与对照组相比差异显著,12 h后又开始下降,且与对照组相比无显著差异;Bcl-2蛋白在下降,与对照组相比差异显著.3)RT-PCR结果显示Caspase-3基因表达量减少;Bax基因表达量逐渐上升;Bcl-2基因表达量波动很大.综上所述,Caspase-3、Bcl-2和Bax三个基因可能参与了镉应激状态下小鼠睾丸组织细胞的凋亡过程.