核转录因子-κB圈套策略对肝癌细胞HepG2凋亡的影响

目的:观察NF-κB圈套寡核苷酸(Decoy ODNs)对NF-κB活性的影响,进一步探讨其对人肝癌细胞HepG2凋亡的作用机制。方法:设计NF-κB圈套寡核苷酸,将FITC标记的NF-κB Decoy ODNs转染HepG2,共聚焦显微镜观察其核内定位,描记生长曲线;凝胶阻滞法(EMSA)检测转染前后NF-κB活性;再分别用流式细胞术和TUNEL法检测细胞凋亡情况。Western blot检测HepG2细胞中Bcl-2和Fas的含量。结果:NF-κB Decoy ODNs转染HepG2细胞后定位于胞核,NF-κB活性明显下降,其显著抑制细胞生长,促进HepG2细胞凋亡;低活性NF-κB可以下调Bcl-2、增加Fas的表达水平。结论:NF-κB圈套寡核苷酸促进肝癌细胞HepG2凋亡的机制可能与其下调NF-κB的活性,使促凋亡蛋白Fas表达增加和降低抑凋蛋白Bcl-2表达有关。

内毒素诱导HepG2凋亡的研究

目的：探讨内毒素对HepG2细胞的直接作用,为肝细胞损害的防治研究提供新的模型.方法：利用HepG2细胞作为体外肝细胞模型,HepG2细胞经不同浓度LPS（0.1、10、20μg/ml、作用24、48小时, 应用流式细胞仪测定细胞凋亡率,分光光度计法测 Caspase-3活性;ELISA 法检测细胞培养上清 TNF-α;结果：不同浓度内毒素刺激后 TNF-α、Caspase-3明显升高.结论：LPS直接引起 HepG2 细胞凋亡,并呈浓度依赖性.

NF-κB,AP-1及caspase-3在SC58125诱导的HepG2细胞凋亡中的作用

目的 :探讨SC5 812 5诱导HepG2细胞凋亡的分子机理。方法 :应用细胞培养、酶联免疫吸附实验、流式细胞术、RT -PCR、Westernblot及电泳迁移率实验等方法研究SC5 812 5诱导HepG2细胞凋亡及其分子机理。结果 :SC5 812 5剂量依赖性地诱导HepG2细胞凋亡 ,并伴有NF -κB结合活性的抑制、caspase - 3的激活、bcl- 2mRNA水平的降低及p5 3mRNA的上调 ,而AP - 1的结合活性则没有明显的改变。结论 :SC5 812 5诱导HepG2细胞的凋亡 ,这种效应可能与抑制NF -κB结合活性、激活caspase - 3、降低bcl- 2mRNA的表达及上调p5 3mRNA的水平有关。

氟西汀通过上调miR-16诱导HepG2细胞凋亡

肝细胞癌（hepatocellular carcinoma，HCC）是世界第二大癌症相关死因，严重威胁着人类的健康和生命。本病发病隐匿，进展迅速，晚期HCC的自然生存期约3～4个月，预后极差。HCC对大部分标准化疗方案不敏感，迄今为止被证实唯一有效的分子靶向药物为索拉菲尼，但其提高患者总体生存率有限（中位生存时间仅延长3个月）。因此，寻找既能够提高治疗效果又能减少全身不良反应的抗HCC药物显得尤为迫切。

MG132诱导HepG_2细胞凋亡及其对p53表达的影响

目的观察蛋白酶体抑制剂MG132对人肝癌细胞系HepG2的致凋亡作用及其对凋亡相关基因p53蛋白表达的影响。方法采用多个浓度(2,5,10μmol/L)的蛋白酶体抑制剂MG132处理HepG2细胞;流式细胞术和Hoechst荧光染色检测细胞凋亡;免疫细胞化学检测HepG2细胞p53蛋白含量。结果对照组HepG2细胞凋亡率低于5%,在2,5,10μmol/LMG132作用下,细胞凋亡率分别为42.9%,66.1%和72.8%,MG132诱导HepG2细胞凋亡具有剂量—效应关系。经Hoechst荧光染色可见细胞染色质浓缩等凋亡改变。免疫组织化学检测HepG2细胞p53蛋白表达水平增高。结论蛋白酶体抑制剂MG132能够诱导HepG2细胞凋亡,其作用呈剂量—效应关系;p53蛋白表达增加与MG132抑制泛素—蛋白酶体系统降解细胞内p53蛋白有关。

熊果酸对HepG2增殖和凋亡的影响及部分机制研究

目的:观察熊果酸对人肝癌细胞株HepG2增殖和凋亡的影响及其部分机制的研究。方法:将不同浓度熊果酸体外培养人肝癌HepG2细胞,通过MTT法检测熊果酸对细胞增殖抑制的情况,利用流式细胞检测术观察熊果酸对细胞凋亡及细胞周期的影响。同时利用Western blot法检测不同浓度熊果酸干预后人肝癌HepG2细胞pERK1/2蛋白、Cyclin D1蛋白的表达情况。结果:不同浓度熊果酸对人肝癌HepG2细胞的增殖均有抑制效应,并呈剂量、时间依赖性(P<0.05);60μmol/L熊果酸作用于人肝癌细胞株HepG2 72 h后达到最大细胞凋亡率(78.723±3.623)%。熊果酸可明显增加G_0/G_1期的细胞含量,诱导人肝癌细胞株HepG2的凋亡。熊果酸可抑制pERK1/2蛋白、Cyclin D1蛋白的表达,并随着浓度、时间逐渐上调抑制作用更加明显。结论:熊果酸可抑制人肝癌细胞株HepG2增殖,阻滞细胞期于G0/G1期,促进癌细胞凋亡,这一过程可能与下调细胞pERK1/2蛋白、Cyclin D1蛋白的表达有关。

茉莉酸甲酯诱导人肝癌细胞HepG2细胞凋亡过程中端粒酶活性变化

目的:探讨在茉莉酸甲酯(MeJA)诱导HepG2细胞凋亡过程中的端粒酶活性变化。方法:RT-PCR检测HepG2细胞中hTERTmRNA表达,免疫细胞化学检测HepG2细胞端粒酶蛋白表达。结果:MeJA作用HepG2细胞48 h后,hTERTmRNA表达水平明显下降(P<0.05);端粒酶蛋白表达水平明显降低(P<0.01)。结论:MeJA通过下调hTERTmRNA及其蛋白的表达,引起HepG2细胞凋亡,从而发挥抗肝癌作用。

Caspase蛋白酶活化在牛磺酸脱氧胆酸诱导HepG2细胞凋亡中的作用

目的 探讨Caspase蛋白酶活化在牛磺酸脱氧胆酸 (TDCA)诱导HepG2细胞凋亡中的作用。 方法 应用苏木精 伊红染色、Hoechst 3 3 2 5 8染色、电镜和DNA电泳对细胞凋亡定性 ;应用流式细胞仪对细胞凋亡定量 ;检测TDCA诱导HepG2细胞凋亡过程中 ,凋亡特异性蛋白酶Caspase 3、8、9活性的变化。 结果 TDCA 40 0 μmol/L孵育 12h可诱导显著HepG2细胞凋亡 ,凋亡率为 (5 0 .3 5± 2 .2 0 ) % ;在TDCA诱导HepG2细胞凋亡过程中 ,Caspase 9、3蛋白酶活性显著提高 ,Caspase 8活性仅轻度增高。 结论 TDCA可能主要是通过激活Caspase 9活化途径 ,启动Caspase级联反应介导细胞凋亡 ,Caspase 8活化在TDCA诱导的细胞凋亡中可能不起主要作用。

幽门螺杆菌通过Bcl-2/Bax机制诱导HepG2细胞凋亡的研究

探讨幽门螺杆菌（H．pylori）对肝细胞HepG2细胞凋亡的影响及其机制。方法：应用HepG2细胞为实验模型．用MTT、荧光染色技术、透射电子显微镜、AO／EB染色、流式细胞仪定性和定量地检测HepG2细胞凋亡情况；WestemBlotting法检测凋亡相关蛋白Bcl－2和Bax蛋白的表达。结果：不同浓度H．pylori处理组（4．0x105、4．0×106、4．0×107和4．0×108CFU／mL）的24h细胞生长抑制率分别为（2．64±0．34）％、（16．35±4．24）％、（27．15±3．19）％和（33．35±2136）％（P〈0．05），抑制率随作用时间的延长而增加（P〈0．05）。HepG2细胞与4×107／mL浓度的NCTCll637共培养24h后，在透射电镜下可见部分细胞呈不同时期的凋亡形态。不同浓度H．pylori的凋亡率分别为（2．50±0．45）％、（6．00±0．35）％、（11．10±0．45）％和（14．65±0．27）％，当H．pylori的浓度≥4．0x10‘CFU／mL，HepG2细胞凋亡率与对照组比较差异有统计学意义（P〈0．05）。H．pylori作用HepG2细胞后，HepG2细胞的Bcl一2蛋白表达明显减少，Bax蛋白表达则与Bcl一2蛋白表达情况相反。结论：H．pylori通过Bcl一2／Bax机制诱导HepG2细胞凋亡。

姜黄素诱导肿瘤细胞凋亡的实验研究

目的:探讨姜黄素诱导HepG2细胞凋亡的作用机制。方法:应用形态学方法、AnnexinV荧光染色和流式细胞仪(FCM)检测HepG2细胞凋亡的发生,应用RT-PCR检测凋亡相关基因P53、bcl-2和Fas的变化。结果:姜黄素对HepG2细胞的生长有明显的抑制作用,并诱导肿瘤细胞发生凋亡。凋亡细胞表现为细胞固缩,核染色质碎裂;流式细胞仪检测凋亡率为11.7%,细胞停在G1和G2期。AnnexinV标记的方法检测凋亡时发现,坏死与凋亡共存。在姜黄素诱导HepG2细胞凋亡过程中,凋亡相关基因Fas转录水平比用药前增强。结论:凋亡为姜黄素抑癌的机制之一,姜黄素诱导HepG2细胞凋亡可能与P53、bcl-2和Fas基因表达有关。

Induction of apoptosis in human liver carcinoma HepG2 cell line by 5-allyl-7-gen-difluoromethylenechrysin

AIM： To investigate the effect of 5-allyl-7-gen-difluoromethylenechrysin （ADFMChR） on apoptosis of human liver carcinoma HepG2 cell line and the molecular mechanisms involved.METHODS： HepG2 cells and L-02 cells were cultured in vitro and the inhibitory effect of ADFMChR on their proliferation was measured by MTT assay. The apoptosis of HepG2 cells was determined by flow cytometry （FCM） using propidium iodide （PI） fluorescence staining. DNA ladder bands were observed by DNA agarose gel electrophoresis. The influence of ADFMChR on the proxisome proliferator-activated receptor γ （PPARγ）, NF-κB, Bcl-2 and Bax protein expression of HepG2 cells were analyzed by Western blotting.RESULTS： MTT assay showed that ADFMChR significantly inhibited proliferation of HepG2 cells in a dose- dependent manner, with little effect on growth of L-02 cells, and when ICs0 was measured as 8.45 μmol/L and 191.55 μmol/L respectively, the potency of ADFMChR to HepG2 cells, was found to be similar to 5-fluorouracil （5-FU, ICso was 9.27 μmol/L）. The selective index of ADFMChR cytotoxicity to HepG2 cells was 22.67 （191.55/8.45）, higher than 5-FU （SI was 7.05 （65.37/9.27）. FCM with PI staining demonstrated that the apoptosis rates of HepG2 cells treated with 3.0, 10.0 and 30.0 μmol/L ADFMChR for 48 h were 5.79%, 9.29% and 37.8%, respectively, and were significantly higher when treated with 30.0 μmol/L ADFMChR than when treated with 30.0 μmol/L ChR （16.0%） （P 〈 0.05） and were similar to those obtained with 30.0 μmol/L 5-FU（41.0%）. DNA agarose gel electrophoresis showed that treatment of HepG2 cells with 10.0 μmol/L ADFMChR for 48 h and 72 h resulted in typical DNA ladders which could be reversed by 10.00 pmol/1 GW9662, a blocker of PPARy. Western blotting analysis revealed that aEer 24 h of treatment with 3.0, 10.0, 30.0 μmol/L ADFMChR, PPARy and Bax protein expression in HepG2 cells increased but Bcl-2 and NF-κB expression decreased; however, pre-incubation with 10.0 μmol/L GW9662 could efficiently antagonize and weaken the regulatory effect of 3.0, 30.0 μmol/L ADFMChR on PPARy and NF-KB protein expression in HepG2 cells.CONCLUSION： ADFMChR induces apoptosis of HepG2 cell lines by activating PPARγ, inhibiting protein expression of Bcl-2 and NF-κB, and increasing Bax expression.

Tea pigments induce cell-cycle arrest and apoptosis in HepG2 cells

AIM： To investigate the molecular mechanisms by which tea pigments exert preventive effects on liver carcinogenesis. METHODS： HepG2 cells were seeded at a density of 5×10^5/well in six-well culture dishes and incubated overnight. The cells then were treated with various concentrations of tea pigments over 3 d, harvested by trypsinization, and counted using a hemocytometer. Flow cytometric analysis was performed by a flow cytometer after propidium iodide labeling. Bd-2 and p21^WAF1 proteins were determined by Western blotting. In addition, DNA laddering assay was performed on treated and untreated cultured HepG2 cells. RESULTS： Tea pigments inhibited the growth of HepG2 cells in a dose-dependent manner. Flow-cytometric analysis showed that tea pigments arrested cell cycle progression at G1 phase. DNA laddering was used to investigate apoptotic cell death, and the result showed that 100 mg/L of tea pigments caused typical DNA laddering. Our study also showed that tea pigments induced upregulation of p21^WAF1 protein and downregulation of Bcl-2 protein. CONCLUSION： Tea pigments induce cell-cycle arrest and apoptosis. Tea pigments may be used as an ideal chemopreventive agent.

Midkine accumulated in nucleolus of HepG2 cells involved in rRNA transcription

AIM： To invesgate the ultrastructural location of midkine （MK） in nucleolus and function corresponding to its location. METHODS： To investigate the ultrastructural location of MK in nucleolus with immunoelectronic microscopy. To study the role that MK plays in ribosomal biogenesis by real-time PCR. The effect of MK on anti-apoptotic activity of HepG2 cells was studied with FITC- conjugated annexin V and propidium iodide PI double staining through FACS assay. RESULTS： MK mainly localized in the granular component （GC）, dense fibrillar component （DFC） and the border between the DFC and fibrillar center （FC）. The production of 45S precursor rRNA level was decreased significantly in the presence of MK antisense oligonucleotide in the HepG2 cells. Furthermore, it was found that exogenous MK could protect HepG2 from apoptosis significantly. CONCLUSION： MK was constitutively translocated to the nucleolus of HepG2 cells, where it accumulated and mostly distributed at DFC, GC components and at the region between FC and DFC, MK played an important role in rRNA transcription, ribosome biogenesis, and cell proliferation in HepG2 cells. MK might serve as a molecular target for therapeutic intervention of human carcinomas.

Octreotide induces caspase activation and apoptosis in human hepatoma HepG2 cells

AIM: To investigate the role of octreotide on cellular proliferation and apoptosis of human hepatoma (HepG2) cells. METHODS: We studied cellular proliferation, apoptosis and the possible internal caspase-mediated apoptosis pathway involved, after treatment of HepG2 carcinoma cells with octreotide in comparison with the apoptosis caused by tumor necrosis factor-α (TNF-α). Activities of caspase-3, caspase-9, caspase-8 and caspase-2 were studied, while apoptosis was investigated through detection of DNA fragmentation and through identification of apoptotic cells with the annexin-V/propidium iodide flow cytometric method. RESULTS: After an initial increase in HepG2 cellular proliferation, a significant inhibition was observed with 10-8 mol/L octreotide, while TNF-α dose-dependently decreased proliferation. Early and late apoptosis was significantly increased with both substances. Octreotide significantly increased caspase-3, caspase-8 and caspase-2 activity. TNF-α signifi cantly increased only caspase-2. Cellular proliferation was decreased after treatment with octreotide or TNF-α alone but, in contrast to TNF-α, octreotide decreased proliferation only at concentrations of 10-8 mol/L, while lower concentrations increased proliferation. CONCLUSION: Our findings are suggestive of caspasemediated signaling pathways of octreotide antitumor activity in HepG2 cells, and indicate that measurements of serum octreotide levels may be important, at least in clinical trials, to verify optimal therapeutic drug concentrations.

Pro-apoptotic effects of tectorigenin on human hepatocellular carcinoma HepG2 cells

AIM:To investigate the effects of tectorigenin on human hepatocellular carcinoma(HCC)HepG2 cells.METHODS:Tectorigenin,one of the main components of rhizome of Iris tectorum,was prepared by simple methods,such as extraction,filtration,concentration,precipitation and recrystallization.HepG2 cells were incubated with tectorigenin at different concentrations,and their viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.Apoptosis was detected by morphological observation of nuclear change,agarose gel electrophoresis of DNA ladder,and flow cytometry with Hoechst 33342,Annexin V-EGFP and propidium iodide staining.Generation of reactive oxygen species was quantified using DCFH-DA.Intracellular Ca2+was monitored by Fura 2-AM.Mitochondrial membrane potential was monitored using Rhodamine 123.Release of cytochrome c from mitochondria to cytosol was detected by Western blotting.Activities of caspase-3,-8 and-9 were investigated by Caspase Activity Assay Kit.RESULTS:The viability of HepG2 cells treated by tectorigenin decreased in a concentration-and timedependent manner.The concentration that reduced the number of viable HepG2 cells by 50%(IC50)after 12,24 and 48 h of incubation was 35.72 mg/L,21.19 mg/L and 11.06 mg/L,respectively.However,treatment with tectorigenin at 20 mg/L resulted in a very slight cytotoxicity to L02 cells after incubation for 12,24 or 48 h.Tectorigenin at a concentration of 20 mg/L greatly inhibited the viability of HepG2 cells and induced the condensation of chromatin and fragmentation of nuclei.Tectorigenin induced apoptosis of HepG2 cells in a time-and dose-dependent manner.Compared with the viability rate,induction of apoptosis was the main mechanism of the anti-proliferation effect of tectorigenin in HepG2 cells.Furthermore,tectorigenininduced apoptosis of HepG2 cells was associated with the generation of reactive oxygen species,increased intracellular[Ca2+]i,loss of mitochondrial membrane potential,translocation of cytochrome c,and activation of caspase-9 and-3.CONCLUSION:Tectorigenin induces apoptosis of HepG2 cells mainly via mitochondrial-mediated pathway,and produces a slight cytotoxicity to L02 cells.

氯唑沙腙对抗HepG2肿瘤细胞的作用研究

目的 研究氯唑沙腙（chlorzoxazone）对HepG2细胞存活和凋亡的影响。方法 采用MTT法检测氯唑沙腙对体外培养的HepG2细胞存活率的影响，通过检测LDH释放观察氯唑沙腙对HepG2细胞的致坏死作用，通过TUNEL法评价细胞凋亡率，透射镜评价细胞的超微结构。结果 氯唑沙腙在50～500μmol·L^-1浓度范围内对UepG2细胞的存活率均有抑制作用，呈明显的剂量依赖效应关系。荧光显微镜和透射电镜下可见典型的肿瘤细胞凋亡改变。氯唑沙腙在100、200、300及500μmol·L^-1浓度下作用48h均可诱导HepG2细胞凋亡，具有明显的剂量效应关系。氯唑沙腙100、200、300及500μmol·L^-1 4种浓度分别作用于HepG2细胞24、48及72h，发现以上各种浓度在48h即可诱导细胞凋亡，作用时间越长，凋亡率越高，有明显的时间效应关系。结论 氯唑沙腙能够抑制HepG2细胞存活和诱导细胞凋亡。

Apoptosis induced by nucleosides in the human hepatoma HepG2

AIM： To investigate the apoptotic effects of nucleosides on the human hepatoma HepG2. METHODS： The nucleosides included inosine （I）, cytidine （C）, uridine （U）, thymidine （T）, adenosine （A）, and guanosine （G）. Cells were incubated by the mediums with or without nudeosides at 37 ℃ in a 50 mL/L CO2 humidified atmosphere. RESULTS： It was found that the cell viabilities were significantly decreased, when cells were treated with 30 mmol/L I, 30 mmol/L C, 30 mmol/L U, 30 mmol/L T, 0.5 mmol/L A, and 0.5 mmol/L G after 12 h incubation （P〈0.05）. About the apoptotic phenomenon, the cell percentages of sub-G1 cells were significantly increased in the mediums containing nucleosides such as C, U, T, A, and G （P〈0.05）. Furthermore, the caspase-3 activity was increased, when the cells were incubated with T （P〈0.05）. The protein expressions of p53 and p21 showed no difference in each group. To investigate the mechanism of apoptosis induced by nucleosides, it was found that the contents of soluble Fas ligand contents were increased in HepG2 cells following I, U, T, and A treatment （P〈0.05）. But, TNF-α and cytochrome cwere undetectable. CONCLUSION： Thymidine may induce the apoptosis in HepG2, but the effective dosages and reactive time must be investigated in the future study. However, the apoptosis-inducing abilities of other nucleosides were still unclear in this study.

Mechanism of RNA interference targeting at survivin gene on apoptosis of hepatoma-cellular carcinoma cell line HepG2

Objective:The aim of this study was to investigate the molecular mechanism of anti-apoptotic action of survivin to the hepatoma-cellular carcinoma cell line HepG2.Methods:Design and synthesize siRNA gene sequence specifically targeting at HepG2 cell.HepG2 cells cultures were divided into five groups:blank control group,negative control group,low dose group,medium dose group and high dose group.HepG2 cells were treated respectively by pshRNA-survivin-387 of different concentrations.The apoptosis index(AI) was determined by flow cytometry(FCM).Cells were stained with rhodomine-123(Rh123) to detect changes of mitochondrial membrane potentials.The concentration of cytoplasmic cytochrome C(Cyt.C) was continuously determined by ELISA.Relative activities of caspase-9 and caspase-3 were assessed by colorimetric assay.Results:Compared with the control group,due to the function of short interference RNAs(SiRNAs) that suppresses the survivin gene expression,the apoptotic index of transfected groups were significantly higher than those of control groups(F = 13568.68,q = 110.47-327.16,P < 0.01),the apoptosis index of high concentration of transfected cells was higher than the low concentration transfected group(q = 39.63-168.22,P < 0.01).The apoptosis index of high concentrations transfected HepG2 cells was 25.54%,higher than that of blank control group,negative control group,low dose group and medium dose group(5.24%,6.61%,12.63% and 15.64%,respectively).HepG2 cells transfected with SiRNA exhibit gradually decreasing mitochondrial membrane potentials,which then lead to the releasing of Cyt C,following it were the activation of caspase-9 and caspase-3.Conclusion:Survivin performs the function of anti-apoptosis to the HepG2 cells via modulating the apoptosis of mitochondrial.HepG2 cells transfected with SiRNA survivin can significantly induce apoptosis.

硝酸酯/齐墩果酸杂合物的合成及HepG2细胞凋亡抑制活性的研究

为寻找疗效更好的抗肝炎药物,本文以齐墩果酸(OA)为先导化合物,设计、合成了系列硝酸酯类NO供体/OA杂合物(10a、10b、11a～11e和12a～12c),其结构经红外、质谱、核磁共振氢谱及元素分析确证。采用LDH法测定了目标化合物体外抗Fas介导的HepG2细胞凋亡活性,其中化合物12a的活性最强,其HepG2细胞凋亡抑制作用及NO释放量均与阳性对照NCX-1000相当。

乙醇诱导HepG_2细胞凋亡模型的建立及Bcl-2对抗作用

目的研究 Ｂｃｌ－２家族蛋白在乙醇引发肝细胞凋亡时的作用。方法以 ＴＵＮＥＬ法、 ＤＮＡ梯度检测乙醇诱导ＨｅｐＧ_２肝细胞凋亡，以免疫组织化学法检测细胞中Ｂｃｌ－２、Ｂａｘ、Ｂａｋ等蛋白表达情况，并以Ｂｃｌ－２腺病毒感染细胞观察其抗凋亡作用。结果体积分数 ０．２％、 １．０％乙醇处理 ２４ ｈ对 ＨｅＰＧ_２细胞无明显细胞毒性作用；３．０％组则有明显的细胞毒性作用，伴有典型的凋亡特征。免疫组织化学发现Ｂａｘ、Ｂａｋ蛋白在３．０％组显著增加；Ｂｃｌ－２腺病毒载体可部分抑制凋亡。结论 Ｂａｘ、Ｂａｋ蛋白加强表达在乙醇诱导ＨｅｏＧ_２细胞凋亡中起一定作用； Ｂｃｌ－２腺病毒载体可有效抑制乙醇诱导的凋亡。